the functional characterisation of human rmnd5 …...5.2.2 rmnd5 proteins colocalise with nkx3.1 in...
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The Functional Characterisation of Human RMND5 Proteins in Normal
Physiology and Prostate Cancer
Alison Louw
Bsc (Hons)
Student Number 10476359
School of Pathology and Laboratory Medicine
This thesis is submitted in fulfilment of the requirements for the award of Doctor of Philosophy at the University of Western Australia
DECLARATION FOR THESES CONTAINING PUBLISHED WORK AND/OR WORK PREPARED FOR PUBLICATION The examination of the thesis is an examination of the work of the student. The work must have been substantially conducted by the student during enrolment in the degree. Where the thesis includes work to which others have contributed, the thesis must include a statement that makes the student’s contribution clear to the examiners. This may be in the form of a description of the precise contribution of the student to the work presented for examination and/or a statement of the percentage of the work that was done by the student. In addition, in the case of co-authored publications included in the thesis, each author must give their signed permission for the work to be included. If signatures from all the authors cannot be obtained, the statement detailing the student’s contribution to the work must be signed by the coordinating supervisor. Please sign one of the statements below.
1. This thesis does not contain work that I have published, nor work under review for publication. Student Signature .........................................................................................................................................................
2. This thesis contains only sole-authored work, some of which has been published and/or prepared for publication under sole authorship. The bibliographical details of the work and where it appears in the thesis are outlined below. Student Signature .........................................................................................................................................................
3. This thesis contains published work and/or work prepared for publication, some of which has been co-authored. The bibliographical details of the work and where it appears in the thesis are outlined below. The student must attach to this declaration a statement for each publication that clarifies the contribution of the student to the work. This may be in the form of a description of the precise contributions of the student to the published work and/or a statement of percent contribution by the student. This statement must be signed by all authors. If signatures from all the authors cannot be obtained, the statement detailing the student’s contribution to the published work must be signed by the coordinating supervisor.
Student Signature …………………………………………………………………………………………. Coordinating Supervisor Signature. ..……………………………………………………………………
Contents
Declaration i Acknowledgements ii Awards iii Publications iv List of Figures vi List of Tables ix Abbreviations x Abstract xvi 1.0 General Introduction 1 1.1 Prostate Cancer 1
1.1.1 Prostate Cancer Incidence and Mortality 1
1.1.2 Prostate Cancer Risk Factors 2
1.1.3 Prostate Cancer Diagnosis and Treatment 2
1.1.4 Castration Resistant Prostate Cancer 4
1.1.5 Molecular Alterations in Prostate Cancer 5
1.2 NKX3.1 7
1.2.1 NKX3.1 Protein Binding Partners 9
1.2.2 NKX3.1 Target Genes 11
1.2.3 Regulation of NKX3.1 Gene Expression 13
1.3 RMND5 Proteins 16
1.4 Ubiquitin and Ubiquitin-like Proteins 16
1.4.1 Ubiquitin Cascade 17
1.5 Ubiquitin Activating Enzymes (E1) 19
1.5.1 Discovery of E1 Enzymes 19
1.5.2 Structure and Function of E1 Enzymes 20
1.6 Ubiquitin Conjugating Enzymes (E2) 22
1.6.1 The Structure of E2 Enzymes 22
1.6.2 Classification of E2 Conjugating Enzymes 25
1.6.3 E2 Conjugating Enzyme Interactions with E1 and E3 Enzymes 26
1.6.4 Roles of E2 Enzymes in Ubiquitin Chain Formation 27
1.6.4.1 Chain Initiating and Chain Elongating E2 Enzymes 27
1.6.4.2 E2 Enzyme Processivity 28
1.3.4.3 E2 Enzyme Ubiquitin Chain Assembly and Linkage Selection 29
1.7 E3 Ubiquitin Ligases 30
1.7.1 HECT Domain E3 Ubiquitin Ligases 31
1.7.1.1 Structure and Mechanisms of Ubiquitin Transfer by HECT
Domains 32
1.7.1.2 Regulation of HECT E3 Ubiquitin Ligases 33
1.7.2 RING Domain E3 Ubiquitin Ligases 35
1.7.2.1 RING Domain Structure 36
1.7.2.2 Mechanism of Ubiquitin Transfer by RING Domain E3s 38
1.7.2.3 Regulation of RING E3 Ubiquitin Ligases 39
1.7.2.4 Single Subunit RING E3 Ubiquitin Ligases 39
1.7.2.5 Multisubunit RING E3 Ubiquitin Ligases 40
1.8 Outcomes of Ubiquitination 42
1.8.1 Ubiquitin Chain Topology Determines Ubiquitinated Protein Fate 42
1.8.2 Ubiquitin Binding Domains Determine Ubiquitinated Protein
Outcome 45
1.9 E3 Ubiquitin Ligases and Cancer 46
1.9.1 E3 Ubiquitin Ligases and the Cell Cycle 46
1.9.2 E3 Ubiquitin Ligases and DNA Damage 47
1.9.2.1 Tumour Suppressor p53 47
1.9.2.2 BRAC1/BARD1 49
1.9.3 E3 Ubiquitin Ligases and Signal Transduction 49
1.10 Statement of Aims 51
2.0 Materials 53
2.1 Reagents 53
2.1.1 Cell Culture 53
2.1.2 Primers 53
2.1.3 Reverse Transcription - Polymerase Chain Reaction (PCR) 54
2.1.4 Plasmids 54
2.1.5 Cloning 55
2.1.6 GST Fusion Protein Production 55
2.1.7 Immunoprecipitation 55
2.1.8 Western Blotting 56
2.1.9 Fluorescence Microscopy 56
2.1.10 General Laboratory Reagents 56
2.2 Commercial Kits 57
2.3 Equipment 59
2.4 Computer Software 60
3.0 Methods 61
3.1 Cell Culture 61
3.1.1 Routine Maintenance of Mammalian Cell Lines 61
3.1.2 Cryopreservation and Thawing of Mammalian Cells 61
3.1.3 Preparation of Cells for Fluorescence Microscopy 62
3.1.4 Transfection of Mammalian Cells 62
3.1.5 Treatment of Mammalian Cells 63
3.2 RNA Extraction and DNase Treatment 64
3.2.1 RNA Extraction 64
3.2.2 DNase Treatment of RNA 65
3.3 Reverse Transcription 65
3.4 Polymerase Chain Reaction (PCR) 65
3.4.1 PCR 65
3.4.2 “A” Tailing of PCR Products 67
3.4.3 Site Directed Mutagenesis 67
3.4.3.1 Mutagenesis PCR 67
3.5 Spectrophotometric Quantitation of RNA/DNA 68
3.6 Agarose Gel Electrophoresis 68
3.7 DNA Purification 68
3.7.1 Purification of DNA 68
3.7.2 Gel Purification of DNA 69
3.8 Cloning of PCR Products 69
3.8.1 Plasmids 69
3.8.2 Restriction Enzyme Digestion of Plasmids 70
3.8.3 Shrimp Alkaline Phosphatase Digestion 70
3.8.4 Ligation Reactions 70
3.8.5 Preparation of Competent Bacterial Cells 75
3.8.5.1 Preparation of Competent Escherichia coli DH5α 75
3.8.5.2 Preparation of Competent Escherichia coli BL21 75
3.8.6 Transformation of Bacterial Cells 75
3.8.7 Preparation of Bacterial/Glycerol Stocks 76
3.9 Small Scale Plasmid Purification 76
3.10 Large Scale Plasmid Purification 77
3.11 GST Fusion Protein Production and Purification 77
3.11.1 Small Scale Production of GST Fusion Proteins 77
3.11.2 Large Scale GST Fusion Protein Production 79
3.11.3 Total Protein Extraction from E. coli BL21 Cells 80
3.12 DNA Sequencing 80
3.13 Immunoprecipitation 81
3.14 Ubiquitin Assays 82
3.14.1 In Vitro Ubiquitin Assay 82
3.14.2 In Vivo Ubiquitin Assay 83
3.15 Western Blotting 84
3.15.1 Preparation of Whole Cell Lysates 84
3.15.2 Polyacrylamide Gel Electrophoresis 84
3.15.3 Western Transfer 85
3.15.4 Immunoblotting (Western Blotting) 85
3.15.5 Coomassie Blue Staining 86
3.16 Microscopic Imaging of Cells 86
3.16.1 Preparation of Slides for Fluorescence Microscopy 86
3.16.2 Preparation of Slides for Immunofluorescence Microscopy 87
3.16.3 Fluorescence Microscopy 87
3.17 Mass Spectrometry 88
4.0 Characterisation of RMND5 E3 Ubiquitin Ligase Activity 90
4.1 Introduction 90
4.1.1 Yeast RMD5/Gid2 90
4.1.2 Human RMND5 Proteins 94
4.1.2.1 RMND5A 94
4.1.2.2 RMND5B 94
4.1.3 Protein Domains 94
4.1.3.1 Lissencephaly 1 Homology Motif (LisH) 94
4.1.3.2 C-Terminal to LisH (CTLH) Domain 98
4.1.3.3 CT11-RanBPM (CRA) Domain 99
4.1.3.4 Really Interesting New Gene (RING) Domain 99
4.1.4 RMND5 Proteins and Cancer 99
4.2 Results 101
4.2.1 Bioinformatics Analyses of RMND5 Protein Architecture 101
4.2.2 Cloning of Full Length RMND5A into pGEX-2TK and Expression of
GST-RMND5 Proteins for In Vitro Ubiquitination Assays 101
4.2.2.1 Cloning of RMND5A into pGEX-2TK 101
4.2.2.2 Small Scale Production of GST, GST-RMND5A and
GST-RMND5B 106
4.2.2.3 Large Scale Production of GST-RMND5A 106
4.2.3 Cloning of RMND5 RING Domains for In Vitro Ubiquitination Assays 107
4.2.3.1 Cloning of RMND5 Proteins into pGEX-2TK 107
4.2.3.2 pGEX-RING Domain Protein Expression 112
4.2.4 In Vitro Auto-Ubiquitination Assays 113
4.2.4.1 Optimisation of In Vitro Auto-Ubiquitination Assays using the
GST-CBL RING Domain 113
4.2.4.2 In Vitro Auto-Ubiquitination Assays Using the GST-RMND5A
and GST-RMND5B RING Domains 116
4.2.4.3 Screening of E2 Conjugating Enzymes in In Vitro Ubiquitination
Assays 116
4.2.4.4 Control In Vitro Auto-Ubiquitination Assays 119
4.2.5 In Vivo Ubiquitination Assays 119
4.2.6 Investigation of the E3 Ubiquitin Ligase Activity of the
RMND5A and RMND5B RING Domains using RMND5A (C356S) and
RMND5B (C358S) RING Domain Mutants 121
4.2.6.1 Introduction of C356S into the RMND5A RING Domain 121
4.2.6.2 Introduction of C358S into the RMND5B RING Domain 127
4.2.6.3 Cloning of the RMND5A (C356S) and RMND5B (C358S) RING
Domains into pGEX-2TK 131
4.2.6.4 Expression and Intracellular Localisation of RMND5A
(C356S) and RMND5B (C358S) 134
4.2.6.5 In Vivo Ubiquitination Activity of RMND5A (C356S) and
RMND5B (C358S) 137
4.2.6.6 In Vitro Auto-Ubiquitination Activity of RMND5A
(C356S) and RMND5B (C358S) RING Domains 139
4.2.7 Examination of the E3 Ubiquitin Ligase Activity of the
RMND5A and RMND5B RING Domains by the Introduction
of Dual Mutations in the RMND5 RING Domain 143
4.2.7.1 Mutation of the RMND5A (C358A/H360A) RING Domain 145
4.2.7.2 Cloning of the RMND5A (C356A/H358A) RING Domain into
pGEX-2TK 150
4.2.7.3 Mutation of the RMND5B (C358A/H360A) RING Domain 152
4.2.7.4 Cloning of the RMND5B (C358A/H360A) RING Domain into
pGEX-2TK 153
4.2.7.5 Expression and Intracellular Localisation of RMND5A
(C356A/H358A) and RMND5B (C358A/H360A) 157
4.2.7.6 In Vivo Ubiquitination Activity of RMND5A
(C356A/H358) and RMND5B (C358A/H360A) Mutant Proteins 157
4.2.7.7 In Vitro Auto-Ubiquitination Activity of RMND5A
(C356A/H358A) and RMND5B (C358A/H360A) RING Domains 163
4.3 Discussion 170
5.0 RMND5 Proteins Ubiquitinate NKX3.1 188
5.1 Introduction 188
5.2 Results 193
5.2.1 RMND5 Proteins Interact with NKX3.1 in LNCaP Prostate Cancer
Cells 193
5.2.1.1 RMND5A Interacts with NKX3.1 193
5.2.1.2 RMND5B Interacts with NKX3.1 193
5.2.2 RMND5 Proteins Colocalise with NKX3.1 in LNCaP Cells 195
5.2.3 Regulation of NKX3.1 Expression in Prostate Cancer Cells 196
5.2.4 RMND5 Protein Effects on NKX3.1 Protein Expression 203
5.2.4.1 RMND5A and RMND5B Reduce NKX3.1 Protein Levels 203
5.2.4.2 RMND5A (C356S and C356A/H358A) and RMND5B
(C358S and C358A/H360A) Reduce NKX3.1 Protein Levels 206
5.2.5 NKX3.1 is Ubiquitinated in LNCaP Cells 208
5.2.5.1 RMND5 Proteins Ubiquitinate NKX3.1 208
5.2.5.2 RMND5A (C356A/C358A) and RMND5B (C358A/H360A)
Ubiquitinate NKX3.1 210
5.3 Discussion 213
6.0 Characterisation of RMND5 Protein Binding Partners 223
6.1 Introduction 223
6.1.1 Characterisation of the CTLH Complex Components 223
6.1.2 Protein Domain Architecture of the CTLH Complex Members 224
6.1.3 The Yeast Vid30 Complex 227
6.1.4 CTLH Complex Components 229
6.1.4.1 Muskelin 230
6.1.4.2 ARMC8α 231
6.1.4.3 RanBPM 232
6.1.4.4 EMP 233
6.2 Results 236
6.2.1 Transcripts Encoding the CTLH Complex Components are
Expressed in Prostate Cancer Cells 236
6.2.2 Cloning of RanBPM 239
6.2.2.1 Cloning of Full Length RanBPM (90kDa) 239
6.2.2.2 Cloning of the RanBPM 55kDa Isoform into pmCherry-C1 243
6.2.3 Interaction between RanBPM and RMND5A/RMND5B 245
6.2.3.1 RMND5A Interaction with RanBPM (55kDa) 245
6.2.3.2 RMND5B Interaction with RanBPM (55kDa) 247
6.2.3.3 RanBPM (55kDa) Interaction with RMND5 proteins 247
6.2.4 Colocalisation of RanBPM with RMND5A and RMND5B 249
6.2.5 Interaction Between RMND5A and RMND5B 251
6.2.5.1 Cloning of RMND5B into pmCherry-C1 251
6.2.5.2 RMND5A and RMND5B Interact in LNCaP Cells 257
6.2.5.3 RMND5A and RMND5B Colocalise in LNCaP Cells 257
6.2.6 Mass Spectrometric Identification of RMND5 Binding Partners 259
6.2.6.1 Identification of RMND5A Binding Proteins 259
6.2.6.2 Investigation of a Putative RMND5A Binding Partner 265
6.2.8.3 Identification of RMND5B Binding Proteins 268
6.3 Discussion 273
7.0 General Discussion 285
7.1 Discussion 285
7.2 Future Directions 293
8.0 References 302 Appendix I: Buffers and Solutions 350 Appendix II: Primer Sequences 366 Appendix III: Sequencing 369 Appendix IV: Mass Spectrometry Mascot Data 381
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Declaration I certify that this thesis does not incorporate, without my acknowledgement, any
material previously submitted for a degree or diploma from any university and that to
the best of my knowledge and belief, does not contain any material previously
published or written by another person except where due reference is made in text.
Candidate: Alison Louw ___________________ Date: ________ Supervisors: Dr Jacqueline Bentel ___________________ Date: ________ Winthrop Professor Jennet Harvey ___________________ Date: ________
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Acknowledgements There are so many people who I would like to thank and without whom this thesis would not have eventuated if not for their support, guidance and distraction! Firstly, I would like to sincerely thank my supervisors Jacky and Jennet. Jacky, I have learned so much from you and I would like to thank you for your mentorship, guidance and advice over the past few years. To Jennet, thank you for your positive advice and much needed perspective when required! To my good friend and lab mate Jasmine, I had a great time shopping, eating great food, drinking cocktails, laughing (and crying) with you! You always provided sound advice and reminded me that I was not alone in this and I hope we will remain good friends for a long time to come. To Lisa, I will remember all of our conversations and the laughs in the bug lab (my refuge!), breakfasts/lunches and your much loved cupcakes. Diana and Dian, thanks for taking the time to listen and offer advice (with Diana’s always starting, “You know Alison, you should just…”) and support – I know I can always count on you! Marc, you were always available to answer any questions and offer much needed advice and perspective – both work and running related! Thank you. To the current lab members, Jamie, Vivian and Abbie, and past members, Darren, Ebony, Agata, Cheryl, Chris, Lily and Ivy thanks for the conversations, including but not limited to experimental advice and entertaining stories about experimental fails (aka “optimisation”) reminding me that I am not the only one to make stupid mistakes (usually essential to the experiment’s success)! I have enjoyed getting to know every one of you and I have learned so much from everyone. I will cherish the good times, which were many – what ever happened to Friday morning coffee (or hot/iced chocolate)? A ritual that definitely needs to be resurrected I think! To my wonderful family, my Moms, Janet, Johan, Amber, Chelsey and my grandparents, I would never have made it this far without your love and never ending support through good and bad. Thank you for listening to my practice talks – even though you probably didn’t know what I was talking about always pointing to smears and bands all the time! – if it was important to me you were always there to help (and agree with me about the all-important colour schemes). Last but not least, the sixth family member my Chippey, thanks for the welcome feathery distraction and chats – even though I know most of the time you were only interested in my food or colourful shoes! I will be forever grateful to pump classes and running training for the chance to vent my frustrations about science in general and certain experiments in particular (which were seemingly endless), I have retained my sanity! To ASOS etc and the city of Perth, I appreciate the evening and lunch time retail therapy that was badly needed to gain some much needed clarity! Thank you too to the Head of School and Graduate Research Coordinator of the School of Pathology and Laboratory medicine and the members of the Research Centre next door, especially Kay for putting up with me always reporting broken equipment (I promise I didn’t break all that stuff!). Again to everyone, I say thank you and I hope to keep in touch in the future - Alison
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Awards During my studies I have been the recipient of a Prescott Postgraduate Scholarship from
the University of Western Australia.
I was the recipient of the Australia and New Zealand Society for Cell and
Developmental Biology Poster Prize for my poster presentation entitled, “ RMND5
Proteins Target the Prostatic Tumour Suppressor, NKX3.1 for Proteasomal
Degradation” at the Combined Biological Sciences Meeting in Perth, Australia , August
2012
I received the Murdoch University Prize for my oral presentation entitled, “RMND5 E3
Ubiquitin Ligases Ubiquitinate the Prostatic Tumour Suppressor, NKX3.1 Targeting it
for Proteasomal Degradation” at the Australian Society for Medical Research (ASMR)
Medical Research Week WA Student Symposium in Perth, Australia, June 2012
I was the recipient a Protein Synthesis, Targeting and Quality Control Best Thematic
Poster Award for my poster presentation entitled, “RMND5 Proteins Function as E3
Ubiquitin Ligases in Prostate Cancer Cells” at the American Society for Biochemistry
for Biochemistry and Molecular Biology in San Diego, USA, April 2012
I received the Murdoch University Prize for my oral presentation entitled, “RMND5
Proteins Function as E3 Ubiquitin Ligases in Prostate Cancer Cells” at the Australian
Society for Medical Research (ASMR) Medical Research Week WA Student
Symposium in Perth, Australia, June 2011
I was the recipient of the Scientific Encouragement Award for my oral presentation
entitled, “The Functional Characterisation of Human RMND5 Proteins in Prostate
Cancer Cells” at the Royal Perth Hospital Young Investigators Day in Perth, Australia,
August 2010
I received the Australian Society for Biochemistry and Molecular Biology Poster Prize
for a poster presentation entitled, “Functional Characterisation of Human RMND5
Proteins in Prostate Cancer Cells” at the Combined Biological Sciences Meeting
(CBSM) in Perth, Australia, August 2010
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Publications Conference Abstracts/Presentations Louw, A. , Thomas, M., Harvey, J., and Jacqueline Bentel (2012) “RMND5 Proteins
are Novel E3 Ubiquitin Ligases that Target the Prostatic Tumour Suppressor,
NKX3.1” ComBio 2012, Adelaide, Australia September 2012
Louw, A. Harvey, J., and Jacqueline Bentel (2012) “RMND5 Proteins Target the
Prostatic Tumour Suppressor, NKX3.1 for Proteasomal Degradation”
Combined Biological Sciences Meeting, Perth, Australia, August 2012
Louw, A, Harvey, J., and Jacqueline Bentel (2012) “RMND5 E3 Ubiquitin Ligases
Ubiquitinate the Prostatic Tumour Suppressor, NKX3.1 Targeting it for
Proteasomal Degradation”, Australian Society for Medical Research WA
Student Symposium, Perth, Australia, June 2012.
Louw, A, Harvey, J., and Jacqueline Bentel (2012) “RMND5 Proteins Function as E3
Ubiquitin Ligases in Prostate Cancer Cells”, American Society for
Biochemistry and Molecular Biology (ASBMB) Annual Meeting, San Diego,
CA, April 2012.
Louw, A, Harvey, J., and Jacqueline Bentel (2011) “RMND5 Proteins Function as E3
Ubiquitin Ligases in Prostate Cancer Cells”, Royal Perth Hospital Young
Investigators Day, Perth, Australia, November 2011
Louw, A, Harvey, J., and Jacqueline Bentel (2011) “Interaction of RMND5 Proteins
with the Prostatic Tumour Suppressor, NKX3.1”, Combined Biological
Sciences Meeting, Perth, Australia, August 2011
Louw, A, Harvey, J., and Jacqueline Bentel (2011) “RMND5 Proteins Function as E3
Ubiquitin Ligases in Prostate Cancer Cells”, Australian Society for Medical
Research WA Student Symposium, Perth, Australia, June 2011.
v
Louw, A, Harvey, J., and Jacqueline Bentel (2011) “Human RMND5 Proteins Function
as E3 Ubiquitin Ligases in Prostate Cancer Cells”, Ubiquitin Satellite Meeting,
Melbourne, Australia, February 2011.
Louw, A, Harvey, J., and Jacqueline Bentel (2010) “The Functional Characterisation of
Human RMND5 Proteins in Prostate Cancer Cells”, 12th IUBMB OZBIO
Conference, Melbourne, Australia, October 2010.
Louw, A, Harvey, J., and Jacqueline Bentel (2010) “The Functional Characterisation of
Human RMND5 Proteins in Prostate Cancer Cells”, Royal Perth Hospital
Young Investigators Day, Perth, Australia, August 2010
Louw, A, Harvey, J., and Jacqueline Bentel (2010) “The Functional Characterisation of
Human RMND5 Proteins in Prostate Cancer Cells”, Combined Biological
Sciences Meeting, Perth, Australia, August 2010
Louw, A, Harvey, J., and Jacqueline Bentel (2010) “Functional Characterisation of
Human RMND5 Proteins in Prostate Cancer Cells”, Australian Society for
Medical Research WA Student Symposium, Perth, Australia, June 2010.
vi
List of Figures
1.0 General Introduction 1
Figure 1.1: Prostate cancer incidence 1
Figure 1.2: Chromosomal losses associated with human prostate cancer
initiation and progression 7
Figure 1.3: The ubiquitination cascade 18
Figure 1.4: Domain structure and sequence conservation of UBE1 and UBA6 21
Figure 1.5: Mechanism of ubiquitin activation by E1 ubiquitin conjugating
Enzymes 21
Figure 1.6: Three dimensional structure of E2 enzyme ubiquitin
conjugating domains 23
Figure 1.7: E2 ubiquitin chain linkage selection model 30
Figure 1.8: HECT domain structure 33
Figure 1.9: Regulation of HECT domain E3 catalytic activity 34
Figure 1.10: Mechanism of ubiquitin transfer by HECT and RING domain
containing E3 ubiquitin ligases 35
Figure 1.11: RING domain structure 36
Figure 1.12: RING and U-box domain amino acid residues involved in E2
enzyme interaction 37
Figure 1.13: Multisubunit E3 ubiquitin ligases 40
Figure 1.14: APC and SCF multisubunit E3 ubiquitin ligases 41
Figure 1.15: The type of ubiquitination determines substrate protein fate 43
Figure 1.16: p53 regulation in response to genotoxic stress 48
Figure 1.17: Roles of CBL in the regulation of receptor tyrosine kinase
Signalling 51
3.0 Methods 61
Figure 3.1: Map of the pGEM®-T Easy cloning vector 71
Figure 3.2: Map of the pEGFP-C2 expression vector 72
Figure 3.3: Map of the pmCherry C1 expression vector 73
Figure 3.4: Map of the pGEX-2TK bacterial expression vector 74
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4.0 Characterisation of RMND5 E3 Ubiquitin Ligase Activity 90
Figure 4.1: Proposed mechanism of action of the Vid30 complex 92
Figure 4.2: Protein domain architecture of RMND5 proteins 102
Figure 4.3: Cloning of full length RMND5A into pGEX-2TK 104
Figure 4.4: Expression and purification of full length GST-RMND5A
and GST-RMND5B 108
Figure 4.5: Cloning of the RING domains of RMND5A, RMND5B and CBL
into pGEX-2TK 110
Figure 4.6: Expression and purification of the GST-RING domains of
RMND5A, RMND5B and CBL 114
Figure 4.7: Optimisation of in vitro ubiquitination assays 117
Figure 4.8: RMND5 RING domains mediate ubiquitin transfer with
specific E2 conjugating enzymes 118
Figure 4.9: Control reactions for in vitro ubiquitination assays 120
Figure 4.10: In vivo ubiquitination activity of RMND5 proteins 122
Figure 4.11: Site directed mutagenesis of the RING domains of
RMND5A and RMND5B 123
Figure 4.12: Preparation of pEGFP-RMND5A (C356S) 125
Figure 4.13: Preparation of pEGFP-RMND5B (C358S) 129
Figure 4.14: Cloning the RMND5A (C356S) and RMND5B (C358S) RING
domains into pGEX-2TK 132
Figure 4.15: Expression and cellular localisation of GFP-RMND5A (C356S)
and GFP-RMND5B (C358S) proteins 135
Figure 4.16: In vivo ubiquitination activity of RMND5A (C356S) and
RMND5B (C358S) 138
Figure 4.17: Expression and purification of GST-RMND5A (C356S) RING
and GST-RMND5B (C358S) RING domains 140
Figure 4.18: In vitro ubiquitination activity of GST-RMND5A (C356S)
and GST-RMND5B (C358S) RING domains 144
Figure 4.19: Site directed mutagenesis of the RING domains of RMND5A
and RMND5B to produce RMND5A (C356A/H358A) and
RMND5B (C358A/H360A) 146
viii
Figure 4.20: Generation of RMND5A (C356A/H358A) by site directed
mutagenesis and cloning of pEGFP-RMND5A (C356A/H358A) 148
Figure 4.21: Cloning of the RMND5A (C356A/H358A) RING domain into
pGEX-2TK 151
Figure 4.22: Generation of RMND5B (C358A/H360A) by site-directed
mutagenesis and cloning into pEGFP-C2 154
Figure 4.23: Cloning of sequences encoding the RMND5B (C358A/H360A)
RING domain into pGEX-2TK 156
Figure 4.24: Expression and cellular localisation of GFP-RMND5A
(C356A/H358A) and GFP-RMND5B (C358A/H360A) 158
Figure 4.25: In vivo ubiquitination activity of GFP-RMND5A (C356A/H358A)
and GFP-RMND5B (C358A/H360A) 161
Figure 4.26: Expression and purification of GST-RMND5A (C356A/H358A)
RING and GST-RMND5B (C358A/H360A) RING 164
Figure 4.27: Optimisation of in vitro ubiquitination assays for
GST-RMND5A (C356A/H358A) RING and GST-RMND5B
(C358A/H360A) RING 168
Figure 4.28: In vitro ubiquitination activity of GST-RMND5A (C356A/H358A)
and GST-RMND5B (C358A/H360A) 169
5.0 RMND5 Proteins Ubiquitinate NKX3.1 188
Figure 5.1: Post-translational modification of NKX3.1 190
Figure 5.2: NKX3.1 interacts with RMND5A and RMND5B in prostate cancer
Cells 193
Figure 5.3: RMND5 proteins colocalise with NKX3.1 in LNCaP cells 197
Figure 5.4: Determination of NKX3.1 half-life 199
Figure 5.5: Degradation of NKX3.1 by the proteasome 200
Figure 5.6: Lysosomal processing of NKX3.1 202
Figure 5.7: Overexpression of RMND5A or RMND5B reduces NKX3.1 levels 204
Figure 5.8: Proteasome inhibition restores NKX3.1 protein levels following
RMND5 overexpression 205
Figure 5.9: Overexpression of wild-type and mutant RMND5 proteins reduces
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NKX3.1 levels 207
Figure 5.10: Ubiquitination of NKX3.1 in vivo by RMND5A and RMND5B 209
Figure 5.11: In vivo ubiquitination of NKX3.1 following overexpression of
wild-type and mutant RMND5 proteins 212
6.0 The CTLH Complex 223
Figure 6.1: Protein domain architecture of the CTLH complex components 225
Figure 6.2: Predicted Vid30 and CTLH complex topology 229
Figure 6.3: Optimisation of RMND5A, RMND5B, RanBPM, muskelin,
Twa1, EMP, ARMC8α and C17orf39 PCR conditions 237
Figure 6.4: The CTLH complex components are expressed in prostate and
breast cancer cells 240
Figure 6.5: Cloning of the RanBPM coding region 241
Figure 6.6: Cloning of RanBPM (55kDa) into the pGEM®-T Easy cloning
Vector 244
Figure 6.7: Preparation of the pmCherry-RanBPM (55kDa) expression
plasmid 246
Figure 6.8: RMND5A and RMND5B interact with RanBPM in LNCaP cells 248
Figure 6.9: Optimisation of immunoprecipitation reactions using
Protein G Sepharose 250
Figure 6.10: Cherry-RanBPM (55kDa) colocalises with GFP-RMND5A and
GFP- RMND5B 252
Figure 6.11: Cloning of RMND5B into pGEM®-T Easy 253
Figure 6.12: Cloning of RMND5B into pmCherry-C1 255
Figure 6.13: RMND5A and RMND5B interact and colocalise in LNCaP cells 258
Figure 6.14: Immunoprecipitation of GFP-RMND5A and its binding partners 261
Figure 6.15: Identification of putative GFP-RMND5A binding partners 267 Figure 6.16: Immunoprecipitation of GFP-RMND5B and its binding partners 271
x
List of Tables
1.0 General Introduction 1
Table 1.1 – Human ubiquitin conjugating enzymes 24
3.0 Methods 61
Table 3.1 – Cell seeding density and reagents for transfection of mammalian
cells 63
Table 3.2 – LNCaP cell treatments 64
Table 3.3 – Addition of DNA polymerases to PCRs 66
Table 3.4 – PCR conditions 66
Table 3.5 – Site directed mutagenesis PCR conditions 67
Table 3.6 – Beads and buffers utilised for immunoprecipitation reactions 82
Table 3.7 – In vitro auto-ubiquitination assay components 83
Table 3.8 – In vivo ubiquitination assay plasmid combinations 84
Table 3.9 – Primary and secondary antibodies and their respective dilutions 86
Table 3.10 – Excitation and emission wavelengths of fluorescent labels 88
4.0 Characterisation of RMND5 E3 Ubiquitin Ligase Activity 92
Table 4.1 – Human LisH domain containing proteins 95
Table 4.2 – Optimisation of in vitro ubiquitination assay enzyme
Concentrations 166
6.0 Characterisation of RMND5 Protein Binding Partners 223
Table 6.1 – The human CTLH complex components and their yeast orthologues 228
Table 6.2 – Candidate RMND5A binding partners identified by mass
spectrometry 263
Table 6.3 – Function of RMND5A binding proteins identified by mass
Spectrometry 266
Table 6.4 – Putative RMND5B binding partners identified by mass
Spectrometry 270
xi
Abbreviations
°C Degrees Celcius aa Amino Acids AJCC American Joint Committee on Cancer ALLN N-Acetyl-Leu-Leu-Nle-CHO APC/C Anaphase Promoting Complex/Cyclosome APS Ammonium Persulphate AR Androgen Receptor ARF ADP Ribosylation Factor ARF-BP1 ARF-Binding Protein 1 (alias MULE/HUWE1) Arg Arginine ARMC8α/β Armadillo Repeat Containing α/β AS Antisense ATP Adenosine Triphosphate BARD1 BRCA1 Associated RING Domain 1 BCL-2 B Cell Leukemia BDNF Brain-Derived Neurotrophic Factor BIRC6 Baculoviral Inhibitor of Apoptosis Repeat Containing
Protein 6 BLAST Basic Local Sequence Alignment bp Base Pairs BRCA1 Breast Cancer Susceptibility Gene 1 BRCC BRCA1-BRCA2 Containing Complex BRET Bioluminescence Resonance Energy Transfer BRUCE Baculovirus Inhibitor of Apoptosis Repeat Containing
Ubiquitin Conjugating Enzyme (alias BIRC6/Apollon) BSA Bovine Serum Albumin β-TrCP β-Transducin Repeat Containing Protein C2 Ca2+ Binding Motif Ca2Cl Calcium Chloride CBL Casitas B-Lineage Lymphoma CCD Catalytic Cysteine Domain Cdc20 Cell Division Cycle Protein 20 Cdh1 Cadherin 1 CDHC Cytoplasmic Dynein Heavy Chain Cdk Cyclin Dependent Kinase cDNA Complementary DNA CDS Coding Sequence CHIP Carboxyl Terminus of Hsc70 Interacting Protein CK2 Casein Kinase 2 c-MDH Cytoplasmic Malate Dehydrogenase CO2 Carbon Dioxide CRA CT11-RanBPM CRL4 Cullin4A-RING E3 Ubiquitin Ligase CSTF1 Cleavage Stimulation Factor 1 C-terminal Carboxy-Terminal CTLH C-Terminal to LisH Cul Cullen Protein Cys Cysteine DAPK Death Associated Protein Kinase DCAF-1 DDB1 and Cul4 associated factor 1
xii
ddH2O Deionised Water DEPC Diethylpolycarbonate DHT 5α-dihydrotestosterone DMSO Dimethylsulphoxide DNA Deoxyribonucleic Acid dNTP Deoxynucleoside Triphosphate DRE Digital Rectal Examination DSB Double Stranded Breaks DUB Deubiquitinating Enzyme E1 Ubiquitin Activating Enzyme E2 Ubiquitin Conjugating Enzyme E3 Ubiquitin Ligase E4 Conjugation Factor/Ubiquitin Chain Elongation Factor EBS ETS1 Binding Site EDTA Ethylenediaminetetraacetic Acid EEAP Early Endosome Associated Protein eGFP Enhanced Green Fluorescent Protein EGFR Epidermal Growth Factor Receptor ElaC2 ElaC Homologue 2 EMP Erythroblast Macrophage Protein (alias MAEA) ERAD Endoplasmic Reticulum Associated Degradation ERG Ets Related Gene ERK Extracellular Signal Regulated Kinase ESE3 Epithelium Specific Ets Transcription Factor 3 ETS Ethrythroblastosis Virus E26 ETS1 Erythroblastosis Virus E26 Oncogene Homolog 1 EZH2 Enhancer of Zeste Homologue 2 FANCD2 Fanconi Anaemia Group D2 FAT10 F Adjacent Transcript 10/ Human Leukocyte Antigen F
Associated FBPase Fructose 1,6 Bisphosphatase FDA US Food and Drug Administration FCS Fœtal Calf Serum FGFR1 Fibroblast Growth Factor Receptor 1 FMRP Fragile X Mental Retardation Protein FOP FGFR1 Oncogene Partner FRET Fluorescence Resonance Energy Transfer G1/2 Growth Phase1/ 2 (Cell Cycle) G2BR Ube2G2 Binding Region GABAAR Gamma-aminobutyric Acid A Receptor GAT domain GGA and Tom1 Domain Gid Glucose Induced Degradation GST Glutathione S Transferase GWAS Genome Wide Association Studies H1 Helix 1 H2O2 Hydrogen Peroxide HACE1 HECT and Ankyrin Repeat Containing E3 Ubiquitin
Ligase 1 HAUSP Herpes-virus Ubiquitin Specific Protease HDAC Histone Deacetylase HDAC1 Histone Deacetylase 1 HECT Homologous to E6-AP Carboxyl Terminus
xiii
HEK Human Embryonic Kidney Cells HeLa Henrietta Lacks Ovarian Carcinoma Cells HERC HECT and RCC1 Domain Containing Protein HGF Hepatocyte Growth Factor His Histidine HPN Hisitidine-Proline-Asparagine HPV Human Papilloma Virus HRP Horse Radish Peroxidase HRS Hepatocyte Growth Factor-Regulated Tyrosine Kinase
Substrate HSMpp8 Matrix Metalloprotease 8 HUWE1 HECT, UBE, WWE Domain Containing Protein 1 (alias
MULE/ARF-BP1) Hxt7p Hexose Transporter Protein 7 IGF1 Insulin-like Growth Factor 1 IGF1R IGF1 Receptor IGFBP3 IGF1 Binding Protein 3 IgG Immunoglobulin G IKK IKappaβ Kinase IP Immunoprecipitation ITCH Ubiquitin Protein Ligase Itchy Homologue JFK Just One F-box and Kelch Domain Containing Protein JNK c-Jun N-terminal Kinase kbp Kilobase Pairs kDa Kilo Daltons L4/7 Loop 4/7 LAMP1/2 Lysosome Associated Membrane Protein 1/2 LB Luria Bertani LHR Linker Helix Region LHRH Luteinising Hormone Releasing Hormone LIS1 Lissencephaly Protein 1 LisH Lissencephaly 1 Homology Domain LOH Loss of Heterozygosity LUBAC Linear Ubiquitin Chain Assembly Complex Lys Lysine MAEA Macrophage Erythroblast Attacher MAPK Mitogen Activated Protein Kinase MCS Multiple Cloning Site MDH2 Malate Dehydrogenase 2 MDM2 Murine Double Minute Homologue 2 MDMX Murine Double Minute Homologue X MEKK1 Mitogen Activated Portien/ERK Kinase Kinase 1 MG132 Carbobenzoxy-Leu-Leu-Leucinal MgCl2 Magnesium Chloride mL Millilitres mM Millimolar MMS2 Methyl Methanesulphonate Sensitive Gene Product 2 MOPS 3(N-Morpholino)propanesulphonic Acid mRNA Messenger Ribonucleic Acid MS Mass Spectrometry MSR1 Macrophage Scavenging Receptor 1 MULE Mcl-1 Ubiquitin Ligase E3 (alias HUWE1/ARF-BP1)
xiv
MYC Myelocytomatosis Viral Oncogene Homologue N Amino N4BP Nedd4-Binding Protein NaOH Sodium Hydroxide NCBI National Centre for Biotechnology Information N-CoR Nuclear Receptor Corepressor NEDD4/8 Neural Precursor Cell-Expressed, Developmentally Down
Regulated 4/8 NEMO NF-κB Essential Modulator NETN NP-40, EDTA, Tris, NaCl Buffer NF-κB Nuclear Factor Kappa Light Chain Enhancer of Activated
B Cells NHS Normal Horse Serum NP-40 Nonidet P-40 NPAT Nuclear Protein of the Ataxia Telangiectasia Mutated
Locus NSLC Non-Small Cell Lung Carcinoma NTD N-Terminal Domain OD Optical Density OFD1 Oral Facial Density ORF Open Reading Frame p75NTR p75 Neurotrophin Receptor PBGD Porphobilinogen Deaminase PBS Phosphate Buffered Saline PBST Phosphate Buffered Saline Tween-20 PCAN Prostate Cancer Gene 1 PCNA Proliferating Cell Nuclear Antigen PCR Polymerase Chain Reaction PEPCK Posphoenolpyruvate Carboxykinase PHD Plant Homeodomain PIN Prostatic Intraepithelial Neoplasia PIPES Piperazinediethanesulphonic Acid PKC Protein Kinase C pmol Pecomole PMSF Polymethylsulphonyl Fluoride POTEE Prostate Ovary Testis Expressed Protein E PPxY Proline-Proline-any amino acid- Tyrosine PRR Proline Rich Repeat PS Penicillin/Streptomcyin PSA Prostate Specific Antigen PTEN Phosphate and Tensin Homologue Deleted on
Chromosome Ten PTM Post-Translational Modification PUF60 Poly (U) Binding Splicing Factor RA Retinoic Acid RanBP9/10 Ran Binding Protein 9/10 RanBPM M Ran Binding Protein in the Microtubule Organising
Centre (RanBP9) RAR Retinoic acid Receptor RCC1 Regulator of Chromosome Condensation 1 RGG box Arginine (R), Glycine (G) Rich Region RHOT Ras homolog family member T2
xv
RING Really Interesting New Gene RIPA Radioimmune Precipitation Buffer RLD RCC1-like Domains RMD5 Required for Meiotic Nuclear Division 5 (Saccharomyces
cerevisiae) RMND5A Required for Meiotic Nuclear Division 5 homologue A RMND5B Required for Meiotic Nuclear Division 5 homologue B RNA Pol II RNA Polymerase II RNA Ribonucleic Acid rpm Revolutions Per Minute RPMI Roswell Park Memorial Medium RTK Receptor Tyrosine Kinase RT-PCR Reverse Transcription-Polymerase Chain Reaction S phase Synthesis Phase (DNA Replication Phase - Cell Cycle) S Sense SAP Shrimp Alkaline Phosphatase SCF Skip-Cullin-F-box SDS Sodium Dodecyl Sulphate SH2 SRC Homology 2 Siah Seven In Absentia Homologue Sic1 S-phase cyclin/cyclin-dependent kinase inhibitor Sif2p Sir4p Interacting Protein 2 SILAC Stable Isotope Labelling with Amino Acids in Culture siRNA Small Interfering RNA SKIP Ski Interacting Protein 1 SKP S-Phase Kinase-Associated Protein SMAC Second Mitochondria-Derived Activator of Caspases SMART Simple Modular Architecture Research Tool SMGA Smooth Muscle Gamma Actin SMRT Silencing Mediator for Retinoic and Thyroid Receptor SMURF SMAD Ubiquitination Regulatory Factor SNP Single Nucleotide Polymorphism SNW1 SNW Domain Containing Protein 1 (alias SKIP) SOCS Suppressor of Cytokine Signalling SPRY Spla and Ryanodine Receptor SRB Suppressor of RNA Polymerase B SRC Rous Sarcoma Viral Oncogene Homoloue SRE Serum Response Element SRF Serum Response Factor SUMO Small Ubiquitin Like Modifier TAD Transcriptional Activation Domains TAF5/6 Transcription initiation factor TFIID subunit 5/6 T-ALL T Cell Acute Lymphoblastic Leukemias Taq Thermus Aquaticus TBL1 X/Y Transducin β Like Protein 1 X/Y TBL1 Transducin β-like Protien 1 TBLR1 Transducin β-like Protein 1 Related Protein TBS Tris Buffered Saline TBST Tris Buffered Saline Tween-20 TCA Tricarboxylic Acid TCOF1 Treacher Collins-Franceschetti Syndrome 1 TCR T Cell Receptor
xvi
TE Tris/EDTA TKBD Tyrosine Kinase Binding Domain TNF Tumour Necrosis Factor TNM Tumour, Node and Metastasis TOPO I Topoisomerase I TOPORS Topoisomerase Binding RING Finger Protein TOR Target of Rapamycin TPR Tetrapeptide Repeat TRAF TNF Receptor Associated Factor TRITC Tetramethylisothiocyanate TSG Tumour Suppressor Gene TSP-1 Thrombospondin 1 Twa1 Two-Hybrid Associated Protein 1 U Units Uae/UBA Ubiquitin Activating Enzyme Ub Ubiquitin UBA Ubiquitin Associated Domain UBC Ubiquitin Conjugating Domain/Enzyme UBD Ubiquitin Binding Domain UBE Ubiquitin-Like Modifier Activator Enzyme Ubl Ubiquitin-Like Protein/Domain UBM Ubiquitin Binding Motif UBZ Ubiquitin Binding Zinc Finger UEV Ubiquitin Enzyme Variants UFD2 Ubiquitin Fusion Degradation UIM Ubiquitin Interacting Motif UPS Ubiquitin Proteasome System USP Ubiquitin Specific Protease UTR Untranslated Region UV Ultraviolet V Volts VEGF-C Vascular Endothelial Growth Factor C VEGF-C Vascular Endothelial Growth Factor C VHL Von Hippel Landau VHL-CBC VHL-cullin 2-elongin B-elongin C Vid Vacuolar Import and Degradation WD40 Tryptophan-Aspartic Acid (W-D) dipeptide WDR26 WD repeat containing protein 26 WW Tryptophan-Tryptophan WWE Tryptophan-Tryptophan-Glutamic Acid WWP1 WW Domain Containing Protein 1 Y/FPPxxP Tyrosine/Phenylalanine-Proline-Proline-any amino acid-
any amino acid-Proline
xvii
Abstract
RMND5A and RMND5B are highly homologous uncharacterised proteins named after
their yeast orthologue, Required for Meiotic Nuclear Division 5 (RMD5), a RING
domain-containing E3 ubiquitin ligase. RMND5B was originally identified in our
laboratory to interact with the prostatic tumour suppressor, NKX3.1, expression of
which is reduced or undetectable in up to 80% of metastatic prostate tumours.
Bioinformatics analyses of the cDNA and translated sequences of RMND5A and
RMND5B identified four protein-protein interaction domains, a Lissencephaly 1
homology (LisH), a C-terminal to LisH (CTLH), a CT11-RanBPM (CRA) and a Really
Interesting New Gene (RING) domain. Alignment of the RING domains of RMND5
proteins with that of yeast RMD5 identified that all eight amino acid residues essential
for RING domain folding and therefore activity were identical between the proteins,
suggesting that RMND5A and RMND5B function as E3 ubiquitin ligases. In vitro
ubiquitination assays carried out using the RING domains of RMND5A and RMND5B
and a panel of 11 E2 conjugating enzymes identified that RMND5A interacted with the
E2 enzymes UbcH2, UbcH5b and UbcH5c, whilst RMND5B associated with UbcH5b
and UbcH5c to mediate ubiquitin transfer to substrate lysine residues. Consistent with
this finding, full length RMND5 proteins were associated with ubiquitinated proteins in
vivo in LNCaP prostate cancer cells and this effect was augmented by proteasome
inhibition. Site-directed mutagenesis reduced the in vitro autoubiquitination activity of
the RMND5A (C356A/H358A) and RMND5B (C358A/H360A) RING domain
mutants, while in vivo, interaction of RMND5B (C358A/H360A) with ubiquitinated
proteins was decreased.
In prostate cancer cells, both RMND5A and RMND5B were found to
coimmunoprecipitate with NKX3.1 and overexpression of either RMND5A or
RMND5B enhanced NKX3.1 ubiquitination, resulting in a dose-dependent decline in
NKX3.1 protein levels that was reversed upon proteasome inhibition. These findings
indicated that RMND5 proteins interact with NKX3.1, promoting its ubiquitination and
proteasome-mediated degradation. RMND5A has been reported to form part of a large,
multi-protein complex, the CTLH complex, human orthologue of the yeast Vid30 E3
ubiquitin ligase complex which contains RMD5. All CTLH complex members were
shown to be expressed in prostate and breast cancer cell lines and RMND5A and
RMND5B were identified to interact and colocalise with RanBPM, a proposed core
xviii
component of the CTLH complex. These findings suggested that the CTLH complex is
able to form in prostate cancer cells potentially containing either or both RMND5
proteins. Mass spectrometry, used to identify proteins that interacted by
coimmunoprecipitation with RMND5A or RMND5B, resulted in the isolation of a
number of candidate binding partners including mitochondrial proteins such as ATPase
synthase subunits α and β, and nuclear proteins such as SNW1 and XRCC6. These
results suggest the involvement of RMND5 proteins in the regulation of other cellular
and metabolic processes, which may be investigated in future studies. This thesis has
therefore determined that both RMND5A and RMND5B function as E3 ubiquitin
ligases in prostate cancer cells and are able to target the prostatic tumour suppressor,
NKX3.1 for ubiquitination and proteasome-dependent degradation. As the RMND5
chromosomal loci are frequently disrupted in cancer, further characterisation of the
biological activity of RMND5A and RMND5B will elucidate their roles in normal
physiological processes and the contribution of their deregulated function to cancer
formation and progression.
Chapter 1 General Introduction
Chapter 1: General Introduction
Chapter 1 General Introduction
1
1.0 General Introduction
The expression of factors involved in the development and maintenance of organs
including the prostate is meticulously regulated at all stages, from transcription to
translation, ensuring the appropriate temporal and spatial expression of each gene. Once
produced, the cell maintains tight control over protein function through a range of post-
translational modifications and protein-protein interactions, ensuring the precise activity
of each protein and its degradation when damaged or unnecessary. Deviation of any of
these regulatory pathways is therefore associated with a number of abnormalities
including aberrant gene expression and improper protein folding, activity or turnover,
potentially leading to the development of pathological states including cancer.
1.1 Prostate Cancer
1.1.1 Prostate Cancer Incidence and Mortality
Prostate cancer is the second most common cause of cancer and the sixth leading cause
of cancer related mortality of men worldwide, however in developed countries, prostate
cancer is typically the most commonly diagnosed cancer of men and the third leading
cause of cancer related mortality (Figure 1.1) (Ferlay et al., 2009). The higher incidence
of prostate cancer in western countries such as Australia, USA, UK, New Zealand and
Europe is well documented, and although the prostate cancer mortality rates in these
countries are declining, the mortality rates in less developed countries in Africa, Asia
and Eastern Europe are rising (Center et al., 2012).
Figure 1.1: Prostate cancer incidence. The incidence of prostate cancer is particularly high in developed countries including Australia/New Zealand and is lowest in Asia and Africa (Ferlay et al., 2010).
Chapter 1 General Introduction
2
It is proposed that differences in prostate cancer incidence and mortality between
developed and non-developed countries are due to genetic and lifestyle factors as well
as differences in disease diagnosis and treatment availability (Center et al., 2012).
1.1.2 Prostate Cancer Risk Factors
A number of risk factors for prostate cancer development have been proposed including
diet, occupation and infection, however the best defined risk factors are age, family
history and ethnicity (Schottenfeld and Fraumeni, 2006; Brawley, 2012).
Approximately 30% of men over the age of 50 and 75% of men over the age of 80 years
exhibit evidence of prostate cancer at autopsy, while the median age at diagnosis of
clinical prostate cancer between 2001 – 2010 was 67 years of age (Brawley, 2012;
Eylert and Persad, 2012). Prostate cancer incidence rates have increased since the mid-
1980s and in 2005, the incidence rates of prostate cancer were 3.64 and 7.23 times
higher among males aged 50-59 and <50, respectively compared to those in 1986
(Brawley, 2012). The younger age at diagnosis is likely to be due in part to improved
screening and detection methods including measurement of circulating prostate specific
antigen (PSA) levels (Section 1.1.3), more sensitive imaging techniques and greater
public awareness of the disease (Damber and Aus, 2008). Although familial prostate
cancer syndromes are relatively rare, accounting for <10% of cases (Section 1.1.5), men
with an affected first degree relative have an estimated 2-3 fold increased risk for
development of the disease themselves, which is likely to result from both genetic and
lifestyle factors, including diet (Powell, 2011). African American men have a 1.4 times
higher risk of being diagnosed with prostate cancer than European American men and
are at a 2-3 times higher risk of dying from the disease, and this has been attributed to a
combination of dietary, genetic and socioeconomic factors (Chornokur et al., 2011;
Powell, 2011).
1.1.3 Prostate Cancer Diagnosis and Treatment
Screening and detection of prostate cancer can involve digital rectal examination (DRE)
and measurement of serum PSA levels, with many men asymptomatic during the early
stages of disease. PSA is present in both normal and malignant prostatic epithelial cells,
and although increased serum PSA levels may indicate the presence of prostate cancer,
other conditions such as prostatitis and benign prostatic hyperplasia may also be
associated with elevated serum levels of PSA (Porter and Brawer, 1993; Hochreiter,
Chapter 1 General Introduction
3
2008). Abnormalities detected using DRE and PSA testing may be further investigated
by transurethral ultrasound guided needle biopsy to sample the prostate tissue for
histopathological analysis and diagnosis of prostate cancer (Greene et al., 2009; Bailey
and Brewster, 2011).
Treatment of prostate cancer depends upon the stage of the tumour, which can be
categorised using the American Joint Committee on Cancer (AJCC) Tumour, Node and
Metastasis (TNM) classification system (Edge et al., 2010). The Gleason score obtained
from histological examination of the tumour tissue is recognised as the preferred
grading system (Gleason and Mellinger, 1974; Greene and Sobin, 2002). Along with
this information, the patient’s age and general health are also important determinants for
assessing potential treatments. Watchful waiting or active surveillance is recommended
for those patients with low grade, small volume tumours and this is due to the side
effects associated with surgery or radiation therapy which include urinary incontinence
and erectile dysfunction (Singer et al., 2012). For organ confined prostate cancer, active
surveillance may also be employed. However in fitter younger men, radical
prostatectomy can be performed to surgically remove the prostate and this treatment is
reported in some studies to be more effective in the longer term for these patients
compared to surveillance (Bill-Axelson et al., 2005; Heidenreich et al., 2008; Wiltz et
al., 2009; Hugosson et al., 2011; Holmberg et al., 2012).
Androgen ablation and anti-androgen therapies may be used in the clinical management
of patients with high-risk localised disease (frequently in combination with
radiotherapy), for patients with elevated PSA levels after local treatment and to treat
patients with metastatic disease (Sharifi et al., 2010). The approach of androgen
depletion is based on the knowledge that prostate tumours are dependent on androgens,
which mediate their effects via the androgen receptor (AR), for their growth and
survival. Androgen deprivation treatment usually involves either surgical
(orchidectomy) or chemical castration using oestrogens (rarely), anti-androgens or
luteinising hormone releasing hormone (LHRH) agonists or antagonists to halt the
production of testosterone by the testes (Harris et al., 2009; Sharifi et al., 2010). LHRH
agonists and antagonists prevent testicular androgen production by antagonising the
release of pituitary gonadotropins, however LHRH agonists are associated with an
initial increase in gonadotropin and therefore testosterone production, leading to acute
growth of the tumour (“tumour flare”) until circulating testosterone levels fall (Harris et
Chapter 1 General Introduction
4
al., 2009). To prevent tumour flare associated with initiation of LHRH agonist
treatment, non-steroidal AR antagonists such as bicalutamide, flutamide and more
recently enzalutamide (MDV3100) are used to block the actions of androgens
(Perlmutter and Lepor, 2007; Connolly et al., 2012; Scher et al., 2012). The
combination of LHRH agonists with anti-androgen treatments, particularly bicalutamide
is known as maximum/combined androgen blockade and is widely used for the
treatment of advanced prostate cancer (Connolly et al., 2012). In addition, treatments
such as ketoconazole which reduce adrenal androgen synthesis may be used in
combination with chemical and surgical castration to further decrease circulating
androgen levels (Perlmutter and Lepor, 2007). Treatments which prevent the synthesis
of androgens such as the CYP17A inhibitor, abiraterone acetate may be used in the
management of prostate cancer, however treatment resistance in the form of upregulated
CYP17A or mutated AR has already been noted (Cai et al., 2011; de Bono et al., 2011).
Inhibitors of 5α-reductase such as finasteride may also be used to block the production
of the potent AR activator 5α-dihdrotestosterone (DHT) from testosterone, reducing
prostate cancer growth (Nacusi and Tindall, 2011).
1.1.4 Castration Resistant Prostate Cancer
Hormonal therapies for prostate cancer are effective in the short term with remissions
typically lasting 2-3 years. However, most patients develop resistance to these
treatments, termed castration-resistant prostate cancer, and due to the lack of effective
therapies, patient survival from the time of progression is ~16-18 months (Pienta and
Bradley, 2006). Although prostate tumours become castration resistant they still make
use of the AR signalling pathway and a number of mechanisms by which prostate
cancer cells survive the androgen deprived environment have been proposed. These
include androgen hypersensitivity, in which prostate tumours circumvent the depletion
of androgens by developing the ability to respond to very low levels of circulating
androgens (Gregory et al., 2001b). Prostate cancer cells achieve this by increasing
expression of the AR, which may be due to gene amplification, by increased AR
sensitivity to androgens due to AR mutations, or in association with the intratumoral
production of androgens (Visakorpi et al., 1995a; Gregory et al., 2001b; Linja et al.,
2001; Chen et al., 2004; Locke et al., 2008). AR mutations, which have been reported to
occur in ~10% of prostate tumours following androgen deprivation therapy, may also
allow the receptor to bind nonandrogenic or nonsteroidal ligands or undergo ligand
independent activation, as evidenced by the identification of AR splice variants lacking
Chapter 1 General Introduction
5
the ligand binding domain which are constitutively active (Veldscholte et al., 1992;
Zhao et al., 2000; Ueda et al., 2002; Taplin et al., 2003; Marques et al., 2005; Bluemn
and Nelson, 2012). Cross-talk between the AR and other pathways that are able to
activate the AR, for example HER2 and MAPK has been observed (Craft et al., 1999;
Ueda et al., 2002). In a proportion of castrate-resistant prostate cancers, the balance
between AR coactivators and repressors is altered and in particular, increased
coactivator levels have been found in these cells (Gregory et al., 2001a; Ngan et al.,
2003). Another mechanism by which castration resistance can arise is by the use of
alternative non-androgen mediated mechanisms, for example upregulation of
antiapoptotic factors including Bcl-2 has been reported in advanced castration resistant
prostate tumours (McDonnell et al., 1992; July et al., 2002; Pootrakul et al., 2006).
Chemotherapeutic agents such as docetaxel and cabazitaxel, immunotherapies such as
sipuleucel-T and CYP17 inhibitors including abiraterone acetate may be used for the
treatment of castrate-resistant prostate cancer, increasing survival by 2-5 months
(Rehman and Rosenberg, 2012; Gerritsen, 2012). Bone metastases are common in men
with castrate-resistant prostate cancer and treatments include radiotherapy to reduce
bone pain, bisphosphonates such as zoledronic acid and denosumab an antibody
treatment targeting RANK ligand (Shore et al., 2012).
1.1.5 Molecular Alterations in Prostate Cancer
In addition to AR signalling, aberrant expression of many other proteins, tumour
suppressor gene or oncogene products and genetic alterations are common in prostate
cancer and as in many cancers, these involve hereditary and sporadic mutations which
accumulate over time, driving prostate tumour initiation and progression or determining
treatment responses (Visakorpi, 2003). A number of susceptibility loci for prostate
cancer development have been identified by genome wide linkage studies in families
with a high prostate cancer incidence, and these include chromosome 1q24-25
(RNASEL), 17p (ElaC homologue 2 (ElaC2)), 20q13 and chromosome Xq (Smith et al.,
1996; Xu et al., 1998; Berry et al., 2000; Tavtigian et al., 2001; Wang et al., 2002b;
Adler et al., 2003; Wiklund et al., 2004). Germline mutations in the AR, BRCA1,
BRCA2, CYP1B1 and MSR1 genes have also been associated with prostate cancer
susceptibility (Dong, 2006). The identification of genes conferring a predisposition to
prostate cancer development has been challenging in part due to the late age of disease
onset, making it difficult to screen two or more generations for susceptibility loci and
therefore to discern which mutations are associated with disease development (Rubin
Chapter 1 General Introduction
6
and De Marzo, 2004, Xu et al., 2005). In addition, the likelihood that mutations or
variants of multiple genes predispose to prostate cancer development rather than one or
two further complicates the identification of susceptibility loci (Rubin and De Marzo,
2004; Xu et al., 2005).
Sporadic chromosomal abnormalities or mutations in specific genes are commonly
present in prostate cancers, with the most common chromosomal abnormalities
including loss of heterozygosity at 8p, 10q, 13q and 17p and chromosomal gains at 7,
8q, 18q and Xq (Cher et al., 1994; Joos et al., 1995; Visakorpi et al., 1995b; Nupponen
et al., 1998; Dong, 2006). Genes within these loci associated with prostate cancer
initiation and progression include the homeobox gene NKX3.1 (8p21.2), the AKT
signalling regulator PTEN (10q), the Retinoblastoma gene Rb (13q), p53 (17p) and the
MYC proto-oncogene (8q24) (Abate-Shen and Shen, 2000; Visakorpi, 2003). More
recently identified common chromosomal aberrations in prostate cancer involve ETS
gene fusions, particularly the TMPRSS2-ERG gene fusion, which is present in
approximately 50% of localised prostate cancers and is proposed to result in the
overexpression of oncogenic transcription factors including ERG (Mosquera et al.,
2009; Tomlins et al., 2009). Consistent with a multistep theory of cancer development,
the loss of specific chromosomal regions is proposed to be associated with particular
stages of prostate cancer formation and disease progression (Figure 1.2) (Abate-Shen
and Shen, 2000). For example, the preneoplastic lesion, prostatic intraepithelial
neoplasia (PIN) is associated with the loss of chromosomal regions such as 8p
(NKX3.1), followed by tumour initiation and progression which are associated with the
loss of 10q (PTEN), 13q (Rb) and TMPRSS2 gene rearrangement, and invasion,
metastasis and progression to castration resistance mediated in part by the loss of 17p
(p53) (Figure 1.2) (Abate-Shen and Shen, 2000).
The advent of newer sequencing technologies including next generation sequencing
techniques has allowed the identification of specific single nucleotide polymorphisms
(SNP) or base substitutions prevalent in prostate tumours. For example, whole exome
sequencing has identified repeated somatic mutations in SPOP, FOXA1 and MED12,
with base substitutions in the cullin based E3 ubiquitin ligase SPOP present in 6-13% of
prostate tumours (Barbieri et al., 2012). In addition to chromosomal abnormalities and
gene mutations, interruption in caretaker gene expression such as the pi class of
Glutathione-S Transferases (GST) via promoter hypermethylation is present in a high
Chapter 1 General Introduction
7
percentage of prostate tumours (He et al., 1997; Millar et al., 1999). Accumulating data
documenting the spectrum of molecular abnormalities present in prostate cancers may
identify prostate cancer subtypes and so increase understanding of the formation and
indolence or progression of individual prostate tumours. Depending on the major
genetic changes present, targeted therapies could be developed for treatment of specific
prostate cancer subtypes.
1.2 NKX3.1
NKX3.1 is a homeodomain transcription factor, expression of which is localised to the
prostatic epithelium and to a lesser extent, the testis (Prescott et al., 1998; Meeks and
Schaeffer, 2011). In mice, Nkx3.1 expression is the earliest marker of prostate
development and is required for the formation and maintenance of the prostate gland,
with disruption of Nkx3.1 expression during embryogenesis resulting in defects in
ductal morphogenesis and the abnormal production of secretory proteins (Sciavolino et
al., 1997; Bhatia-Gaur et al., 1999; Tanaka et al., 2000; Matusik et al., 2008). Nkx3.1
expression is maintained in adulthood and adult heterozygous (Nkx3.1+/-) and
homozygous (Nkx3.1-/-) mutant mice exhibit prostatic epithelial hyperplasia and
dysplasia which increases in severity with age to resemble the preneoplastic lesion, PIN
(Bhatia-Gaur et al., 1999; Abdulkadir et al., 2002; Kim et al., 2002a; Abdulkadir, 2005;
Abate-Shen et al., 2008). An interesting finding is that PIN lesions in heterozygous
Nkx3.1 mutant mice express undetectable levels of Nkx3.1 protein, suggesting that
Figure 1.2: Chromosomal losses associated with human prostate cancer initiation and progression. Each stage in prostate cancer formation and progression is associated with the disruption of specific chromosomal regions containing tumour suppressor genes and the accumulation of gene mutations or dysregulation of gene expression (Abate-Shen and Shen, 2000).
Chapter 1 General Introduction
8
prostate carcinogenesis is facilitated by loss of Nk3.1 expression (Abdulkadir et al.,
2002). This is supported in cell culture studies where Nkx3.1 overexpression inhibits
cell proliferation, indicating a role for Nkx3.1 in tumour suppression (Kim et al.,
2002a). Although loss of Nkx3.1 expression alone is not sufficient for prostate tumour
formation, Nkx3.1 cooperates with the loss of expression of other tumour suppressor
gene products such as p27 and Pten to promote the development of invasive prostate
adenocarcinomas and tumour metastasis (Kim et al., 2002b; Abate-Shen et al., 2003;
Gary et al., 2004).
In humans, the NKX3.1 gene is located at the chromosomal locus, 8p21.2, a region that
undergoes loss of heterozygosity (LOH) in approximately 80% of advanced prostate
tumours, with NKX3.1 protein levels reduced or undetectable in up to 80% of
metastatic tumours (Emmert-Buck et al., 1995; Vocke et al., 1996; He et al., 1997;
Bowen et al., 2000; Aslan et al., 2006; Barnabas et al., 2011). LOH at 8p21.2 has also
been reported in 20—80% of high grade PIN lesions, indicating that NKX3.1 loss is an
early event in prostate carcinogenesis (Emmert-Buck et al., 1995; Haggman et al.,
1997; Bowen et al., 2000; Asatiani et al., 2005; Aslan et al., 2006; Bethel et al., 2006).
Despite the frequent loss of NKX3.1 expression, epigenetic changes such as classical
hypermethylation of the NKX3.1 promoter or mutations in the NKX3.1 coding region
that would account for the lack of NKX3.1 expression are not commonly reported and
do not correlate with NKX3.1 levels in prostate tumours (Section 1.2.3) (Voeller et al.,
1997; Xu et al., 2000; Ornstein et al., 2001; Asatiani et al., 2005). Additionally, NKX3.1
mRNA levels may not be reduced in prostate tumours, whilst a discordance between
NKX3.1 mRNA and protein levels has been observed in human prostate cancer,
suggesting that translational or post-translational control of NKX3.1 expression may be
altered in prostate tumour cells (Xu et al., 2000; Ornstein et al., 2001; Bethel et al.,
2006). In the prostate tumours of Nkx3.1+/-/Pten+/- mice, Nkx3.1 mRNA expression is
maintained, however Nkx3.1 is mislocalised to the cytoplasm in a subset of prostate
tumour cells, indicating its inability to perform transcriptional regulatory functions
(Kim et al., 2002b; Mimeault and Batra, 2011). Thus, in addition to NKX3.1 LOH in
prostate tumours, altered NKX3.1 gene transcription, potentially involving epigenetic
alterations, and translational or posttranslational dysregulation may contribute to the
reduced or lack of function of NKX3.1 in prostate cancer cells.
Chapter 1 General Introduction
9
Recent studies have shown that NKX3.1 is involved in the DNA damage response by
regulating the activation of the kinases ATM and ATR and thereby their
phosphorylation of histone 2AX (termed γH2AX), an indicator of DNA damage
(Bowen and Gelmann, 2010; Erbaykent-Tepedelen et al., 2011). Upon DNA damage,
NKX3.1 also colocalised with ATM, ATR and γH2AX at DNA damage foci and in the
longer term, NKX3.1 expression reduced the accumulation of γH2AX foci in DNA
damage induced cells, indicative of lower levels of DNA damage (Bowen and Gelmann,
2010). Erbaykent-Tepedelen et al. (2011) determined that the more efficient response
of cells expressing high levels of NKX3.1 to DNA damage is likely to be due to higher
basal levels of phosphorylation of proteins involved in the DNA damage response such
as ATM, CHK2 and H2AX, indicating a constitutively active DNA damage response in
these cells (Erbaykent-Tepedelen et al., 2011). Given the involvement of NKX3.1 in the
DNA damage response, loss of NKX3.1 may result in a defective response to DNA
damage, thereby contributing to prostate tumour initiation and progression.
1.2.1 NKX3.1 Binding Partners
A number of NKX3.1 binding partners have been identified and of interest is the finding
that the majority of these are also transcription factors with which NKX3.1 interacts to
regulate gene expression (Section 1.2.2). In addition, NKX3.1 is able to associate with
other factors that influence gene expression such as histone deacetylase 1 (HDAC1) and
topoisomerase I.
The first NKX3.1 binding partner identified was serum response factor (SRF), a
transcription factor that plays important roles in embryogenesis and in the adult is
required for the growth and differentiation of skeletal muscle (Carson et al., 2000; Li et
al., 2005). The binding of NKX3.1 and SRF has been shown to require the NKX3.1
tinman motif (amino acids 29-35), the acidic domain (amino acids 88-96) and the SRF
interacting motif (amino acids 99-105) located in the amino–terminal region as well as
the carboxy-terminal amino acids 216-234 of NKX3.1, which interact with the SRF
MADS box (Ju et al., 2006; Zhang et al., 2008b). The MADS box of SRF is a DNA
binding and dimerisation motif and has been documented to facilitate interactions of
SRF with the transcription factor, TEL1 (Gupta et al., 2001), as well as the
homeodomain transcription factor, Nkx2.5 (Chen and Schwartz, 1996). The interaction
between SRF and Nkx2.5 requires the homeodomain of Nkx2.5, which is traditionally
characterised as a DNA binding domain, and although the homeodomain had previously
Chapter 1 General Introduction
10
been reported to mediate NKX3.1 interaction with SRF, more recent studies have
shown that it is not necessary for this interaction (Chen and Schwartz, 1996; Carson et
al., 2000; Ju et al., 2006; Zhang et al., 2008b). The amino- and carboxy-terminal
regions of NKX3.1 facilitate its interaction with other proteins (Ju et al., 2006; Zhang et
al., 2008b; Chen et al., 2002), however fine mapping of the specific protein domains
involved has only been performed in the above-mentioned studies.
Other NKX3.1-interacting transcription factors include PDEF, the SP transcription
factors and MYC. Prostate derived ETS factor (PDEF) is a recently isolated member of
the ETS transcription factor family and its expression is confined to epithelial cells of a
number of tissues, including the prostate and breast, where it is purported to function as
a tumour suppressor and in some studies as an oncogene (Oettgen et al., 2000; Steffan
and Koul, 2011; Sood et al., 2012). The interaction between NKX3.1 and PDEF was
shown to be dependent on the NKX3.1 homeodomain and a tyrosine rich sequence
carboxy-terminal to the homeodomain, which interacts with the PDEF ETS domain and
linker region (Chen et al., 2002a; Chen and Bieberich, 2005). Nkx3.1 and SP family
members form complexes both in vitro and in vivo that require the homeodomain and
amino-terminal of Nkx3.1 and the DNA-binding domain of SP proteins (Simmons and
Horowitz, 2006). Interestingly, Nkx3.1 DNA binding is not required for its interaction
with SP family members (Simmons and Horowitz, 2006). Recently, direct interaction of
NKX3.1 with both AR and FOXA1 was reported, with the novel transcriptional
complex forming in an androgen dependent manner (Tan et al., 2012). Nkx3.1 has also
been identified to interact with the oncoprotein Myc, with the process requiring the Myc
II box but occurring in a DNA independent manner (Anderson et al., 2012).
In addition to transcription factors, NKX3.1 has been shown to interact with HDACI, an
enzyme that plays a role in transcriptional repression by deacetylating histones and
thereby aiding in chromatin condensation (Doetzlhofer et al., 1999). As such, NKX3.1
recruits HDAC1 to NKX3.1 response elements to downregulate target gene expression
(Zhang et al., 2008a). HDAC1 is also able to deacetylate (acetylated) proteins, for
example p53, resulting in p53 ubiquitination and degradation (Ito et al., 2002) and it has
been proposed that NKX3.1 interaction with HDAC1 recruits it away from MDM2-p53
complexes, thereby stabilising p53 protein levels and activity (Li et al., 2006). NKX3.1
interaction with the DNA resolving enzyme topoisomerase I, which requires the
homeodomain, enhances topoisomerase activity on DNA, and this effect is supported in
Chapter 1 General Introduction
11
vivo by the observation that prostate tissue from Nkx3.1-/- and Nkx3.1+/- mice exhibits
reduced topoisomerase I activity (Bowen et al., 2007). Given the cellular functions of
topoisomerase I, this interaction, and therefore NKX3.1, have the potential to play a role
in the regulation of transcription, DNA replication or DNA repair.
1.2.2 NKX3.1 Target Genes
Using its homeodomain, NKX3.1 is able to bind to its ‘TAAGTA’ consensus sequence
located in the regulatory regions of target genes, and in cooperation with cofactors,
modulates gene expression. NKX3.1 functions predominantly as a transcriptional
repressor, with analysis of gene expression profiles in the prostates of conditional
Nkx3.1-/- mice identifying that the majority (~80%) of differentially expressed genes
were overexpressed in PIN lesions compared to the percentage of genes exhibiting
reduced expression (~20%) (Song et al., 2009). Although NKX3.1 has typically been
documented to act as a transcriptional repressor, it upregulates the expression of another
prostate specific protein, prostate cancer gene 1 product (PCAN1/GDEP) via binding to
two NKX3.1 binding sites (NBS) located in the PCAN1 promoter (Olsson et al., 2001;
Liu et al., 2008). In mice, Nkx3.1 interaction with Srf activates transcription from the
smooth muscle gamma-actin (SMGA) promoter in a mechanism that is proposed to
involve Nkx3.1 binding to an Nkx3.1 binding site in the SMGA promoter and
recruitment of Srf to nearby Srf binding elements, thereby increasing transcription
(Carson et al., 2000).
NKX3.1 interacts with steroid hormone receptors in the hormonal regulation of gene
expression, competing with the oestrogen receptor for binding sites in the promoter
regions of a subset of oestrogen responsive genes, and thereby acting as a
transcriptional repressor of oestrogen responsive genes (Holmes et al., 2008). In
addition, NKX3.1 represses expression of the AR via binding to an NKX3.1 responsive
element in the AR promoter, in vitro results that are supported by in vivo observations
that prostatic tissue in Nkx3.1-/- mice exhibits higher levels of AR expression (Lei et al.,
2006). However, other studies have reported that AR expression is correlated with
NKX3.1 protein levels in human prostate tumours and that knockdown of NKX3.1
reduces AR expression (Xu et al., 2000; Tan et al., 2012). It has also recently been
shown that NKX3.1 and the AR co-operate to regulate the expression of a number of
androgen-responsive genes and that AR and NKX3.1 are able to bind both the AR and
NKX3.1 promoter regions, implicating the transcription factors in auto-regulation and
Chapter 1 General Introduction
12
coregulation (Tan et al., 2012). Tan et al. (2012) determined that in the presence of
androgens, NKX3.1 binds to an intragenic region 79kb downstream of the AR
transcriptional start site where it is suggested to enhance AR gene expression (Tan et al.,
2012). Similarly, the oncoprotein Myc and Nkx3.1 have been determined to bind to
regulatory sites within the promoters of many common target genes, thereby suggesting
that these proteins coregulate gene expression, although Nkx3.1 was reported to
antagonise the transcriptional activity of Myc on a subset of coregulated target genes
including Hk2 (Anderson et al., 2012). Nkx3.1 loss cooperates with Myc
overexpression in the promotion of prostate carcinogenesis, highlighting the importance
of interactions between these transcriptional regulators (Anderson et al., 2012).
In genome-wide screens performed to identify genes differentially expressed in Nkx3.1
null prostates during various stages of prostate carcinogenesis it was observed that the
differentially expressed gene signatures were similar in Nkx3.1-/- and Pten-/- mouse
prostate tissues (Song et al., 2009). Further, these gene signatures were similar to those
of prostate tumours in mice with prostate restricted expression of constitutively active
Akt (Song et al., 2009). The differential expression of a common subset of genes in
these mouse models was dependent on the loss of Nkx3.1, which occurs early during
prostate carcinogenesis in Pten-/- mice (Song et al., 2009). NKX3.1 has also been
implicated in the regulation of PI3K/Akt signalling by antagonising the expression of
ligands responsible for activating the signalling pathway (Sarker et al., 2009). In its
negative regulation of expression of one of these ligands, vascular endothelial growth
factor C (VEGF-C), NKX3.1 binds to an NKX3.1 response element located at -997 of
the VEGF-C promoter and recruits HDAC1, with this effect abrogated upon siRNA
mediated knockdown of NKX3.1 expression or the use of HDAC1 inhibitors (Zhang et
al., 2008a). By binding to its cognate receptor, VEGFR3, VEGF-C enhances
lymphangiogenesis, and increased levels of VEGF-C are correlated with lymph node
metastasis in prostate cancer (Zhang et al., 2008a). Therefore, given its negative
regulation of VEGF-C expression, NKX3.1 loss may aid in lymphangiogenesis, and this
is supported by the finding that deletion of 8p21.1-21.2 is associated with the
development of lymph node metastases (Oba et al., 2001).
An alternative mechanism by which NKX3.1 negatively regulates PI3K/Akt signalling
is by its downregulation of the expression and function of IGF1, a ligand for the IGF1
receptor (IGF1-R) (Muhlbradt et al., 2009; Zhang et al., 2012). In the PC-3 prostate
Chapter 1 General Introduction
13
cancer cell line, ectopic expression of NKX3.1 was found to reduce IGF1 expression at
both the mRNA and protein level, implicating NKX3.1 in the transcriptional regulation
of IGF1 expression (Zhang et al., 2012). In addition, by upregulating the expression of
IGF1 binding protein 3 (IGFBP3) which antagonises IGF1 activity, NKX3.1 acts to
reduce IGF1 signalling and suppress IGF1 induced cell proliferation (Muhlbradt et al.,
2009). NKX3.1 overexpression was also found to reduce the phosphorylation of
downstream IGF1 signalling factors such as IGF1-R and AKT, whilst in the presence of
an IGF1 variant which is unable to bind IGFBP3, NKX3.1 mediated inhibition of IGF1
signalling was reduced (Muhlbradt et al., 2009; Zhang et al., 2012). Overall, these
studies have demonstrated that by modifying IGF1 signalling, NKX3.1 is able to
downregulate cell growth, thus providing a potential mechanism by which NKX3.1 loss
leads to deregulated cell proliferation in prostate cancer.
NKX3.1 interaction with transcription factors can also modulate their transcriptional
activity, thereby indirectly regulating gene expression. For example, NKX3.1 binding to
PDEF and SP family members is able to repress their transcriptional activation function,
thereby negatively regulating expression of their target genes, including PSA (Chen et
al., 2002a; Simmons and Horowitz, 2006). Although numerous putative SP recognition
elements are located in the human PSA promoter, only those at the distal end were
necessary for NKX3.1 mediated suppression of promoter activity (Simmons and
Horowitz, 2006). In addition to its role as an intracellular transcription factor, NKX3.1
has been investigated as a paracrine transcription factor, and was recently shown to be
secreted from prostate epithelial cells, regulating gene expression in nearby cells (Zhou
et al., 2012). Supporting this finding, the effect was abolished in the NKX3.1 (T164A)
mutant, a germline NKX3.1 mutation reported to cosegregate with hereditary prostate
cancer, which is not able to be secreted (Zhou et al., 2012). Although the NKX3.1
(T164A) mutant has been documented to disturb the NKX3.1 homeodomain and
therefore reduce DNA binding, in a human prostate epithelial cell line the NKX3.1
(T164A) mutant was able to repress transcription from the PSA promoter (Zheng et al.,
2006; Zhou et al., 2012). This indicated that the NKX3.1 (T164A) mutant was still able
to function as a transcription factor, perhaps by its interaction with other transcriptional
regulators or cofactors and therefore that the mutation affected expression of a specific
subset of NKX3.1 target genes (Zhou et al., 2012).
Chapter 1 General Introduction
14
1.2.3 Regulation of NKX3.1 Gene Expression
NKX3.1 expression is regulated by a number of factors including androgens (AR),
retinoids (RAR/RXR) and the ETS transcription factors, ETS1, ERG and ESE3. These
transcription factors include NKX3.1 target genes which NKX3.1 regulates in a
feedback loop. The androgen regulated expression of NKX3.1 is well characterised in
vitro and in vivo (He et al., 1997; Prescott et al., 1998), with the original report of the
cloning of Nkx3.1 identifying that following castration of mice, Nkx3.1 mRNA levels
rapidly declined but were restored by the administration of androgens (Bieberich et al.,
1996). Similarly in human androgen-responsive prostate cancer cell lines, NKX3.1
expression is markedly upregulated upon treatment with androgens and decreases
following androgen withdrawal (He et al., 1997; Prescott et al., 1998). The effects of
androgens on NKX3.1 expression are mediated at least in part by an androgen response
element located in the NKX3.1 5’ promoter (-3013) which negatively regulates NKX3.1
expression and two androgen responsive elements in the NKX3.1 3’UTR which enhance
NKX3.1 expression (Yoon and Wong, 2006; Thomas et al., 2010). The AR is recruited
in a ligand dependent manner to the NKX3.1 promoter (+2 and +39 from the
transcriptional start site), with siRNA-mediated knockdown of AR expression reducing
NKX3.1 mRNA and protein levels (Tan et al., 2012). Thus NKX3.1 and AR coregulate
each other’s expression by binding to consensus sequences located in their own
promoter regions, as well as common target genes (Tan et al., 2012).
The tumour suppressor p53 reduces NKX3.1 expression by preventing androgen-
induced NKX3.1 promoter transactivation, and this effect is opposed by AR
overexpression (Jiang et al., 2006a). p53 was hypothesised to repress NKX3.1
expression by preventing AR binding to an (uncharacterised) androgen response
element located in the NKX3.1 promoter (Jiang et al., 2006a). This is in contrast to the
role of NKX3.1 in stabilising p53 protein levels (Lei et al., 2006). The relationship
between p53 and NKX3.1 is proposed to be linked via PTEN which regulates both p53
stabilisation and transcriptional activity and is suggested to regulate NKX3.1 as Nkx3.1
is down regulated in Pten null prostate tissues (Wang et al., 2003b). Therefore, several
transcription factors with which NKX3.1 cooperates in the regulation of gene
expression and whose expression NKX3.1 regulates also modulate NKX3.1 expression
in a feedback loop. This has also been suggested for Myc, where PIN lesions
overexpressing Myc exhibit low levels of Nkx3.1 expression in comparison to normal
Chapter 1 General Introduction
15
epithelial tissue, indicating a role for Myc in the regulation of Nkx3.1 expression (Iwata
et al., 2010).
NKX3.1 expression is induced by retinoids (9-cis retinoic acid (9-cis RA) and all-trans
retinoic acid (tRA)) which are present at high levels in the prostate (Jiang et al., 2006b;
Thomas et al., 2006). The effects of 9-cis RA are reported to be mediated via two RA
response elements located in the NKX3.1 promoter, while the tRA responsive regions in
the NKX3.1 promoter have not been defined (Jiang et al., 2006b; Thomas et al., 2006).
NKX3.1 expression is regulated both negatively and positively by ETS transcription
family members. ETS1 overexpression induces an increase in NKX3.1 mRNA and
protein levels in prostate cancer cells, and these effects are mediated in part by an ETS1
binding site (EBS) located in the NKX3.1 5’ promoter (Preece et al., 2011). The ETS
transcription factors ESE3 and ERG mediate opposing effects on NKX3.1 expression,
with ERG reducing NKX3.1 expression directly by binding to ERG binding sites in the
NKX3.1 promoter and indirectly by inducing the polycomb group protein, EZH2, which
mediates H3K27 methylation of the NKX3.1 promoter (Kunderfranco et al., 2010).
Conversely, ESE3 is a positive regulator of NKX3.1 expression, binding to the same
response element in the NKX3.1 promoter and activating transcription as well as binding
to the EZH2 promoter, reducing its expression and therefore downregulating NKX3.1
promoter methylation (Kunderfranco et al., 2010). ERG displaces ESE3 binding on the
NKX3.1 and EZH2 gene promoters, indicating that overexpression of ERG has a
negative effect on NKX3.1 expression and therefore promotes cell proliferation
(Kunderfranco et al., 2010). This finding may have implications for prostate cancers
with TMPRSS2-ERG gene fusions that result in ERG overexpression, thus providing a
mechanism for the reduction in NKX3.1 expression in cancers where NKX3.1 is not
disrupted (Clark and Cooper, 2009). Interestingly, transgenic mice overexpressing
either ERG or a TMPRSS2-ERG fusion protein do not develop prostate cancer but
develop PIN. However, simultaneous loss of Pten in these mice results in the
development of invasive carcinoma (Carver et al., 2009; King et al., 2009). As
mentioned previously, mice exhibiting overactive PI3K/Akt signalling display low or
undetectable Nkx3.1 protein levels, suggesting that loss of Nkx3.1 expression caused by
increased PI3K/Akt signalling or ERG overexpression may aid in part the initiation or
progression of prostate cancers in these mice.
Overexpression of the transcription factor SP-1 was found to enhance NKX3.1
expression at both the mRNA and protein level, an effect mediated by SP-1 binding to
Chapter 1 General Introduction
16
two SP-1 recognition elements (+29 to +43, and -60 to -46 from the transcription start
site), which are located in the NKX3.1 promoter (Yu et al., 2009). A SNP in the NKX3.1
5’UTR associated with prostate cancer susceptibility has been determined to encode a
SP-1 binding site, with the susceptible G allele associated with lower levels of NKX3.1
expression in patients carrying this variant (Akamatsu et al., 2010). Thus, patients with
this polymorphism may be predisposed to the development of prostate cancer. NKX3.1
LOH and dysregulation of NKX3.1 regulators provides one mechanism by which
NKX3.1 expression is reduced or lost in prostate tumours, however studies have
reported that NKX3.1 mRNA levels are high in prostate tumours and that there is a
discordance between NKX3.1 mRNA and protein levels (Xu et al., 2000; Ornstein et al.,
2001; Bethel et al., 2006; Kim et al., 2002b). Additionally, Nkx3.1 is reported to be
mislocalised to the cytoplasm in a proportion of prostate tumours, suggesting that in
these cases, aberrant NKX3.1 function is not due to transcriptional abnormalities but
may be due to dysfunctional translation or post-translational control of NKX3.1.
1.3 RMND5 Proteins
In order to identify novel NKX3.1 binding partners, yeast two hybrid assays were
carried out in our laboratory, which identified the FLJ22318 gene product as a potential
NKX3.1 interacting protein. This protein was later renamed RMND5B (NM_022762.3)
and together with RMND5A (NM_022780.3) it forms the RMND5 protein family
(Section 4.1.2). RMND5 proteins share 70% amino acid homology and are highly
conserved between diverse mammalian species, suggesting that they play similar
cellular roles. Named after their yeast orthologue, RMD5, a RING domain E3 ubiquitin
ligase, both RMND5 proteins also possess carboxy-terminal RING domains, suggesting
that they too possess this enzymatic activity (Section 4.1.1, 4.2.1) (Santt et al., 2008).
The aims of this thesis included characterisation of the biological activity of RMND5
proteins and the functional consequences of the interaction between NKX3.1 and
RMND5B, which had been confirmed previously (Dawson, 2006), and potentially
RMND5A. These studies were based on the investigation of RMND5 proteins as E3
ubiquitin ligases.
1.4 Ubiquitin and Ubiquitin-like Proteins Post-translational modifications (PTMs) add a layer of complexity and diversity to the
proteome and currently more than 200 types of PTMs have been described, most of
Chapter 1 General Introduction
17
which are carried out by enzymes (Walsh, 2005). These protein modifications include
the covalent linkage of small molecules such as phosphoryl or glycosyl groups or the
attachment of whole proteins, such as ubiquitin to amino acid side chains. Their
presence may alter the function, stability and cellular localisation of the modified
protein (Walsh, 2005).
The best characterised example of a protein PTM is the small 76 amino acid protein
ubiquitin which was first identified in the 1970s (Ciechanover et al., 1980; Hershko et
al., 1980; Hershko et al., 1981). Ubiquitin, which adopts a characteristic β-grasp or
ubiquitin-like fold, is highly conserved in eukaryotes but is absent from bacteria and
archaea (Vijay-Kumar et al., 1987a; Vijay-Kumar et al., 1987b; Hochstrasser, 2009).
Following the characterisation of ubiquitin as a PTM, a family of small related proteins,
termed ubiquitin-like proteins (Ubl) were identified, all of which are activated and
conjugated to their specific substrates via a similar mechanism, with each containing a
ubiquitin-like fold, even when their sequences are not conserved with that of ubiquitin
(Kerscher et al., 2006). The Ubls, which include small ubiquitin related modifier
(SUMO), neural precursor cell expressed, developmentally down-regulated 8 (NEDD8)
and human leukocyte antigen F adjacent transcript 10 (FAT10), all contain a carboxy-
terminal glycine residue which is the site of conjugation of ubiquitin or the Ubl to its
target site (Kerscher et al., 2006; Hanzelmann et al., 2012). Ubiquitin and most Ubls
must undergo processing to expose the carboxy-terminal glycine residue as they are
produced as inactive precursors, and deubiquitinating enzymes (DUBs) or Ubl-specific
proteases (USPs) cleave ubiquitin or Ubls, respectively before they can be attached to
substrate proteins (Pickart and Rose, 1985; Monia et al., 1989; Kamitani et al., 1997;
Layfield et al., 1999).
1.4.1 Ubiquitin Cascade
In order for the carboxy-terminal glycine residue of ubiquitin and Ubls to be attached
covalently to a target lysine residue, it must undergo activation via an enzymatic
cascade consisting of three steps (Figure 1.3). In the first step, a ubiquitin activating
enzyme (E1) activates free ubiquitin in an ATP dependent manner, forming an E1-
ubiquitin thioester linkage with the carboxy-terminal glycine residue of ubiquitin
(Ciechanover et al., 1981; Haas et al., 1982). The ubiquitin is then passed from the
active site cysteine of the E1 enzyme to that of the next enzyme in the cascade, the E2
or ubiquitin conjugating enzyme (Hershko et al., 1983). The activated ubiquitin
Chapter 1 General Introduction
18
molecule is then attached to the ε-amino group of a lysine residue in the substrate or
previously attached ubiquitin, or in some cases the ubiquitin is linked to the α-amino
group of the amino-terminal residue of ubiquitin or cysteine side-chain of the substrate
protein with the aid of a ubiquitin ligase, the E3 enzyme, which is responsible for
substrate protein recognition and binding (Hershko et al., 1983; Ciechanover and
Schwartz, 1989; Ciechanover and Ben-Saadon, 2004; Cadwell and Coscoy, 2005).
Two main types of E3 ubiquitin ligases have been identified, the homologous to E6-AP
carboxy-terminus (HECT) proteins and the really interesting new gene (RING) proteins,
which are classified according to the domain that is required for interaction with the E2
conjugating enzyme and thus ubiquitin transfer (Deshaies and Joazeiro, 2009). In
addition to this three-step reaction, complexity is added in the form of E4 and DUB
enzymes. E3 enzymes that elongate polyubiquitin chains in cooperation with other E3
enzymes are known as E4 enzymes (Koegl et al., 1999; Hoppe, 2005), and DUB
enzymes remove ubiquitin by hydrolysing the ubiquitin-substrate bond thereby
recycling ubiquitin chains (Reyes-Turcu et al., 2009). The ubiquitin enzymatic cascade
is hierarchical, there are two E1 enzymes, approximately 40 E2 enzymes and over 600
Figure 1.3: The ubiquitination cascade. The ubiquitin conjugating enzyme, E1 activates free ubiquitin in an ATP dependent manner, forming a ubiquitin thioester with the carboxy-terminal glycine residue of ubiquitin. The E1 then associates with the E2 ubiquitin conjugating enzyme resulting in the transfer of the activated ubiquitin molecule to the active site cysteine residue of the E2. The E2 enzyme next binds to an E3 ubiquitin ligase, which recognises and binds the substrate, resulting in the transfer of ubiquitin to the target lysine residue of the substrate, with the carboxy-terminal glycine of ubiquitin linked via an isopeptide bond to the lysine residue of the substrate protein (Adapted from (Fang and Weissman, 2004)).
Chapter 1 General Introduction
19
ubiquitin protein ligases, and therefore it is the E3 enzymes that confer the most
specificity to the pathway (Deshaies and Joazeiro, 2009; Schulman and Harper, 2009).
1.5 Ubiquitin Activating Enzymes (E1)
1.5.1 Discovery of E1 Enzymes
In order for ubiquitin to become attached to its substrate protein or another ubiquitin
molecule, the carboxy-terminal glycine residue must be activated and this initial
activation step is carried out by a ubiquitin activating enzyme, E1 (UBE1) which was
originally described in the early 1980s (Ciechanover et al., 1982; Haas and Rose, 1982).
UBE1 is present in the cell as two isoforms, E1a (117kDa) and E1b (110kDa) that are
produced from alternative translation start sites, with E1b lacking the amino-terminal 40
amino acid residues (Cook and Chock, 1992; Shang et al., 2001). E1a and E1b are
ubiquitously expressed in equal amounts, their levels are steady during all stages of the
cell cycle, both proteins have a half-life of ~20 hours and both appear to possess the
same E2 enzyme charging ability (Handley et al., 1991; Stephen et al., 1996; Shang et
al., 2001). E1a contains a nuclear localisation signal and is itself subject to post-
translational modification by serine phosphorylation, which causes its nuclear
translocation during the G2 phase of the cell cycle (Cook and Chock, 1995). As E1b
remains unphosphorylated and localised in the cytoplasm, E1a is the predominant
isoform in the nucleus, where it is involved in cell cycle progression, with E1 mutant
cell lines undergoing cell cycle arrest at S/G2 and G2 (Finley et al., 1984; Cook and
Chock, 1995).
Plants, marsupials and mice contain more than one Ube1 or Ube1-like gene, and in mice
and marsupials the second Ube1-like gene encodes Ube1y, a protein that shares 90%
amino acid homology to UbeE1x and is a testis-specific enzyme, the gene for which is
encoded on the Y chromosome (Kay et al., 1991; McGrath et al., 1991). In 2007, three
groups reported the existence of a second mammalian E1 enzyme, UBA6/E1-
L2/UBE1L2, encoded by a gene on human chromosome 4 and sharing 40% amino acid
homology with UBE1 (Chiu et al., 2007; Jin et al., 2007; Pelzer et al., 2007). This
enzyme, now termed UBA6 is found in vertebrates, but not in worms, plants or yeast
(Pelzer et al., 2007). It is able to link covalently with ubiquitin both in vitro and in vivo,
and transfers activated ubiquitin onto a subset of E2 enzymes, including UbcH5b
(Section 1.6) (Jin et al., 2007; Pelzer et al., 2007). Interestingly, Chiu et al (2007)
Chapter 1 General Introduction
20
reported that UBA6 can activate the ubiquitin-like protein, FAT10 whose cognate E1
enzyme had not previously been identified, and that RNAi against UBA6 reduced the
formation of FAT10 conjugates in cells (Chiu et al., 2007). FAT10, also known as
diubiquitin, contains two ubiquitin-like domains and its expression is induced by
tumour necrosis factor α (TNFα) and interferon γ (IFNγ), however the cognate E2 and
E3 enzymes for FAT10 are yet to be reported as UBA6 was not able to transfer FAT10
to any E2 enzymes tested (Chiu et al., 2007).
UBA6, like UBE1, is widely expressed in tissues and cell lines, with UBE1 exhibiting
an approximately ten-fold higher level of expression, although UBA6 exhibits higher
expression levels in the testis, suggesting a role for this E1 enzyme in testis
development and function (Pelzer et al., 2007). Jin et al. (2007) reported an E2 enzyme
specific for UBA6 interaction and ubiquitin transfer, USE1, which is coexpressed with
UBA6 in most human tissues, implicating UBA6 in the activation of a distinct
ubiquitination pathway with its own E2 and perhaps E3 enzymes. Uba6 homozygous
knockout mice are embryonically lethal, indicating UBA6 involvement in development,
however, since Fat10 knockout mice show no abnormalities in development, this
lethality is likely not due to defects in the FAT10 pathway (Chiu et al., 2007).
Therefore, although UBE1 is the most abundant of the E1 enzymes, suggesting that it is
responsible for mediating the activation of most ubiquitin dependent pathways, the
newly discovered UBA6 may be responsible for other as yet uncharacterised ubiquitin
and FAT10 pathways in embryonic and/or adult tissues. In addition, UBA6 may be able
to contribute its activity to a proportion of the same regulatory pathways as UBE1, as
both enzymes are able to transfer activated ubiquitin to a subset of the same E2
conjugating enzymes (Jin et al., 2007).
1.5.2 Structure and Function of E1 Enzymes
All E1 enzymes contain a similar three domain architecture, an adenylation domain
made up of two ThiF-homology motifs, a catalytic cysteine domain (CCD), and a
carboxy-terminal ubiquitin fold domain or β-grasp fold which is responsible for E2
binding (Figure 1.4) (Lake et al., 2001; Walden et al., 2003; Huang et al., 2005a). Each
domain plays a role in ubiquitin activation and the transfer of ubiquitin to cognate E2
conjugating enzymes.
Chapter 1 General Introduction
21
The mechanism of catalysis of E1 enzymes, for example UBE1, has been well studied
(Haas and Rose, 1982; Haas et al., 1982). Using their adenylation domain, E1 enzymes
activate ubiquitin by initially binding Mg-ATP and catalysing the formation of a
ubiquitin adenylate with the carboxy-terminal glycine residue of ubiquitin (Figure 1.5)
(Ciechanover et al., 1982; Haas et al., 1982).
Figure 1.5: Mechanism of ubiquitin activation by E1 ubiquitin conjugating enzymes. Ubiquitin is activated in an ATP dependent manner resulting in the formation of a ubiquitin-adenylate, Ub(A) and the release of inorganic phosphate. The E1 active site cysteine residue then attacks the ubiquitin-adenylate, resulting in the formation of a ubiquitin thioester, S~Ub(T) leaving the E1 enzyme able to load a second ubiquitin molecule as a ubiquitin-adenylate Ub(A) before the transfer of the ubiquitin thioester to the ubiquitin conjugating enzyme, E2 (Schulman and Harper, 2009).
Figure 1.4: Domain structure and sequence conservation of UBE1 and UBA6. The E1 enzymes UBE1 and UBA6 contain an adenylation domain, consisting of two ThiF motifs which between UBE and UBA6 share 43% and 57% amino acid homology. The catalytic cysteine domain (CCD) and ubiquitin fold domain of UBE1 and UBA6 share 41%, 32% amino acid homology, respectively (Jin et al., 2007).
Chapter 1 General Introduction
22
This ubiquitin-adenylate is subject to attack by the E1 active site cysteine residue in the
CCD domain, resulting in the formation of a high energy E1-ubiquitin linkage, with the
cysteine residue of the E1 attached to the carboxy-terminal glycine of ubiquitin (Figure
1.5) (Haas and Rose, 1982; Haas et al., 1982). The E1 enzyme is then able to load a
second ubiquitin molecule by the formation of another ubiquitin-adenylate on its
adenylation domain (Figure 1.5), with one ubiquitin covalently bound at the active
cysteine site and the other non-covalently bound at the adenylation active site (Haas et
al., 1982). A thioester transfer reaction subsequently occurs whereby the ubiquitin-
thioester on the E1 is transferred to the active cysteine residue of its cognate E2 enzyme
(Figure 1.5) (Haas et al., 1982).
1.6 Ubiquitin Conjugating Enzymes (E2)
1.6.1 The Structure of E2 Enzymes
The human genome encodes at least 37 ubiquitin conjugating enzymes (E2) (Table 1.1),
however for many of these, the particular E1 enzymes with which they are able to
associate in order to become charged with ubiquitin or Ubl, and the E3 enzymes with
which they interact to transfer activated ubiquitin/Ubl molecules to the substrate remain
poorly characterised (Michelle et al., 2009). All E2 conjugating enzymes contain a
conserved ~150 – 200 amino acid ubiquitin conjugating domain (UBC) (Hofmann and
Falquet, 2001; Burroughs et al., 2008). Structurally, the UBC is composed of four α-
helices, a four stranded anti-parallel β-sheet and a 310 helix located near the active site,
which contains a catalytically active cysteine residue located in a shallow groove (Lin et
al., 2002, Ozkan et al., 2005, Eddins et al., 2006). This is surrounded by conserved
amino acid residues involved in E2~Ub thioester formation and ubiquitination of target
lysine residues (Lin et al., 2002; Ozkan et al., 2005; Eddins et al., 2006).
Within the UBC, the conserved active site catalytic cysteine residue is responsible for
the formation of the ubiquitin thioester (Wu et al., 2003b), while a tripeptide histidine-
proline-asparagine (HPN) motif is located 7-10 amino acid residues to the amino-
terminal of the active cysteine residue (Wenzel et al., 2011). Within this motif, the
histidine residue is believed to be involved in maintaining the structure of the active
site, and the asparagine residue stabilises the oxyanion intermediate formed when the
target lysine residue of the substrate attacks the E2~Ub thioester, resulting in the
formation of an isopeptide linkage between the Ub molecule and target lysine residue
Chapter 1 General Introduction
23
(Figure 1.6) (Wu et al., 2003b; Cottee et al., 2006). Mutation of the asparagine residue
does not impede E1 and E3 interactions with the E2 enzyme or ubiquitin transfer
between the enzymes, suggesting that it is only important for ubiquitin transfer to target
lysine residues (Wu et al., 2003b). Another motif present in the UBC of many E2
enzymes, the Y/FPPxxP*
Martinez-Noel et al., 2001
motif, is found 7 to 11 amino acids amino-terminal to the
HPN motif (Figure 1.6). Structural studies have shown that in the folded protein, the
carboxy-terminal proline residues are located close to a highly conserved tryptophan
residue, positioned carboxy-terminal to both the HPN motif and the catalytic cysteine
residues and this interaction may stabilise the L7 loop and aid in the positioning of the
L4 and L7 loops (Figure 1.6) ( ; Michelle et al., 2009).
* Y = tyrosine, F = phenylalanine, P = proline, x = any amino acid
Figure 1.6: Three dimensional structure of E2 enzyme ubiquitin conjugating domains. The generalised E2 enzyme structure consists of loops labelled L1 to L8, four β-sheets (S1 to S4), four α-helices (H1 to H4), and the 310 helix (h). Conserved amino acid residues are the HPN motif, the catalytic cysteine (C) residue, a conserved tryptophan residue (W) and a PxxPP motif (Michelle et al., 2009).
Chapter 1 General Introduction
24
Table 1.1 – Human ubiquitin conjugating enzymes
Gene Name
Alternative Name(s)
Class Cognate Ub/Ubl and Chain Specificity (in vitro/in vivo)
Biological Role
UBE2A HR6A, RAD6A I Ub (mono, K11, K48,) DNA repair
UBE2B HR6B, RAD6B I Ub (K11, K48, K63) DNA repair
UBE2C UBCX, UbcH10 II Ub (K11) DNA repair
UBE2D1 UbcH5A, E2-17K1 I Ub (mono, K11, K48) Cell cycle regulation
UBE2D2 UbcH5B, UBC4/5, E2-17K2
I Ub (mono, K11, K48, K63) p53, immune
UBE2D3 UbcH5C, E2-17K3 I Ub (mono, K11, K48) DNA repair, apoptosis
UBE2D4 HBUCE1 I Ub (K11, K48) Unknown
UBE2E1 UbcH6 II Ub (K48) Unknown
UBE2E2 UbcH8 II Ub (K11, K48, K63) Unknown
UBE2E3 UBCE4, UbcH9, UbcM2, E2-23K
II Ub (K11, K48, K63) Growth regulation
UBE2F NCE2 II NEDD8 SCF regulation
UBE2G1 Ubc7 I Ub (K48, K63) ER
UBE2G2 Ubc7 I Ub (K48) ER
UBE2H UbcH2, E2-20K III Ub (K11, K48) Unknown
UBE2I Ubc9 I SUMO Cell cycle, chromosome segregation
UBE2J1 NCUBE1 III Ub (K11) ER
UBE2J2 NCUBE2 III Ub (K48) ER
UBE2K Ubc1, HIP-2, E2-25K
III Ub (K48) Protein quality control
UBE2L3 UbcH7, E2-F1, L-UBC, UBCE7
I Unknown Cell Cycle, transcription
UBE2L6 UbcH8, RIG-B I Ub, ISG15 IFN signalling
UBE2M Ubc12 II NEDD8 SCF regulation
UBE2N Ubc13, BLU I Ub (K63) NK-κB signalling, DNA repair
UBE2NL UEV Unknown
UBE2O E2-230K IV Unknown Haematopoiesis
UBE2Q1 NICE-5, Ube2Q II Unknown Unknown
UBE2Q2 II Unknown Mitosis
Chapter 1 General Introduction
25
UBE2R1 Cdc34, Ubc3, E2-32K
III Ub (K48) Cell cycle regulation
UBE2R2 Cdc34B, Ubc3B III Ub (K48) Cell cycle regulation
UBE2S E2-EPF5, E2-24K III Ub (K11) Cell cycle regulation
UBE2T HSPC150 III Ub (mono, K11, K48, K27, K63))
DNA repair
UBE2U III Unknown Unknown
UBE2V1 UEV-1 II UEV, Ub (K63) NK-κB signalling, DNA repair
UBE2V2 MMS2, EDPF-1, UEV2
I UEV, Ub (K63) NK-κB signalling, DNA repair
UBE2V3 UEVLD, UEV-3 Unknown Unknown
UBE2W I Ub (MONO, K11) DNA repair
UBE2Z Use1, HOYS7 IV Acts with UBA6 Apoptosis
AKTIP FTS Unknown Apoptosis, vesicular transport
BIRC6 Bruce, Apollon IV Unknown Apoptosis, cytokinesis
1.6.2 Classification of E2 Conjugating Enzymes
E2s can be classified into four groups depending on their domain architecture. Class I
E2 enzymes contain only the UBC, the minimal region required for E2 activity and the
UbcH5 E2 conjugating enzymes are included in this class (Hofmann and Falquet, 2001;
Winn et al., 2004). Class II E2 enzymes are those which contain the UBC and an
amino-terminal domain, while those containing the UBC with a carboxy-terminal
extension are classified as Class III E2s (Hofmann and Falquet, 2001; Winn et al., 2004;
van Wijk and Timmers, 2010). The amino- and carboxy-terminal extensions of E2
enzymes are important for their activity and regulation of ubiquitination (Kolman et al.,
1992; Silver et al., 1992; Summers et al., 2008). Class IV E2 enzymes possess both
amino- and carboxy-terminal extensions and include two large E2 enzymes, E2-230K
(UBE2O) and apollon (BIRC6/BRUCE). E2-230K is composed of 1292 amino acids, it
plays a role in reticulocyte maturation and haematopoiesis, and is able to ubiquitinate its
substrates directly without the need for an E3 enzyme (Klemperer et al., 1989; Berleth
and Pickart, 1996). The second large E2 enzyme in this class, apollon (BIRC6/BRUCE)
is a 528kDa protein which plays an anti-apoptotic role in the cell by mediating the
ubiquitination and subsequent degradation of SMAC, a proapoptotic protein (Hao et al.,
Chapter 1 General Introduction
26
2004; Sekine et al., 2005). Apollon is also able to ubiquitinate its target proteins directly
without the association of an E3 ubiquitin ligase (Hao et al., 2004).
Alternatively, Michelle et al., (2009) have proposed a different E2 classification system
based on phylogenetic data (Michelle et al., 2009). By this system, E2 enzymes may be
subdivided into 17 subfamilies which do not resemble the abovementioned
classification system, and although the newer proposed classification method takes into
account the functional domains and structure of the E2 enzymes, the 17 families mostly
contain only one or two E2 enzymes, making it more complex compared to the initial
method.
1.6.3 E2 Conjugating Enzyme Interactions with E1 and E3 Enzymes
The function of E2 enzymes in the ubiquitin pathway is to interact with both E1 and E3
enzymes in order to facilitate the transfer of activated ubiquitin to target lysine residues.
Initially the E2 enzyme must interact with a ubiquitin E1 activating enzyme that is
loaded with two ubiquitin molecules, and transfer the ubiquitin to its own cysteine
residue (He and Rape, 2009). The E1 enzyme undergoes conformational alterations
upon ubiquitin binding, which make available negatively charged sites within the UBC
domain of the E1 that can be recognised by lysine residues within the E2 α-helix 1
(H1), with these residues are only present in E2 enzymes that bind ubiquitin (Lee and
Schindelin, 2008). As such, the high affinity of the E2 for the E1 enzyme occurs only
when the E1 is charged with two ubiquitin molecules, thus ensuring specificity of the
E1-E2 interaction and ubiquitin transfer (Huang et al., 2007; Lee and Schindelin, 2008).
The mechanisms by which individual E2 enzymes are able to distinguish between the
two human E1 enzymes, UBE1 and UBA6 are not well understood (Jin et al., 2007).
Following the formation of the E2~Ub thioester, the E2 enzyme must associate with the
E3 ubiquitin ligase to allow substrate ubiquitination. The E1 and E3 interaction sites of
the E2 enzyme are overlapping, requiring the amino-terminal H1 and loops L4 and L7
and as such, the E2 enzyme must disengage from the E1 enzyme before it is able to
interact with the E3 enzyme to enable ubiquitin transfer. Furthermore, the E2 cannot be
recharged with ubiquitin whilst bound to the E3 (Figure 1.6) (Eletr et al., 2005). E2
enzymes can interact with either of the two classes of E3 ubiquitin ligases, HECT and
RING domain containing proteins, with these domains of the E3s interacting with
similar regions of the E2 UBC domain (L4 and L7) (Section 1.6.1). The L4 hydrophobic
Chapter 1 General Introduction
27
phenylalanine amino acid residues appear to be more important for E2-HECT domain
interactions compared to E2-RING interactions, whilst hydrophobic residues in L7 are
required for both HECT and RING interactions with the E2 (Nuber and Scheffner,
1999; Christensen et al., 2007).
The ability of an E2 enzyme to interact with several E3 ubiquitin ligases relates to
multiple E3 interacting residues in the UBC. For example, UBE2N uses the amino acids
arginine 6 and lysine 10 in its recognition of the E3 TRAF6, but residues arginine 7 and
lysine 10 when interacting with CHIP (Zhang et al., 2005; Yin et al., 2009). E2
enzymes are additionally able to recognise and associate with residues outside of the
RING or HECT domains, for example the E2 enzyme UBE2G2 binds the E3 gp78 at its
RING domain and Ube2G2 Binding Region (G2BR) motif, enhancing the interaction
between this E2 and E3 enzyme pair (Chen et al., 2006a; Li et al., 2009). Although the
binding of the E2 and E3 are weak, E2-E3 binding facilitates the release of the ubiquitin
molecule from the E2 active site. This is thought to be due to a conformational change
that takes place upon E3 binding, where the asparagine residue in the HPN motif is
positioned close to the E2 active site, thus stabilising the oxyanion intermediate formed
between the acceptor lysine residue and ubiquitin carboxy-terminal glycine, described
above (Wu et al., 2003b; Ozkan et al., 2005). The low E2 affinity for the E3 is also
hypothesised to favour ubiquitin chain formation because the E2 must disengage from
the E3 before it can become recharged with ubiquitin (Eletr et al., 2005; Ye and Rape,
2009).
1.6.4 Roles of E2 Enzymes in Ubiquitin Chain Formation
1.6.4.1 Chain Initiating and Chain Elongating E2 Enzymes
Ubiquitination of a substrate is initiated when a ubiquitin molecule is attached to the
target lysine residue of the substrate and this monoubiquitination can be built upon by
the attachment of multiple ubiquitin molecules to produce a polyubiquitin chain. E2
enzymes show a preference for ubiquitin chain initiation or elongation (Windheim et
al., 2008), with the E2 enzymes UBE2W and UBE2E2 involved in ubiquitin chain
initiation when they associate with the E3 heterodimer breast cancer type 1
susceptibility protein (BRCA1)- associated RING domain 1 (BARD1), performing
monoubiquitination of the target lysine residue of the substrate (Christensen et al.,
2007). In contrast, the E2s Ubc13-Mms2 and UBE2K are involved in ubiquitin chain
Chapter 1 General Introduction
28
elongation, forming lysine 48 or lysine 63 linked ubiquitin chains upon association with
this E3 complex (Christensen et al., 2007). Some E2 enzymes which are involved in
chain initiation are not specific for the lysine residues in the target which become
ubiquitinated. For example, UbcH5 (UBE2D) family members are able to ubiquitinate
various substrates on multiple lysine residues to initiate chain formation (Kirkpatrick et
al., 2006; Windheim et al., 2008). However, the E2 UBE2T preferentially
monoubiquitinates FANCD2 on specific lysine residues (Alpi et al., 2008). Many chain
elongation E2 enzymes are only able to recognise ubiquitinated substrates, and interact
with the ubiquitin bound to the substrate to attach more ubiquitin molecules, thereby
creating specific chain topologies. UBE2S specifically forms lysine 11 linked
polyubiquitin chains, whereas UBE2K shows a preference for lysine 48 linked ubiquitin
chains, however neither of these E2 enzymes are able to initiate chain formation
(Haldeman et al., 1997; Hofmann and Pickart, 1999; Windheim et al., 2008). As such,
when associated with the E3, TRAF6, UbcH5 (UBE2D) initiates ubiquitin chain
formation and the E2 UBE2N-UBE2VI attaches additional ubiquitin molecules through
lysine 63, activating NF-κB (Petroski et al., 2007). A few E2 enzymes such as yeast
Cdc34 are able to initiate the formation and elongation of ubiquitin chains, and Cdc34
interacts with the E3 SCF to ubiquitinate S-phase cyclin/cyclin-dependent kinase
inhibitor (Sic1), forming lysine 48 linked polyubiquitin chains and targeting Sic1 for
degradation (Verma et al., 1997). Other E2 enzymes are able to initiate chain formation
and catalyse the formation of short ubiquitin chains before a second, chain elongation
E2 extends the short chains (Ye and Rape, 2009).
1.6.4.2 E2 Enzyme Processivity
Ubiquitin chain length and therefore the ultimate fate of the ubiquitinated protein are
dependent on the processivity of ubiquitin chain formation, which is defined as “the
number of ubiquitin molecules transferred to a growing chain during a single round of
substrate association with an E3” (Ye and Rape, 2009). Factors influencing the
processivity of ubiquitin chain formation are the affinity of the E3 for the substrate and
the rate at which the E2 catalyses ubiquitin transfer (Ye and Rape, 2009). For a protein
to be targeted for degradation, a lysine 11 and 48 linked polyubiquitin chain of at least
four ubiquitin molecules is required, and some E2 enzymes have developed novel
mechanisms to increase their processivity. The human anaphase promoting complex
(APC) binds the substrate securin, resulting in its rapid polyubiquitination through
lysine 11, a process catalysed by the E2s UBE2C and UBE2S that results in its
Chapter 1 General Introduction
29
degradation (Rape and Kirschner, 2004). UBE2C recognises a specific region in securin
and ubiquitin known as a TEK box, thereby allowing the E2 to rapidly catalyse
ubiquitin chain initiation by ubiquitinating securin. Elongation occurs by attaching
multiple ubiquitin molecules through UBE2C recognition of the TEK box in both
proteins which, along with the chain elongating E2 UBE2S, increases processivity (Jin
et al., 2008; Williamson et al., 2009). In order to increase processivity, some E2
enzymes, for example UBE2G2, pre-assemble polyubiquitin chains and the chains are
then transferred en bloc to the target lysine residue (Li et al., 2007b; Ravid and
Hochstrasser, 2007). Others form E2-Ub complexes, bound both covalently to the
catalytic cysteine residue of the active site and non-covalently to the backside or β-sheet
of the E2 . This results in the concentration of active E2 enzymes, loaded with ubiquitin,
in close proximity to the E3 enzyme, thereby increasing processivity as occurs (for
example) when the BRCA1-BARD1 complex is autoubiquitinated using UbcH5
(UBE2D) family members (Brzovic et al., 2006).
1.6.4.3 E2 Enzyme Ubiquitin Chain Assembly and Linkage Selection
The ultimate fate of the ubiquitinated protein is dependent on the types of ubiquitin
linkages formed and it is the E2 enzyme which plays a major role in determining not
only the lysine residue on the substrate that is ubiquitinated (Section 1.7) but also the
type of polyubiquitin chains that are formed (Ye and Rape, 2009). Some E2 enzymes
are specific for the types of polyubiquitin chains that they synthesise, with UBE2S
synthesising lysine 11 linked polyubiquitin chains whilst UBE2K and UBE2RI catalyse
the formation of lysine 48 linked ubiquitin chains. A number of E2 enzymes are
additionally able to synthesise ubiquitin chains of these topologies even in the absence
an E3 enzyme (Chen and Pickart, 1990; Haas et al., 1991; Ye and Rape, 2009). In order
to achieve this linkage specificity, the E2 enzyme positions the lysine residue of the
acceptor ubiquitin so that it is exposed to the active site of the E2 which contains the
activated donor ubiquitin, thereby ensuring that specific lysine linkages are formed
between ubiquitin molecules (Figure 1.7) (Petroski and Deshaies, 2005; Eddins et al.,
2006).
Not all E2s are able to confer linkage specificity and members of the UbcH5 (UBE2D)
E2 family which only contain the core UBC domain, do not form specific ubiquitin
chains but rather are able to catalyse the formation of ubiquitin chains of all linkages,
Chapter 1 General Introduction
30
leading to the hypothesis that in this case linkage specificity may be determined by the
E3 enzyme (Brzovic et al., 2006, Ye and Rape, 2009).
Recently, it has been reported that although the E2 enzyme plays a major role in
determining which lysine residues of ubiquitin are linked upon polyubiquitin chain
formation, the E3 enzymes are important in the determination of the type of
ubiquitination (mono- or poly-ubiquitination), ubiquitin linkage type and the identity
and specificity of target lysine residue of the substrate by directing the E2 enzyme
(David et al., 2011). E2 enzymes are therefore an integral part of the ubiquitin
proteasome system and do not merely function to transfer ubiquitin. They are important
for the determination of the length and topology of ubiquitin chains and the
determination of the target lysine residue of the substrate protein thereby determining
the fate of the ubiquitinated substrate.
1.7 E3 Ubiquitin Ligases E3 ubiquitin ligases are involved in the final step of the ubiquitin cascade and are
responsible for bringing together the substrate and activated ubiquitin molecule by
recognising and binding the substrate protein as well as the E2 enzyme loaded with the
Figure 1.7: E2 ubiquitin chain linkage selection model. The E2 enzyme bound to the donor ubiquitin is able to orient the acceptor ubiquitin exposing the correct lysine residue to the active site (Ye and Rape, 2009).
Chapter 1 General Introduction
31
activated ubiquitin. As mentioned previously (Section 1.4.1), E3 enzymes can be
classified according to the domain required for interaction with the ubiquitin-charged
E2 enzyme and ubiquitin transfer. HECT and RING domains are the two main groups
of E3 enzymes, however other domains similar to the RING domain are also involved in
ubiquitination, including U-box and plant homeodomain (PHD) motifs. Another protein
that is able to ubiquitinate proteins without the presence of a recognisable HECT or
RING domain is the deubiquitinating enzyme UCH-L1, which is able to
monoubiquitinate α-synuclein by reversing its deubiquitinating action, thereby
bypassing the need for E1 and E2 enzymes (Liu et al., 2002).
1.7.1 HECT Domain E3 Ubiquitin Ligases
The HECT domain was initially identified in the human papilloma virus E6-associated
protein (E6-AP), which was shown to ubiquitinate p53, targeting it for proteasomal
degradation (Huibregtse et al., 1994; Scheffner et al., 1994). The domain functions by
interacting with the E2 enzyme, which is followed by transfer of the activated ubiquitin
to the HECT domain, resulting in the formation of a HECT~Ub intermediate. The
activated ubiquitin is then transferred to the target lysine residue of the substrate
protein, which is bound to amino-terminal domains of the HECT domain protein
(Scheffner et al., 1995). The HECT domain has been identified in 28 human proteins
and these HECT domain proteins can be divided into three subfamilies, the Nedd4
family consisting of 9 family members, the HERC family (6 members) and finally other
HECT domain containing proteins comprising 13 members (Huibregtse et al., 1995;
Harvey et al., 1999; Rotin and Kumar, 2009).
In addition to the HECT domain, all Nedd4 family members contain an amino-terminal
C2 domain and between two and four WW domains, which regulate substrate binding
and cellular localisation (Ingham et al., 2004). C2 domains are able to bind
phospholipids and proteins and recruit proteins to membranes, thereby regulating
protein localisation (Plant et al., 2000; Dunn et al., 2004). WW domains contain two
conserved tryptophan residues and recognise proline rich motifs including the PPxY†
Sudol et al., 1995
motif, which is recognised by most Nedd4 WW motifs ( ; Staub and
Rotin, 1996). Nedd4 family members include NEDD4, SMURF1, SMURF2 and ITCH
(Rotin and Kumar, 2009). HERC family members consist of proteins ranging from † P = proline, Y = tyrosine, x = any amino acid
Chapter 1 General Introduction
32
100kDa to 500kDa in size dependent on the number of regulator of chromosome
condensation 1 (RCC1)-like domains (RLDs) that they contain. Although most HERC
family members contain only the RLD and HECT domains, some large HERC proteins
such as HERC1 also contain other protein-protein interaction domains such as Spla and
Ryanodine receptor (SPRY) domains and WD40 repeats (Cruz et al., 2001; Garcia-
Gonzalo et al., 2005; Edwin et al., 2010). Other HECT domain containing proteins
contain several different domains located in their carboxy terminus, including HUWE1
(MULE/ARF-BP1) which contains a WWE (Tryptophan-Tryptophan-Glutamine) and a
ubiquitin associated domain (UBA), and HACE1 which contains ankyrin repeats
(Anglesio et al., 2004).
1.7.1.1 Structure and Mechanisms of Ubiquitin Transfer by HECT Domains
The HECT domain, which is always located towards the carboxy terminus of the
protein, is composed of ~350 amino acid residues and can be divided into amino (N)
and carboxy (C) terminal lobes which are joined by a linker region (Huang et al., 1999).
The N terminal lobe associates with the E2 conjugating enzyme whilst the C terminal
lobe contains an active site cysteine residue to which the active ubiquitin molecule is
transferred from the E2 conjugating enzyme active site in a transthiolation reaction
(Huang et al., 1999). The last 60 amino acid residues of the C terminal lobe also appear
to direct ubiquitin chain linkage specificity (Kim and Huibregtse, 2009). Structural
studies of HECT domain E3s bound to E2 enzymes have shed light on the mechanism
of action of these proteins. Crystal structures of the HECT E3s E6-AP and WWP1 in
complex with UbcH7 revealed distances of 41Å or ~16Å, respectively separating the
HECT domain C lobe cysteine of the E3 and the UbcH7 active site cysteine suggesting
that, as this distance is too far for transthiolation to occur, the flexible linker region must
permit conformational changes to reduce the gap to within 6Å (Figure 1.8) (Huang et
al., 1999;(Verdecia et al., 2003).
The type of polyubiquitin chain that is formed upon ubiquitination of a substrate
molecule is important as this determines the ultimate fate of the ubiquitinated protein.
As HECT domain E3 ubiquitin ligases form a thioester intermediate with the activated
ubiquitin molecule prior to the transfer of ubiquitin to the substrate, it is the HECT E3
that determines the ubiquitin chain linkage attached to the substrate, and this is in
contrast to the mechanism of action of RING domain E3 enzymes (Section 1.7.2, 1.8)
(Wang and Pickart, 2005). The mechanism by which HECT E3s synthesise ubiquitin
Chapter 1 General Introduction
33
chains differs, for example, the HECT E3, E6-AP is able to build Lys48 linked ubiquitin
chains on its active site cysteine in the HECT domain in vitro, whereas the HECT E3,
KIAA10 synthesises Lys29 and Lys48 linked free ubiquitin chains (Wang and Pickart,
2005).
1.7.1.2 Regulation of HECT E3 Ubiquitin Ligases
HECT E3 ligases are able to regulate themselves by autoubiquitination as well as being
regulated by other interacting proteins which either enhance or inhibit the HECT E3
activity by releasing autoinhibition, aiding in E2-E3 interaction or blocking E3 substrate
binding. For example, ITCH is regulated by autoinhibition mediated by the interaction
of its WW domain containing a proline rich region (PRR) and its HECT domain
(Gallagher et al., 2006). ITCH becomes activated upon phosphorylation of the PRR
motif by JNK1, which interacts with the HECT domain of ITCH on three residues
(Ser199, Thr222, Ser232) thereby disrupting the intramolecular interactions between the
ITCH domains (Figure 1.9) (Gallagher et al., 2006; Bruce et al., 2008). ITCH activity is
also regulated by binding of N4BP1 to the ITCH WW2 domain, which inhibits the
binding of ITCH to target proteins such as p73α, JUN and p63, thus inhibiting their
ubiquitination (Oberst et al., 2007). SMURF2 is also regulated by auto-inhibition,
Figure 1.8: HECT domain structure. (A) Crystal structure of the HECT domain of E6-AP and UbcH7 showing the N terminal lobe (red) and C terminal lobe (blue) structure of the HECT domain in complex with UbcH7 (yellow) and a 41Å distance between the catalytic cysteine residues (blue squares). (B) Structure of WWP1 bound to UbcH7 showing a distance of only 16Å between the catalytic cysteine residues due to the orientation of the C lobe of WWP1 (Huang et al., 1999, Verdecia et al., 2003, adapted from Rotin and Kumar, 2009)
Chapter 1 General Introduction
34
Figure 1.9: Regulation of HECT domain E3 catalytic activity. (A) SMAD7 activates SMURF2 by using its N-terminal domain (NTD) to interact with both SMURF2 and the E2 UbcH7, bringing them into close proximity. This interaction increases the affinity of SMURF2 for UbcH7. (B) Auto-inhibition of ITCH, mediated by the interaction of its HECT and WW-PRR domains, is relieved by JNK phosphorylation of the PRR domain (Adapted from Kee and Huibregtse, 2007).
thereby protecting it from auto-ubiquitination and degradation (Figure 1.9). In this
process, the C2 and HECT domains of SMURF2 associate to inactivate the enzyme,
with this mechanism of auto-regulation common among HECT E3 ligases including
Nedd4L, NEDD4 and WWP2 (Wiesner et al., 2007; Bruce et al., 2008; Wang et al.,
2010). Interestingly, the auto-inhibition of NEDD4 is released by increasing
intracellular calcium levels, which disrupts the binding of its C2 and HECT domains
(Wang et al., 2010).
The E3 ubiquitin ligase activity of the HECT domain protein SMURF2 is regulated by
the adaptor protein SMAD7, which is required as the conformation of SMURF2 and the
E2 UbcH7 in complex is suboptimal, with the catalytic cysteine residues separated by
50Å (Ogunjimi et al., 2005). SMAD7 uses its amino-terminal to bind both SMURF2
and UbcH7, thereby acting as an adaptor and bringing the two enzymes into close
Chapter 1 General Introduction
35
proximity to allow ubiquitin transfer (Figure 1.9) (Ogunjimi et al., 2005). The low
affinity of SMURF2 for UbcH7 is due to the presence of two hydrophilic amino acids
(His547, Tyr581) within the E2 binding pocket and replacement of the two hydrophilic
amino acids with hydrophobic residues critical for mediating E6-AP-UbcH7 interaction
results in the constitutive activation of SMURF2 (Figure 1.9) (Ogunjimi et al., 2005;
Kee and Huibregtse, 2007).
1.7.2 RING Domain E3 Ubiquitin Ligases
The second major family of E3 ubiquitin ligases possesses a RING domain or RING-
like domain such as the U-box or PHD domain. The RING domain was first described
in 1993 however the function of the domain was not characterised until 1999
(Freemont, 1993; Lorick et al., 1999) and in the intervening years the RING domain
was hypothesised to function as a DNA binding motif (Freemont, 1993; Lovering et al.,
1993; Lorick et al., 1999). RING domains have been identified in over three hundred
proteins and are therefore the most common type of E3 ubiquitin ligase with five times
more members than the HECT family (Fang et al., 2003). RING domain proteins differ
from HECT ligases in the manner in which the charged ubiquitin molecule is transferred
to the target lysine residue of the substrate molecule (Figure 1.10).
Figure 1.10: Mechanism of ubiquitin transfer by HECT and RING domain containing E3 ubiquitin ligases. (a) HECT domains accept the ubiquitin from the ubiquitin conjugating enzyme (E2), forming a ubiquitin thioester before transfer of the activated ubiquitin molecule to the target lysine residue of the substrate. (b) RING domain proteins bind the E2 enzyme and facilitate the direct transfer of the activated ubiquitin molecule to the target lysine residue of the substrate (Adapted from Rotin and Kumar, 2004).
Chapter 1 General Introduction
36
RING domain proteins associate with both the E2 enzyme and the substrate, and
essentially act as a scaffold bringing the E2 and substrate into close proximity and
thereby allowing the transfer of the ubiquitin molecule (Deshaies and Joazeiro, 2009).
In contrast, following interaction with the E2 enzyme, HECT domain E3s form a
HECT~Ub intermediate before the ubiquitin molecule is transferred to the target lysine
residue of the substrate or acceptor ubiquitin (Figure 1.10) (Rotin and Kumar, 2009).
1.7.2.1 RING Domain Structure
The RING domain consists of a sequence of distinctively placed cysteine (C) and
histidine (H) residues which chelate two zinc (Zn2+) ions, thereby forming a cross brace
structure which promotes correct conformation of the domain (Figure 1.11) (Lorick et
al., 1999).
The first, second, fifth and sixth C/H amino acids coordinate the first Zn2+, while the
third, fourth, seventh and eighth C/H residues bind the second Zn2+ ion (Lorick et al.,
1999). The two main RING domain variants present are the C3HC4 (RING-HC) and
C3H2C3 (RING-H2) types, which vary as to the number and placement of their
Figure 1.11: RING domain structure. (A) The canonical RING-HC sequence consists of seven Cysteine (C) residues and one Histidine (His) residue where X is any amino acid and the number refers to the number of amino acids in the linker regions. (B) The cysteine (C) and histidine (H) amino acid residues coordinate two Zinc (Zn2+) ions, forming a cross brace structure (Adapted from Deshaies and Joazeiro, 2009).
Chapter 1 General Introduction
37
histidine and cysteine amino acids. The RING domain, which is catalytically inactive, is
able to associate with the E2 enzyme, acting as a scaffold to facilitate ubiquitin transfer.
Variations in the canonical RING domain sequence have been identified in functional
E3 ubiquitin ligases, for example RBQ1 contains an aspartate at position eight and
RBX1 contains an asparagine residue at position 4 (Zheng et al., 2002; Deshaies and
Joazeiro, 2009). Other conserved amino acid residues in the RING domain that are
important for E2 enzyme binding have been identified with most positioned close to the
zinc coordinating residues and corresponding to the characterised points of contact
between the RING domain and E2 (Figure 1.12) (Deshaies and Joazeiro, 2009).
Many of the three hundred RING domain containing proteins have not yet been
characterised, and although it is believed that the majority of these proteins possess E3
ubiquitin ligase activity, a number of RING domain containing proteins are known to be
non-functional as E3s on their own. For example, the RING domain containing proteins
BARD1 and MDMX do not possess intrinsic E3 activity, but interact with other RING
domain containing proteins, BRCA1 and MDM2, respectively through their RING
domains, enhancing the E3 activity of their interacting E3 (Hashizume et al., 2001;
Linares et al., 2003). In addition to their ability to heterodimerise, RING domain
Figure 1.12: RING and U-box domain amino acid residues involved in E2 enzyme interaction. Amino acid residues important for mediating RING/HECT domain-E2 interactions (red) are distinct from the Zinc coordination residues (blue) of the RING domain proteins c-Cbl and cIAP2. The corresponding residues are marked in the U-box ubiquitin ligase CHIP (Deshaies and Joazeiro, 2009).
Chapter 1 General Introduction
38
proteins are able to form homodimers, as has been reported for the E3 ubiquitin ligases
Siah and c-Cbl (Polekhina et al., 2002; Kozlov et al., 2007). These E3 ubiquitin ligase
dimers can be formed by interaction of the RING domains, as is the case for the
MDM2-MDMX heterodimer (Poyurovsky et al., 2007), however amino acid residues
carboxy-terminal to the RING domain are also involved in dimerisation, and are
involved for example in cIAP2 homodimerisation (Linke et al., 2008; Mace et al.,
2008). The ubiquitin ligase c-Cbl homodimersises using its UBA domain, which is
located distal to the RING domain and is also required for c-Cbl/Cbl-b
heterodimerisation (Kozlov et al., 2007).
1.7.2.2 Mechanisms of Ubiquitin Transfer by RING Domain E3s
Following E2-E3 binding, the RING domain is thought to aid in ubiquitin transfer by
acting as a scaffold bringing the E2 and substrate into close proximity and it is also
hypothesised that upon E2-E3 binding, conformational change is effected in the E2
enzyme, thereby augmenting ubiquitin transfer from the E2 active site to the substrate
lysine (Seol et al., 1999; Deshaies and Joazeiro, 2009). Although generally it is the E2
enzyme that determines substrate lysine specificity and ubiquitin chain linkage
topology, the RING E3 ubiquitin ligase does play a role in this determination. Recent in
vitro studies have ascertained that without an E3 enzyme, E2 enzymes were not
selective of the substrate lysine residue or the type of ubiquitin linkage formed in
studies using multiple well characterised E2-E3 pairs and characterised E3 substrates
including E2G2-gp78 and the gp78 substrate HERP (David et al., 2011). The role of the
E3 enzyme in determining which lysine residue of the substrate was ubiquitinated and
the type of ubiquitin chain linkages formed was hypothesised to be due to the
positioning of the E2 and substrate upon binding by the E3 enzyme (David et al., 2011).
It is important to note that the ubiquitin chain linkage topologies formed by particular
E2-E3 pairs are constrained to the particular chain topologies preferred by the E2
conjugating enzyme, therefore the E3 does not seem to direct new chain topologies to
be formed, but merely guides the E2 enzyme (David et al., 2011). This result highlights
the importance of the E2-E3 complex in directing ubiquitination and specificity.
Additionally, one E3 enzyme is typically able to interact with a number of E2 enzymes,
for example BRCA1 has been reported to interact with ten E2 enzymes (Christensen et
al., 2007). Therefore depending on the type of E2 enzyme with which the E3 interacts,
the ubiquitin chain topology and substrate lysine residues that are ubiquitinated may
Chapter 1 General Introduction
39
change, resulting in different outcomes of ubiquitination of a single substrate.
Furthermore, substrates such as p53 may be coregulated by a number of E3 enzymes
depending upon the cellular environment, resulting in different outcomes for the
substrate depending upon the E3 enzyme responsible for its ubiquitination (Brooks and
Gu, 2006).
1.7.2.3 Regulation of RING E3 Ubiquitin Ligases
The expression and activity of RING E3 ubiquitin ligases are regulated by a variety of
PTMs including ubiquitination, sumoylation, neddylation and phosphorylation which
can be attached to the substrate, E2 or E3 to regulate their activity or recognition by the
E3 enzyme. RING domain containing proteins are able to regulate their own activity by
autoubiquitination, for example, the E3 ubiquitin ligase TRAF6 undergoes site-specific
lysine 63 autoubiquitination in response to interleukin 1 treatment, resulting in the
activation of Iκβ kinase (IKK), whilst MDM2 autoubiquitinates its own lysine residues
in response to DNA damage, resulting in its proteasome-dependent degradation, thus
leaving its target p53 to participate in the DNA damage response (Stommel and Wahl,
2004; Lamothe et al., 2007). MDM2 autoubiquitination is regulated by the
deubiquitinating enzyme, HAUSP which deubiquitinates MDM2 and p53, thereby
additionally regulating cellular p53 protein levels (Li et al., 2002; Cummins et al., 2004;
Cummins and Vogelstein, 2004). As mentioned above, RING domain proteins may be
regulated by other PTMs such as phosphorylation. Many E3 ubiquitin ligases are only
able to recognise their substrates once they are phosphorylated, as is the case for c-CBL,
which binds and ubiquitinates phosphorylated RTKs, propagating both the RTK signal
as well as receptor endocytosis and degradation by the lysosome (Waterman et al.,
1999; Garcia-Guzman et al., 2000). E3 ubiquitin ligases can also be regulated by the
attachment of Ubls, for example the Cullin subunit of Skip-Cullin-F-box E3 complexes
is neddylated, thereby activating the complex by enhancing the recruitment of the E2
enzyme Ubc4 (Kawakami et al., 2001).
1.7.2.4 Single Subunit RING E3 Ubiquitin Ligases
As the name suggests, single subunit E3 ubiquitin ligases are able to ubiquitinate target
proteins themselves, and this is due to the presence of a substrate recognition
component and E2 binding module on the same protein. For example, MDM2 contains
an amino-terminal p53 binding domain and a carboxy-terminal RING domain and is
Chapter 1 General Introduction
40
therefore able to recruit p53 and the E2 enzymes, UbcH5 and E2-25K to ubiquitinate
p53, leading to its proteasome dependent degradation (Fang et al., 2000; Honda and
Yasuda, 2000; Saville et al., 2004).
1.7.2.5 Multisubunit RING E3 Ubiquitin Ligases
Multisubunit E3 ubiquitin ligases are complexes consisting of multiple proteins each
playing a distinct role within the complex. These include the E3 ubiquitin ligase
responsible for E2 recruitment and binding, the substrate recognition element and
adaptors. Three E3 ubiquitin ligase complexes that have been well studied are the APC
and SCF complexes, which regulate each other in a cell cycle dependent manner, and
the structurally related von Hippel Lindau-Cul2/elongin B/elongin C (VHL-CBC)
complex (Figure 1.13).
The APC is a large E3 ubiquitin ligase complex that consists of eleven core subunits
which associate in a cell cycle dependent manner with two different activators and
substrate adaptors, Cdc20 and Cdh1 (Thornton et al., 2006; McLean et al., 2011). The
APC has over 100 substrates, many of which are recognised and recruited to the
complex by Cdc20 and Cdh1 owing to the presence of short motifs such as the
Figure 1.13: Multisubunit E3 ubiquitin ligases. Well characterised multisubunit E3 ubiquitin ligases include SCF (left panel), VCB-CUL2 (middle) and APC (right panel) (Weissman, 2001)
Chapter 1 General Introduction
41
destruction box (RxxL(x)nN/D/E‡) and KEN(x)nP§
Barford, 2011
box in the substrates (Figure 1.13)
( ; Meyer and Rape, 2011).
The APC has been proposed to consist of two subcomplexes, joined by a linker protein
APC1 (Thornton et al., 2006). The catalytic core of the APC consists of the cullin
protein, APC2 and the RING protein APC11, and together these two proteins are able to
bind the E2 activating enzyme and in vitro can ubiquitinate proteins but with little
substrate specificity (Figure 1.14A) (Tang et al., 2001). Also forming part of the core is
APC10, another substrate recognition component of the complex (Kurasawa and
Todokoro, 1999). The second subunit of the complex contains the tetrapeptide repeat
(TPR) containing proteins APC3, APC6, APC6 and APC8, which recruit Cdc20 and
Cdh1 to the complex (Figure 1.14A) (Vodermaier et al., 2003).
Another E3 ubiquitin ligase complex that is involved in cell cycle regulation is the SCF
E3 complex, which causes the degradation of cyclins, cyclin dependent kinase inhibitors
and transcription factors (Marti et al., 1999; Sutterluty et al., 1999; Nakayama et al.,
2004). SCF complex E3s consist of an invariable core containing the RING E3 Rbx1,
‡ R = arginine, L = leucine, N = asparagine, D = aspartic acid, E = glutamic acid x = any amino acid
§ K = lysine, E = glutamic acid, N = asparagine, P = proline
Figure 1.14: APC and SCF multisubunit E3 ubiquitin ligases. (A) The APC consists of 11 components including the RING domain protein Apc11 which binds the E2 conjugating enzyme and the activation components Cdc20 or Cdh1. (B) The SCF family of E3 ubiquitin ligases which contain the Rbx1 RING domain protein, Cul1 and the adaptor Skp1 that recruits F-box proteins, the substrate recognition components of the complex (Adapted from (Willems et al., 2004).
Chapter 1 General Introduction
42
the cullin protein Cul1 and the adaptor Skp1, with the cullin protein interacting with
linker proteins in order to recruit substrates (Figure 1.13, 1.14B) (Willems et al., 2004).
The variable unit in the complex is the substrate recognition component, the F-box
protein, and over 70 F-box proteins have been identified in humans with many able to
recognise multiple substrates. Three of the best characterised SCF complex F-box
proteins are Skp2, Fbw7 and βTrCP (Skowyra et al., 1997; Yu et al., 1998). SCF E3
complexes ubiquitinate a multitude of proteins, usually phosphorylated proteins,
targeting them for degradation (Willems et al., 2004). For example SCFFbw, SCFβTrCP
and SCFSkp2 complexes recognise phosphorylated cyclin E, β-catenin and p27, with F-
box proteins of these complexes interacting directly through the phosphoresidues
(Orlicky et al., 2003; Wu et al., 2003a; Hao et al., 2005; Hao et al., 2007).
1.8 Outcomes of Ubiquitination
1.8.1 Ubiquitin Chain Topology Determines Ubiquitinated Protein Fate
Although the attachment of ubiquitin to a substrate is usually associated with its
proteasomal degradation, this is not the only function of ubiquitination, which can lead
to a variety of different outcomes for the substrate, depending on the type of ubiquitin
linkages (Figure 1.15) (Ikeda et al., 2010). Ubiquitin itself contains seven lysine
residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, and Lys63) all of which can form
chain linkages, whereby the carboxy-terminal glycine residue of the donor ubiquitin is
attached to the ε amino group of a specific lysine residue of the acceptor ubiquitin
molecule (Ikeda and Dikic, 2008). Additionally ubiquitin molecules can be linked in a
linear fashion (Tokunaga et al., 2009; Walczak et al., 2012). Ubiquitin chain formation
through lysine 48 and 63 of ubiquitin are referred to as canonical ubiquitination whereas
polyubiquitination through the remaining lysine residues and linear ubiquitination are
often referred to as atypical or non-canonical ubiquitination as much less is understood
about these types of ubiquitin linkages (Wickliffe et al., 2011).
Attachment of a single ubiquitin molecule at one or multiple sites on the substrate is
known as mono- or multi-monoubiquitination, and this signal does not target proteins
for degradation but rather is associated with a change in protein-protein interactions,
subcellular localisation or modified function (Figure 1.15) (Kerscher et al., 2006;
Deribe et al., 2010). For example, the transcription factor Met4 is polyubiquitinated by
the SCFMET30 complex E3 leading to its inability to associate with its cofactor Cbf1,
Chapter 1 General Introduction
43
resulting in its transcriptional inactivation (Kaiser et al., 2000). The E3 ubiquitin ligase
MDM2 polyubiquitinates p53, targeting it for proteasome dependent degradation,
however under low MDM2 conditions, MDM2 monoubiquitinates p53, causing its
cytoplasmic accumulation (Li et al., 2003).
Polyubiquitination of a substrate through lysine 11 and 48 targets the protein for
degradation by the 26S proteasome (Chau et al., 1989; Kirkpatrick et al., 2006; Jin et
al., 2008). Mutation of lysine 48 of ubiquitin in yeast is lethal, with protein degradation
and therefore cell cycle progression disturbed, highlighting the importance of this
ubiquitin residue in proteasomal degradation (Chau et al., 1989; Xu et al., 2009).
Lysine 11 polyubiquitin chain linkages are involved in endoplasmic reticulum
associated degradation (ERAD), as evidenced by ER stress resulting in the
accumulation of lysine 11 linked ubiquitin chains (Adhikari and Chen, 2009; Xu et al.,
2009). The lysine 11 mutants lead to an increase in the unfolded protein response,
which is activated upon the accumulation of unfolded proteins in the ER lumen
(Adhikari and Chen, 2009; Xu et al., 2009).
Figure 1.15: The type of ubiquitination determines substrate protein fate. A single ubiquitin can be attached to a substrate protein (mono or multi-monoubiquitination) or multiple ubiquitin molecules can be attached via specific lysine residues, forming a polyubiquitin chain, with the topology of the ubiquitin chain determining the outcome of the ubiquitinated protein (Ye and Rape, 2009).
Chapter 1 General Introduction
44
Ubiquitin linkages through lysine 63 are typically not associated with protein
degradation and mutation of yeast ubiquitin lysine 63 leads to abnormalities in DNA
repair (Spence et al., 1995). Ubiquitination through this lysine residue plays a role in
receptor endocytosis, DNA damage repair, protein trafficking and signalling (Spence et
al., 1995; Deng et al., 2000; Zhong et al., 2005; Sorrentino et al., 2008; Yang et al.,
2009; Boname et al., 2010). Lysine 63 ubiquitination is important for the activation of
IKK by TRAF6 in response to proinflammatory cytokines, resulting in the activation of
the transcription factor NF-κβ which regulates diverse processes such as inflammation,
apoptosis and immunity (Section 1.7.2.3) (Chen et al., 1996; Deng et al., 2000). The
most recent type of ubiquitin linkage to be discovered, linear ubiquitination, also plays a
role in the activation of the NF-κβ signalling pathway. Upon genoxotic stress or TNFα
exposure, the linear ubiquitination chain assembly complex, LUBAC ubiquitinates
NEMO, a regulator subunit of the IKK complex, resulting in the activation of the NF-κβ
pathway (Kirisako et al., 2006; Tokunaga et al., 2009; Niu et al., 2011).
Few studies have addressed the fate of proteins modified by the remaining ubiquitin
linkages (Figure 1.15). Lysine 6 ubiquitination has been associated with the prevention
of ubiquitin-mediated protein degradation in a study using biotinylated lysine 6 and
lysine 6 mutants in vitro and in mammalian cells respectively, in which lysine 6 linked
(biotinylated or K6W mutants) were resistant to proteasomal degradation (Shang et al.,
2005). Additionally, in response to DNA damage, BRCA1 has been associated with the
attachment of polyubiquitin linkages through lysine 6 to proteins in foci containing
BRCA1/BARD1 heterodimers (Morris and Solomon, 2004). The transcription factor
Jun, which together with c-Fos forms the transcription factor AP1, may be ubiquitinated
through lysine 27, resulting in its lysosomal localisation and degradation (Ikeda and
Kerppola, 2008). The attachment of lysine 29 polyubiquitin chains to proteins is
associated with both lysosomal and proteasomal protein degradation (Lindsten et al.,
2002; Chastagner et al., 2006). The E3 ubiquitin ligases Cbl-b and Itch induce T cell
receptor-ζ (TCR-ζ) lysine 33 polyubiquitination, modifying its phosphorylation and
protein interactions without targeting the TCR for degradation either by the proteasome
or lysosome (Huang et al., 2010). Aytpical or branched ubiquitin chains, which consist
of ubiquitin molecules linked through various ubiquitin lysines as well as mixed
ubiquitin and Ubl chain linkages have been described, however the cellular roles of
these linkages have not yet been elucidated (Ikeda and Dikic, 2008; Trempe, 2011).
Chapter 1 General Introduction
45
1.8.2 Ubiquitin Binding Domains Determine Ubiquitinated Protein
Outcome
The reason for the variation in outcomes of ubiquitinated proteins is due to the presence
of specialised motifs known as ubiquitin binding domains (UBDs) in many cellular
proteins, which interact with ubiquitinated proteins through these motifs, thereby
interpreting the ubiquitin signal (Hurley et al., 2006). There are currently over 20 types
of UBDs found in over 150 proteins which utilise the ubiquitin β-sheet for their
interaction with ubiquitinated proteins (Dikic et al., 2009). UBDs differ in the type of
ubiquitin linkages that they recognise and therefore have different structures which can
be categorised according to the types of folds that they form, including α-helical, zinc
finger, ubiquitin conjugating domains and plekstrin homology folds (Dikic et al., 2009).
UBDs are found in a variety of proteins including those associated with the proteasome,
E2 conjugating enzymes, ERAD, DNA repair, endocytosis, multivesicular body
biogenesis, kinase regulation and cell signalling (Young et al., 1998; Hofmann and
Falquet, 2001; VanDemark et al., 2001; Fisher et al., 2003; Swanson et al., 2003; Prag
et al., 2005; Stamenova et al., 2007; Fu et al., 2009).
UBDs that recognise monoubiquitin include ubiquitin interacting motifs (UIMs),
ubiquitin binding zinc finger (UBZ) and ubiquitin-associated domains (UBA). For
example, all Y-family translesion synthesis family polymerases, which function in
translesion synthesis upon DNA damage, contain UBM and UBZ domains which
recognise monoubiquitinated PCNA (Lehmann et al., 2007). PCNA monoubiquitination
following DNA damage recruits Y-family polymerases to sites of DNA damage,
allowing the switch from DNA replication polymerases to translesion synthesis
polymerases (Bienko et al., 2005).
Proteins targeted to the proteasome are recognised by ubiquitin receptors, which are
associated with the proteasome, either stably or transiently. These receptors bind the
polyubiquitin chain through their UBDs and bind the proteasome through their
ubiquitin-like domain (Ubl), exhibiting a high affinity for lysine 48 linked ubiquitin
chains (Schauber et al., 1998; Verma et al., 2004). Proteins containing domains that
recognise ubiquitin play an integral part in the ubiquitin system by determining the
consequences of protein ubiquitination. As such the existence of diverse UBDs is
essential for the correct interpretation of the ubiquitin signal and the regulation of
Chapter 1 General Introduction
46
ubiquitinated proteins, leading to their degradation or their function in novel cellular
roles.
1.9 E3 Ubiquitin Ligases and Cancer
Given the involvement of the ubiquitin system in many different cellular processes such
as DNA repair, the cell cycle, proteasomal and lysosomal degradation, cell signalling,
protein trafficking and ERAD, it is not surprising that abnormalities in proteins that
form part of the ubiquitin system are implicated in pathological states including cancer.
The substrate recognition components of the ubiquitin system including E3 ubiquitin
ligases and DUBs are often dysregulated in cancer and are therefore potential targets for
drug development. It is not surprising when their expression is disrupted in cancer, that
several E3 ubiquitin ligases are considered oncogenes or tumour suppressor genes
depending upon the function of their cellular substrates (Lipkowitz and Weissman,
2011). A few E3 ubiquitin ligases may play either role in malignancy due to the diverse
roles of a single substrate or the roles of multiple substrates (Lipkowitz and Weissman,
2011).
1.9.1 E3 Ubiquitin Ligases and the Cell Cycle
Mutation or abnormal expression of members of the APC and SCF E3 ubiquitin ligase
complexes which regulate the cell cycle have been documented in cancers (Section
1.7.2.5). Several members of the APC are mutated in colon cancer cells and
overexpression of an APC8/CDC23 mutant in colon epithelial cells leads to abnormal
levels of cyclin B1 and dysregulated cell cycle progression (Wang et al., 2003a).
Knockout of the Cdc20 substrate adaptor of the APC in mouse embryos results in cell
cycle inhibition at mitosis when embryos are at the two-cell stage (Li et al., 2007a).
Mice lacking Cdh1 (Cdh1-/-), the second APC substrate adaptor, exhibit genomic
instability and do not survive past embryonic day 12.5, while aged Cdh1+/- heterozygous
mice (25 months) develop epithelial neoplasias in a number of tissues, suggesting that
Cdh1 functions as a tumour suppressor gene (Li et al., 2007a; Garcia-Higuera et al.,
2008).
High levels of SKP2, the substrate recognition element in SCFSKP2 E3 ubiquitin ligase
complexes have been detected in a number of cancers (Hershko et al., 2001; Latres et
al., 2001). High SKP2 levels are predicted to be oncogenic due its control of the G1/S
Chapter 1 General Introduction
47
checkpoint by ubiquitinating and targeting for degradation the CDK inhibitor p27
(Skaar and Pagano, 2009). In high grade lymphomas, breast and colorectal tumours,
high SKP2 levels inversely correlate with p27 levels suggesting that a direct effect of
SKP2 on p27 leads to a failure in G1/S checkpoint control and aberrant cellular
proliferation (Hershko et al., 2001; Latres et al., 2001; Traub et al., 2006). The
oncogenic potential of SKP2 is supported by several observations in Skp2 mouse
models, with Skp2-/- mice expressing increased p27 levels and exhibiting reduced cell
growth (Lin et al., 2010).
1.9.2 E3 Ubiquitin Ligases and DNA Damage
1.9.2.1 Tumour Suppressor p53
The transcription factor p53 regulates cellular responses to DNA damage by inducing
cell cycle arrest or apoptosis (Figure 1.16) (Livingstone et al., 1992; Shaw et al., 1992;
O'Connor et al., 1993). p53 is negatively regulated by the E3 ubiquitin ligases MDM2
and MDMX, with MDMX enhancing MDM2 E3 ubiquitin ligase activity toward p53
(Honda et al., 1997; Stad et al., 2001). MDM2 is the primary regulator of p53, with
MDM2 ubiquitinating p53 and itself, targeting both proteins for proteasomal
degradation (Haupt et al., 1997; Honda et al., 1997; Honda and Yasuda, 2000). The
MDM2 gene is disrupted in a number of cancers, with its amplification documented in
osteosarcomas, oesophageal carcinomas and breast cancers (Oliner et al., 1992; Quesnel
et al., 1994; Momand et al., 1998). Mdm2-/- knockout mice exhibit embryonic lethality
due to p53 dysregulation, whilst crossing Mdm2-/- mice with p53-/- mice rescues this
phenotype (Jones et al., 1995; Montes de Oca Luna et al., 1995). Mice expressing low
levels of Mdm2 display reduced tumour formation due to higher levels of p53, and in
Mdm2 null mice, expression of inducible p53 results in growth arrest (Mendrysa et al.,
2006; Ringshausen et al., 2006).
These studies highlight the importance of the balance between MDM2 and p53 which is
supported by the finding that approximately 33% of human sarcomas that display wild-
type p53 exhibit MDM2 amplification, thereby suggesting that MDM2 overactivation
represents one mechanism by which cells escape growth regulation (Leach et al., 1993;
Marine and Lozano, 2010). Several compounds have been designed to reduce MDM2
activity or MDM2-p53 binding, including the small molecule inhibitor Nutlin-3a.
Nutlin-3a disrupts MDM2-p53 interaction, causing cell cycle arrest or apoptosis in
Chapter 1 General Introduction
48
tumour cells, with the apoptotic response strongest in cells overexpressing MDM2
(Tovar et al., 2006). MDM2 activity is regulated by other proteins such as the tumour
suppressor, ARF and posttranslational modifications such as neddylation,
phosphorylation and acetylation of p53, MDM2 and MDMX (Pise-Masison et al., 1998;
Pomerantz et al., 1998; Luo et al., 2004; Xirodimas et al., 2004; Wade et al., 2010).
Additionally, p53 is regulated by several other E3 ubiquitin ligases including CHIP,
PIRH2 and TOPORS (Leng et al., 2003; Rajendra et al., 2004; Esser et al., 2005).
Therefore, although dysregulated expression of MDM2 is present in a percentage of
human tumours that express wild-type p53, potentially representing a therapeutic target
in these tumours, abnormal p53 levels or function in tumours exhibiting wild-type p53
may be due to the altered function of other E3 ubiquitin ligases and proteins regulating
p53 activity.
Figure 1.16: p53 regulation in response to genotoxic stress. Upon genotoxic stress, p53 and MDM2 undergo post-translational modification, disrupting their association and preventing MDM2 mediated p53 ubiquitination. Additionally, ARF and ribosomal proteins interact with MDM2 and inhibit its ability to bind p53, leading to an increase in p53 activity and the induction of proteins involved in cell cycle arrest or apoptosis (Lipkowitz and Weissman, 2011).
Chapter 1 General Introduction
49
1.9.2.2 BRAC1/BARD1
The E3 ubiquitin ligase BRCA1 is frequently mutated in familial breast and ovarian
cancers and plays an important role in the sensing of DNA damage and in DNA damage
repair (Welcsh and King, 2001). Sporadic breast tumours can also exhibit a reduction or
loss of BRCA1 expression, with BRCA1 levels and tumour grade being negatively
correlated (Taylor et al., 1998; Wilson et al., 1999). BRCA1 functions as a heterodimer
with BARD1 in several complexes which are involved in DNA damage detection, cell
cycle checkpoint regulation and recruitment of DNA damage repair proteins (Cantor et
al., 2001; Yarden et al., 2002; Wang et al., 2007). For example, BRCA1 interacts with
serine 406 phosphorylated abraxas and receptor associated protein 80 (RAP80), which
contains a UIM (Wang et al., 2007). Following DNA damage, BRCA1/BARD1-
abraxas-RAD80 complexes are localised to foci of DNA damage (Wang and Elledge,
2007, Wang et al., 2007). This is due to interaction of the UIM of RAP80 with proteins
such as histones that become ubiquitinated as a consequence of DNA damage repair
signalling pathways involving the E3 ubiquitin ligases RNF8 and RNF168 (Wang and
Elledge, 2007; Wang et al., 2007; Yan et al., 2007). Although the precise role of
BRCA1/BARD1 heterodimers in DNA repair once localised to DNA double strand
breaks (DSB) is unknown, it is hypothesised that BRCA1/BARD1 ubiquitinates
proteins at these sites. This is evidenced by the requirement of BRCA1/BARD1 for the
accumulation of ubiquitinated proteins at sites of DNA damage (Mallery et al., 2002;
Morris and Solomon, 2004; Polanowska et al., 2006). Members of the BRCA1/BARD
complex also form part of the BCRA1/BARD1-abraxas-RAD80BRCC36-BRCC45
complex required for regulation of the G2/M checkpoint, which ensures that
chromosomal segregation has occurred before entry into mitosis (Kim et al., 2007;
Sobhian et al., 2007).
1.9.3 E3 Ubiquitin Ligases and Signal Transduction
The CBL proteins, Cbl (also known as c-Cbl), Cbl-b and Cbl-c are a family of E3
ubiquitin ligases that also function as adaptor proteins, thereby acting as both positive
regulators propagating RTK downstream signalling or as negative regulators by
ubiquitinating RTKs, resulting in the internalisation and trafficking of the receptor for
recycling or degradation by the lysosome (Figure 1.17) (Levkowitz et al., 1998;
Ettenberg et al., 1999; Joazeiro et al., 1999; Kales et al., 2010). Mutations in RTKs
causing constitutive activation of the receptor in the absence of ligand or amplification
Chapter 1 General Introduction
50
of RTK genes are common in cancers (Blume-Jensen and Hunter, 2001).
Approximately 5% of human myeloid neoplasms contain mutations that inactivate the
E3 ubiquitin ligase function of CBL implicating a role for CBL as a tumour suppressor
gene (Kales et al., 2010). Mutations in Cbl that inactivate its E3 ubiquitin ligase activity
typically occur within the RING domain and linker region (Lipkowitz and Weissman,
2011). The linker region which is located immediately amino-terminal to the RING
domain is phosphorylated, resulting in a conformational change in CBL and the
activation of the RING domain (Kassenbrock and Anderson, 2004). CBL mutants are
proposed to act via two mechanisms. Firstly, they no longer ubiquitinate RTKs which
are therefore not internalised and degraded or recycled. Secondly, the RING domain
inactive mutants are hypothesised to function in a dominant negative manner by binding
to the activated RTK and thereby blocking the binding of other CBL proteins. The
observation that Cbl and Cbl-b double knockout mice develop early onset
myeloproliferative disease provides support for the tumour suppressor role of CBL
(Naramura et al., 2010).
CBL also exhibits oncogenic activity under specific circumstances as evidenced by the
observation that CBL RING domain mutants which have therefore lost their E3
ubiquitin ligase activity do not induce cellular transformation and that mutations in both
RING domain and linker region are required for transformation (Thien et al., 2001).
This is supported by the finding that a deletion encompassing parts of the linker region
and RING domain of Cbl results in the activation of the epidermal growth factor
receptor (EGFR) and Flt3 in the absence of ligand, and in the presence of ligand
enhances EGFR activity (Thien and Langdon, 1997; Sargin et al., 2007; Sanada et al.,
2009). Furthermore, the oncogene v-Cbl (amino acids 1-355 of CBL), the transforming
gene from the Cas NS-1 murine retrovirus, which lacks the Cbl RING domain, induces
the development of murine lymphomas and leukaemias (Langdon et al., 1989). Studies
in NIH 3T3 cells have demonstrated that a CBL mutant containing the first 357 amino
acids of the CBL protein (similar to v-Cbl), functions by associating with RTKs and
activating RTK mediated signalling (Bonita et al., 1997). The transforming properties
of Cbl mutations are therefore hypothesised to result from the loss of the tumour
suppressor functions of Cbl and unmasking of its oncogenic activity facilitated by
amino-terminal regions which mediate RTK signalling (Kales et al., 2010).
Chapter 1 General Introduction
51
By targeting proteins for ubiquitin-dependent degradation, E3 ubiquitin ligases control
the levels of oncogene and tumour suppressor gene products. Signal transduction
regulators, DNA repair related proteins and cell cycle regulators are all regulated by E3
ubiquitin ligases, with aberrant expression of the E3s hypothesised to result in the
enhanced degradation of tumour suppressors and accumulation of oncogene products
(Kitagawa et al., 2009). Depending on the ubiquitination substrate of the E3 ubiquitin
ligase and its function, the E3 itself can act as an oncogene or tumour suppressor. From
these examples it is evident that disruption of E3 ubiquitin ligases plays a role in the
initiation and/or progression of multiple cancers, highlighting the diverse roles of these
proteins in the regulation of cellular functions.
1.10 Statement of Aims
The homeodomain containing transcription factor NKX3.1 regulates prostatic
development and is expressed in the luminal epithelial cells of the adult prostate (He et
al., 1997; Bhatia-Gaur et al., 1999; Bowen et al., 2000; Chen et al., 2002a). NKX3.1
expression is reduced or undetectable in up to 80% of prostate tumours and it is
proposed to function as a prostate-specific tumour suppressor (Bowen et al., 2000;
Asatiani et al., 2005). Evidence of discordance between NKX3.1 mRNA and protein
levels and its aberrant cytoplasmic mislocalisation have suggested that translational or
post-translational dysregulation may contribute to a loss of NKX3.1 function in prostate
tumours (Kim et al., 2002b; Bethel et al., 2006; Bethel and Bieberich, 2007). Few
Figure 1.17: Roles of CBL in the regulation of receptor tyrosine kinase signalling. CBL ubiquitinates RTKs resulting in their internalisation and lysosomal degradation and thereby functioning as a tumour suppressor. CBL also functions as an oncogene by acting as an adaptor and activating downstream RTK signalling pathways (Kales et al., 2010).
Chapter 1 General Introduction
52
NKX3.1 interacting proteins that regulate NKX3.1 post-translational modification have
been identified, therefore in order to identify NKX3.1 binding partners that potentially
modify its activity or expression, a yeast two hybrid analysis was performed in our
laboratory which determined the FLJ22318 gene product, later renamed RMND5B to
be an NKX3.1 interacting protein (Dawson, 2006). RMND5B and its homologue
RMND5A are named after their yeast orthologue, RMD5 a RING domain containing E3
ubiquitin ligase (Santt et al., 2008) and as RMND5A and RMND5B each contain
putative RING domains, it is feasible that they too function as E3 ubiquitin ligases. To
determine the biological activity of human RMND5A and RMND5B, the aims of this
thesis were:
1. To characterise the E3 ubiquitin ligase activity of RMND5 proteins in prostate cancer
cells.
2. To determine whether RMND5A and/or RMND5B ubiquitinate NKX3.1 and to
characterise the outcome of NKX3.1 ubiquitination by RMND5 proteins.
3. To identify additional RMND5A and RMND5B interacting proteins.
Chapter 2 Materials
Chapter 2: Materials
Chapter 2 Materials
53
2.1 Reagents 2.1.1 Cell Culture Item Supplier Ammonium Chloride Sigma Aldrich, USA Charcoal Stripped Serum Sigma Aldrich, USA Chloroquine Sigma Aldrich, USA Cycloheximide AG Scientific, USA Dihydrotestosterone Sigma Aldrich, USA DU145 Prostate Cancer Cell Line American Type Culture Collection, USA Foetal Calf Serum Trace Scientific Ltd, Australia Lactacystin Sigma Aldrich, USA LNCaP Prostate Cancer Cell Line American Type Culture Collection, USA Metafectine PRO® Reagent Biontex Laboratories, GmbH MG132 AG Scientific, USA Penicillin/Streptomycin Gibco® Life Technologies, Australia RPMI 1640 with L-Glutamine Thermo Electron Corporation, Australia Sodium Hydrogen Carbonate Merck Pty Ltd., Germany Trypsin/EDTA (0.25%) Gibco® Life Technologies, Australia 2.1.2 Primers Primer sequences are located in Appendix II Item Supplier ARMC81103-S Geneworks, Australia ARMC81668-AS Geneworks, Australia β Actin146-S Geneworks, Australia β Actin464-AS Geneworks, Australia C17orf39298-S Geneworks, Australia C17orf39814-AS Geneworks, Australia CBLRING1141-S Geneworks, Australia CBLRING1257-AS Geneworks, Australia EMP1083-AS Geneworks, Australia EMP580-S Geneworks, Australia Muskelin1675-S Geneworks, Australia Muskelin2120-AS Geneworks, Australia pEGFP1266-S Geneworks, Australia M13-S Geneworks, Australia M13-AS Geneworks, Australia pGEX-AS Geneworks, Australia pGEX-S Geneworks, Australia RanBPM1-S Geneworks, Australia RanBPM1029-S Geneworks, Australia RanBPM1259-AS Geneworks, Australia RanBPM1550-S Geneworks, Australia RanBPM2190-AS Geneworks, Australia RanBPM687-S Geneworks, Australia RMND5A(C356A)1045-S Geneworks, Australia RMND5A(C356A)1083-AS Geneworks, Australia RMND5A(C356A/H358A)1045-S Geneworks, Australia RMND5A(C356A/H358A)1083-AS Geneworks, Australia RMND5A(C356S)1045-S Geneworks, Australia RMND5A(C356S)1083-AS Geneworks, Australia
Chapter 2 Materials
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RMND5A1-S Geneworks, Australia RMND5A1176-AS Geneworks, Australia RMND5A490-AS Geneworks, Australia RMND5A603-S Geneworks, Australia RMND5ABamHI1-S Geneworks, Australia RMND5ARING1006-S Geneworks, Australia RMND5ARING1131-AS Geneworks, Australia RMND5B(C358A/H360A)1083-AS Geneworks, Australia RMND5B(C358A/H360S)1054-S Geneworks, Australia RMND5B(C358S)1051-S Geneworks, Australia RMND5B(C358S)1057-S Geneworks, Australia RMND5B(C358S)1080-AS Geneworks, Australia RMND5B(C358S)1089-AS Geneworks, Australia RMND5B790-S Geneworks, Australia RMND5BBamHI 1-S Geneworks, Australia RMND5BBamHI 1182-AS Geneworks, Australia RMND5BRING1012-S Geneworks, Australia RMND5BRING1137-AS Geneworks, Australia RMND5BSalI 1-S Geneworks, Australia RMND5BSalI 1182-AS Geneworks, Australia RMND5BTOPO 1182-AS Geneworks, Australia Twa1335-S Geneworks, Australia Twa1681-AS Geneworks, Australia 2.1.3 Reverse Transcription - Polymerase Chain Reaction (PCR) Item Supplier AmpliTaq Gold® 360 DNA Polymerase Applied Biosystems, Australia AmpliTaq Gold® Mastermix Applied Biosystems, Australia DMSO Thermo Fischer Scientific, Finland DTT (0.1M) Invitrogen, Australia dNTP 100mM (dATP,dGTP,dCTP,dTTP) Promega, Australia 5x First Strand Buffer Invitrogen, Australia MgCl2 (50mM) Invitrogen Technologies, Australia MgCl2 (25mM) Applied Biosystems, Australia Oligo(dT) Primer 0.5µg/µL Promega, Australia 10x PCR Buffer (No MgCl2) Invitrogen, Australia 5x Phusion® HF Buffer Thermo Fischer Scientific, Finland Phusion® High-Fidelity DNA Polymerase Thermo Fischer Scientific, Finland 5x Phusion® High GC Buffer Thermo Fischer Scientific, Finland Platinum Taq Polymerase 5U/µL Invitrogen, Australia Sterile ddH2O Baxter Healthcare Pty Ltd, Australia Superscript II Reverse Transcriptase® Invitrogen, Australia 2.1.4 Plasmids Item Supplier pcDNA3.1-V5 Invitrogen, Australia pCMV-HA-Ubiquitin Gift from Professor Wallace Langdon,
University of Western Australia pGEX-2T-CBL Gift from Professor Wallace Langdon,
University of Western Australia pEGFP-C2 Clontech, USA
Chapter 2 Materials
55
pGEM®-T Easy Cloning Vector Promega, Australia pGEX-2TK GE Healthcare Life Sciences, Australia pmCherry C1 Gift from Dr Archa Fox, West Australian
Institute for Medical Research 2.1.5 Cloning Item Supplier Agar Bacteriological Amresco®, USA Ampicillin Amresco®, USA BamHI-HF Restriction Enzyme New England Biolabs, USA 10x Buffer 4 Promega, Australia 10x Buffer L Invitrogen, Australia DpnI Restriction Enzyme New England Biolabs, USA EcoR1-HF Restriction Enzyme New England Biolabs, USA Escherichia coli BL21 GE Healthcare Life Sciences, Australia HindIII Restriction Enzyme New England Biolabs, USA Escherichia coli DH5α Invitrogen Technologies, Australia Kanamycin CSL Limited, Australia KpnI Restriction Enzyme Invitrogen, Australia NdeI Restriction Enzyme Invitrogen, Australia 10x React Buffer 6 Invitrogen, Australia RNaseA Roche Diagnostics, Germany Rosetta Escherichia coli BL21 Gift from Dr Evan Ingley, West Australian
Institute for Medical Research SalI-HF Restriction Enzyme New England Biolabs, USA SAP Dephosphatase Buffer Roche Diagnostics, Germany Shrimp Alkaline Phosphatase (SAP) Roche Diagnostics, Germany T4 DNA Ligase New England BioLabs, UK 10x T4 DNA Ligase Reaction Buffer New England BioLabs, UK Tryptone Amresco®, USA Yeast Extract Becton Dickinson, Australia 2.1.6 GST Fusion Protein Production Glutathione S Transferase Agarose Beads GE Healthcare Life Sciences, Australia
Isoproyl β-D-1-thiogalactopyranoside(IPTG) Astral Scientific, Australia Lysozyme (10mg/mL) Amresco®, USA
Dithiothreitol (DTT) Sigma Aldrich, USA L-Glutathione, reduced Sigma-Aldrich, USA Coomassie Brilliant Blue G Sigma Aldrich, USA 2.1.7 Immunoprecipitation Item Supplier Anti-GFP Microbeads Miltenyi Biotec, Germany Complete Protease Inhibitor Tablets Roche Diagnostics, Germany Protein A Microbeads Miltenyi Biotec, Germany Protein A Sepharose Beads GE Healthcare, Australia Protein G Microbeads Miltenyi Biotec, Germany Protein G Sepharose Beads GE Healthcare, Australia Sodium Deoxycholate Sigma Aldrich, USA Sodium Orthovanadate Sigma Aldrich, USA
Chapter 2 Materials
56
2.1.8 Western Blotting Item Supplier 40% Acrylamide Amresco®, USA Ammonium Persulphate BioRad, Australia BSA Fraction V High-Grade Fatty Acid Free Roche Diagnostics, Germany ColorPlus Prestained Protein Marker New England Biolabs, USA Donkey Anti-Goat HRP Conjugate Santa Cruz Biotechnology Inc., USA ECLTMWestern Blotting Detection Reagent GE Healthcare, Australia Goat Anti-Actin IgG Santa Cruz Biotechnology Inc., USA Goat Anti-NKX3.1 IgG Santa Cruz Biotechnology Inc., USA Goat Anti-Rat IgG Jackson ImmunoResearch Europe, UK HybondTMC Extra Nitrocellulose Membrane GE Healthcare, Australia 2-Mercaptoethanol BDH Chemicals, Australia Mouse Anti-GFP IgG Clontech, USA Mouse Anti-HA HRP Conjugate Cell Signaling Technology, USA Mouse Anti-RFP IgG Clontech, USA Mouse Anti-V5 IgG Invitrogen, Australia 4-12% Precast Bis-Tris Acrylamide Gels Invitrogen, Australia 12% Precast Polyacrylamide Gels BioRad, Australia Rat Anti-Cherry IgG ChromoTek, Germany Sheep Anti-Mouse HRP Conjugate Chemicon, Australia Skim Milk Powder Bonlac Foods Inc, Australia Tetramethylethylenediamine (TEMED) BioRad, Australia Tween-20 Sigma Aldrich, USA 2.1.9 Fluorescence Microscopy Item Supplier Anti-goat Alexa Fluor® 546 Molecular Probes, USA Chlorobutanol Sigma Aldrich, USA 40% Formaldehyde BDH Chemical, Australia Hœchst 33258 Dye Sigma Aldrich, USA Normal Horse Serum GIBCO® , Australia Immersion Oil for Fluorescence Microscopy Leitz Wetzlar, Germany Phalloidin TRITC 77418 Sigma Aldrich, USA Polyvinyl Alcohol Sigma Aldrich, USA Sodium Azide BDH Chemicals, Australia 2.1.10 General Laboratory Reagents Item Supplier Ammonium Sulphate Sigma Aldrich, USA Agarose ITM Amresco®, USA Big DyeTM Terminator Sequencing Buffer Applied Biosystems, USA Big DyeTM Terminator Applied Biosystems, USA Bromophenol Blue Sigma Aldrich, USA Calcium Chloride Ajax Chemicals, Australia Chloroform Ajax Chemicals, Australia Diethylpyrocarbonate (DEPC) Sigma Aldrich, USA Disodium Hydrogen Orthophosphate BDH Chemicals, Australia Dimethyl Sulfoxide (DMSO) Thermo Fischer Scientific, Finland Ethanol Rowe Scientific, Australia Ethidium Bromide ICN Biochemicals, USA
Chapter 2 Materials
57
Ethylenediaminotetraacetic Acid (EDTA) BDH Chemicals, Australia Glacial Acetic Acid BDH Chemicals, Australia Glycerol Sigma Aldrich, USA Glycine BioRad, Australia Hydrochloric Acid BDH Chemicals, Australia Isopropanol BDH Biochemicals, England Magnesium Chloride BDH Chemicals, Australia Methanol Biolab, Australia Igepal CA630 (NP-40) Sigma Aldrich, USA Orthophosphoric Acid Ajax Finechem, Australia Piperazinediethanesulfonic acid (PIPES) Amresco®, USA Phenylmethanesulfonylfluoride (PMSF) Sigma Aldrich, USA Potassium Acetate BDH Chemicals, Australia Potassium Chloride BDH Chemicals, Australia Potassium Dihydrogen Orthophosphate BDH Chemicals, Australia RQ1 RNase Free DNase Promega, Australia RQ1 Stop Solution Promega, Australia Sodium Acetate BDH Chemicals, Australia Sodium Chloride Rowe Scientific, Australia Sodium Dodecyl Sulphate BioRad, Australia Sodium Fluoride Sigmal Aldrich, USA Sodium Hydroxide Sigma Aldrich, USA Sodium Phosphate BDH Chemicals, Australia Sodium Vanadate Sigmal Aldrich, USA Sucrose Sigma Aldrich, USA Tris Amresco®, USA Triton X-100 Sigma Aldrich, USA Zinc Chloride Sigma Aldrich, USA 2.2 Commercial Kits Item Supplier PureLinkTM HiPure Plasmid Midiprep Life Technologies, Australia Kit (Catalog Number K210004) Equilibration Buffer, EQ1 Resuspension Buffer, R3 Lysis Buffer, L7 Precipitation Buffer, N3 Wash Buffer, W8 Elution Buffer, E4 TE Buffer QIAquick Gel Purification Kit Qiagen, Australia (Catalog Number 28106) QIAquick Spin Columns Buffer QG Buffer PB Buffer EB Collection Tubes (2mL) Loading Dye
Chapter 2 Materials
58
QIAquick PCR Purification Kit Qiagen, Australia (Catalog Number 28706) QIAquick Spin Columns Buffer PBI Buffer PB Buffer EB Collection Tubes (2mL) Loading Dye Quant-iTTM dsDNA BR Assay Kit Life Technologies, Australia (Catalog Number Q32853) Quant-iTTM dsDNA BR Reagent (Component A) Quant-iTTM dsDNA BR Buffer (Component B) Quant-iTTM dsDNA BR Standard #1 (Component C) Quant-iTTM dsDNA BR Standard #2 (Component D) Ubiquitinylation Kit Enzo Life Sciences, USA (Catalog Number BML-UW9920-001) 20X Ubiquitin Activating Enzyme Solution (E1) 10X Ubiquitin Conjugating Enzyme Solutions (E2) - UbcH1 - UbcH2 - UbcH3 - UbcH5a - UbcH5b - UbcH5c - UbcH6 - UbcH7 - UbcH8 - Ubc13/Mms2 - UbcH10 20x Biotinylated Ubiquitin Solution (Bt-Ub) 20x Mg-ATP Solution 2x Non-reducing Gel Loading Buffer 10x Ubiquitinylation Buffer UltraspecTM RNA Isolation System Fischer Biotech, Australia (Biotecx Laboratories, Catalog NumberBL-10200) UltraspecTM RNA µMACSTM GFP Purification Kit Miltenyi Biotech, Germany (Catalog Number 130-091-125) anti-GFP microbeads Lysis Buffer Wash Buffer 1 Wash Buffer 2 Elution Buffer Vectastain Elite ABC Streptavidin-HRP Kit Vector Laboratories, USA (Catalog Number PK-6100) Solution A
Chapter 2 Materials
59
Solution B 2.3 Equipment Item Supplier 0.2µM Filters Pall Corporation, USA 23G Needles Becton Dickinson, USA AGFA CP-1000 Film Developer AGFA-Gevaert NV, Belgium Avanti J-25I Centrifuge Beckman Coulter Inc., Australia Avanti JA-25-5 Rotor Beckman Coulter Inc., Australia C1000 Thermal Cycler Bio-rad Laboratories, USA CellStar® Tissue Culture 10cm Dishes Greiner Labortechnik, Germany DNA Sub CellTM Electrophoresis Tank Bio-rad Laboratories, USA Dry Block Heater Thermoline, Australia Eppendorf 0.5, 1.5 mL Microcentrifuge Eppendorf, Germany Tubes 5mL, 10mL, 50mL Tubes Sarstedt, Germany Eppendorf 5415R Centrifuge Eppendorf, Germany Eppendorf 5804R Centrifuge Eppendorf, Germany Glass Coverslips Menzel-Glaser, Germany Haemocytometer Hawksley, UK Heidolph MR 100 Magnetic Stirrer John Morris Scientific Pty Ltd, Australia Hoefer Mini VE Electrophoresis Unit Hoefer Inc., USA L420S Precision Balance Sartorius, Germany Laminar Flow Hood Email-Westinghouse, Australia Microscope Coverslips Menzel-Glaser®, Germany Mini Sub DNA CellTM Electrophoresis Tank Bio-rad Laboratories, USA NanoDrop® ND-1000 Spectrophotometer Biolab Australia, Australia Nikon Eclipse TS100 Microscope Nikon, Japan Nikon Eclipse TiE Inverted Microscope Nikon, Japan Nunc Cryotubes Inter Med, Denmark Olympus IX71 Inverted Microscope Olympus, USA Orbital Mixer Incubator Ratek Instruments Pty.Ltd., Australia Parafilm American National CanTM, USA PHM 83 AutoCal pH Meter Radiometer, Copenhagen pH Cube pH Meter TPS, Australia Pipetman® Automatic pipettes Gilson, USA Pipette Tips Sarstedt, USA PowerPacTM 300 and 3000 Bio-rad Laboratories, USA PTC-100TM Programmable Thermal Cycler MJ Research Inc, Australia Protean® 3 Cell Acrylamide Gel Apparatus Bio-rad Laboratories, USA Red Rotor Orbital Shaker Hoefer Scientific, Australia Rotator 360º Ratek Instruments Pty.Ltd., Australia Sanyo Incubator Sanyo Electric Co., Japan Sharp Microwave Sharp, Australia Syringes (1mL) Becton Dickinson, USA Tissue Culture Flasks (T75, T25) Sarstedt, USA Tissue Culture Plates Becton Dickinson, USA Transfer Apparatus Bio-rad Laboratories, USA UV Sterile Cabinet Starkeys, Australia UV Transilluminator Hoefer Scientific, Australia Whatman Paper 3mm Whatman International, Ausltralia Zx3 Vortex VELP® Scientifica, Italy
Chapter 2 Materials
60
X-ray film (CL-XPosure) Thermo Fischer Scientific, Finland 2.4 Computer Software Item Supplier Adobe Illustrator Adobe Systems Inc., USA Adobe Photoshop CS Adobe Systems Inc., USA EndNote X1 ISI ResearchSoft, USA Image Pro Plus (Autoquant) Media Cybernetics, USA Microsoft Office® Microsoft® Corporation, USA Nanodrop 1000 Software Thermo Scientific, USA NIS Elements Nikon, Japan Quantity One® Imaging and Quantitation Bio-rad Laboratories, USA Software
Chapter 3 Methods
Chapter 3: Methods
Chapter 3 Methods
61
3.1 Cell Culture
3.1.1 Routine Maintenance of Mammalian Cell Lines
The human prostate cancer cell lines, DU145 and LNCaP obtained from the American
Tissue Culture Collection (ATCC) were used in these studies. Cells were maintained in
75cm2 culture flasks in ~10mL RPMI 1640 medium71*
supplemented with 10% (v/v)
foetal calf serum (FCS), 100U/mL penicillin and streptomycin (RPMI/PS/10%FCS74) in
humidified incubators at 37 ºC and 5% CO2. Medium was replaced every 2-3 days and
cells were passaged every 5-7 days as required.
To passage cells, RPMI/PS/10%FCS74 was aspirated, cells were rinsed with ~2mL
PBS56, then 1.5mL trypsin/EDTA was added to each flask and the flasks were incubated
at 37°C for ~2 minutes to dislodge the cells. RPMI/PS/10%FCS74 medium was added to
inactivate the trypsin and the cells were aliquoted into fresh 75cm2 flasks or used as
required (Section 3.1.3, 3.1.4, 3.1.5). For routine maintenance of cells, DU145 cultures
were passaged at a 1:10 dilution while LNCaP cells were passaged at a 1:5 dilution.
3.1.2 Cryopreservation and Thawing of Mammalian Cells
Cell lines growing in 75cm2 flasks were trypsinised when ~90% confluent (Section
3.1.1) and RPMI/PS/10%FCS74 was added to inactivate the trypsin. Cell suspensions
were centrifuged at 1000rpm for 5 minutes at room temperature, the supernatant
removed and the cells resuspended in 1mL per 75cm2 flask
RPMI/PS/10%FCS/10%DMSO75. Cell suspensions were aliquoted into pre-cooled 2mL
cryotubes, frozen at -80ºC in insulated containers then transferred to liquid nitrogen
storage.
Mammalian cells stored in liquid nitrogen were rapidly thawed in a waterbath at 37ºC
and the thawed cell suspensions pipetted into 75cm2 flasks containing pre-warmed
RPMI/PS/10%FCS74. Flasks were incubated overnight at 37ºC and 5% CO2, the
following morning the medium was replaced with fresh RPMI/PS/10%FCS74 and the
cells were cultured as usual (Section 3.1.1).
* Buffers and Solutions referenced by superscript numbers are described in Appendix I.
Chapter 3 Methods
62
3.1.3 Preparation of Cells for Fluorescence Microscopy
For microscopic imaging, cells were grown on glass coverslips in 6 well plates. Prior to
commencement of the experiments, coverslips were wiped with 70% ethanol25, placed
one per well into 6-well tissue culture plates then exposed to UV light in a laminar flow
hood for 45 minutes to sterilise. To wet the coverslips, ~200µL RPMI/PS/10%FCS74
was placed on the centre of each coverslip and culture plates were incubated at 37°C
and 5% CO2 for 1-2 hours prior to seeding of the cells. Cells were trypsinised, counted
using a haemocytometer, medium was removed from the coverslips and the appropriate
volume of cell suspension was added to each coverslip (Sections 3.1.1, 3.1.5) (Table
3.1). Cells were incubated overnight and the following day were transfected with the
appropriate plasmid DNA (Section 3.1.4), then incubated a further 48 hours before
processing for microscopy (Section 3.16). To preserve fluorescence, coverslips were
processed in minimal light.
3.1.4 Transfection of Mammalian Cells
Transient transfection of the cell lines was carried out using MetafectineTM PRO
(Biontex). For these experiments, cells were trypsinised (Section 3.1.1), counted using a
haemocytometer, the appropriate number of cells per well was added to each culture
plate in RPMI/PS/10%FCS74 (Table 3.1) and the plates were incubated overnight. The
following day, medium was replaced with the appropriate volume of
RPMI/PS/10%FCS74 (Table 3.1), plasmid for each well was made up to the appropriate
volume with RPMI 1640 medium71, MetafectineTM PRO reagent was made up to the
same volume with RPMI 1640 medium71 (Table 3.1), and the two solutions were
combined and incubated for 20 minutes to allow liposomes to form around the DNA.
The solution was added dropwise to each well or coverslip and cultures were incubated
at 37ºC, 5% CO2 for 48 hours. For cells growing on coverslips, medium on the
coverslips was replaced 6 hours post-transfection with fresh RPMI/PS/10%FCS74 and
the coverslips were incubated for a further 42 hours.
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Table 3.1 – Cell seeding density and reagents for transfection of mammalian cells
12 well plate
6 well plate Coverslips in 6 well plate
10 cm petri dish
Total volume 1.1mL 2.2mL 2.2mL 11.4mL RPMI/PS/10%FCS74 1mL 2mL 2mL 10mL MetafectineTM PRO 3µL 6µL 6µL 42µL
Amount of DNA transfected
2µg 4µg 4µg 10-30µg
Total transfection volume
2x 50µL 2x100µL 2x100µL 2x700μL
Number of DU145 cells seeded
1.5 x105
cells/well 4x105
cells/well 0.75x105
cells/coverslip 2x106
cells/well Number of LNCaP cells
seeded 2 x105
cells/well 5x105
cells/well 2x105
cells/coverslip 4x106
cells/well
3.1.5 Treatment of Mammalian Cells
For experiments involving the depletion or addition of androgens to cultures, LNCaP
cells were trypsinised (Section 3.1.1), aliquoted into the required culture dishes (Table
3.1) in RPMI/PS/10%FCS74 and incubated overnight at 37ºC and 5% CO2. The
following morning the medium was replaced with RPMI/PS/5% charcoal treated FCS
(CSS)73. For experiments examining the effect of androgens on NKX3.1 protein levels,
the cells were cultured in RPMI/PS/5%CSS73 for 24 hours (androgen depletion) before
the addition of 10-8M Dihydrotestosterone (DHT)18. Cells were cultured for 8-48 hours
at 37ºC and 5% CO2 prior to cell lysis. For experiments examining the effects of
androgen depletion on NKX3.1 protein levels, the cells were cultured in
RPMI/PS/5%CSS73 at 37ºC and 5% CO2 for 8-24 hours prior to cells lysis.
For treatment with proteasome or lysosome inhibitors, cells were trypsinised (Section
3.1.1) and the appropriate numbers of cells were seeded into 12 or 6 well plates (Table
3.1) in RPMI/PS/10%FCS74 then incubated at 37°C and 5% CO2 overnight. Where cells
were treated for 24-48 hours with proteasome or lysosome inhibitors, the medium was
replaced with RPMI/PS/10%FCS74 containing the inhibitor and the cells were cultured
for up to 48 hours prior to harvest in Whole Cell Lysis Buffer112 (Table 3.2) (Section
3.15.1). Cells treated for shorter period of time were grown to ~80% confluency prior to
2-8 hours of treatment with the inhibitor and harvest in Whole Cell Lysis Buffer112
(Table 3.2) (Section 3.15.1). Cells treated with inhibitors and 10-8M DHT18 were
prepared as described above, with culture of cells in RPMI/PS/5%CSS73 for 24 hours
prior to the addition of DHT and/or inhibitors. Additionally, where cells were depleted
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of androgens as described above by culture in RPMI/PS/5%CSS73, inhibitors were
added at the time the medium was changed for 8-24 hours prior to cell lysis.
Table 3.2 –LNCaP cell treatments
Treatment Final Concentration Hours of Treatment
NH4Cl3 10mM – 20mM 8 - 24
Chloroquine 25µM - 100µM 8-48
Lactacystin37 10µM 2-8
MG13250 10µM 3-8
DHT18 10-8 M 24-48
Cycloheximide13 10µg/mL or 20µg/mL 0.25-4
For treatment of cultures with cycloheximide13, LNCaP cells were grown in 6 well
plates in RPMI/PS/10%FCS74 until ~80% confluent, either 10µg/mL or 20µg/mL
Cycloheximide13 was added to the medium and the cells were cultured for a further 15-
240 min prior to harvesting in Whole Cell Lysis Buffer112.
3.2 RNA Extraction and DNase Treatment
3.2.1 RNA Extraction
For RNA extraction, cells cultured in 75cm2 flasks were trypsinised (Section 3.1.1),
passaged as required and the remaining cell suspensions were transferred into sterile
10mL tubes, centrifuged at 5000rpm for 5 minutes at room temperature, the
supernatants removed and the cell pellets stored at -80ºC until use. To extract RNA, cell
pellets were lysed in 1mL of Ultraspec® RNA and pipetted to disperse the solution.
Lysates were transferred to fresh 1.5mL microcentrifuge tubes, incubated on ice for 5
minutes at 4ºC, 0.2 volumes of chloroform was added to each tube and the tubes were
shaken vigorously for 15 seconds then immediately placed on ice for 5 minutes. Tubes
were centrifuged at 12000rpm for 15 minutes and the upper aqueous layer containing
the RNA was transferred to a fresh 1.5mL microcentrifuge tube, an equal volume of
isopropanol was added to each tube and the tubes were shaken to mix, then incubated
on ice for 10 minutes. Tubes were centrifuged at 12000rpm and 4ºC for 10 minutes to
precipitate the RNA, the supernatants were removed, the pellets were washed twice with
1mL 75% ethanol25 per 1mL Ultraspec® RNA followed by centrifugation at 7500rpm
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for 5 minutes at 4ºC. After the final centrifugation, supernatants were removed and
pellets were air dried then dissolved in 10-100µL DEPC-treated ddH2O15 and stored at
-80ºC.
3.2.2 DNase Treatment of RNA
For DNase treatment of RNA (Section 3.2.1), 10μL RNA, 20μL RQ1 RNase Free
DNase, 4μL 10x Reaction buffer and ddH2O to 40μL were added to a 0.5mL tube and
the solution was incubated at 37ºC for 30 minutes. Tubes were immediately placed on
ice, 6μL RQ1 Stop buffer was added then the tubes were heated at 95ºC to inactivate the
DNase. For ethanol precipitation of RNA, 6.6μL 3M Sodium Acetate pH4.679 (1/10th
sample volume) and 156μL 100% ethanol (2.5-3 times sample volume) were added to
each tube, the tubes were vortexed, placed on dry ice for one hour then centrifuged at
12000rpm for 30 minutes and 4ºC. Supernatants were removed, the RNA pellets washed
with 100μL 75% ethanol25 and the tubes again centrifuged at 12000rpm and 4ºC for 5
minutes. The supernatants were discarded, tubes were heated at 60ºC for ~5 minutes to
dry the pellets and the RNA was dissolved in ~10μL DEPC-treated ddH2O15. RNA
concentrations were measured using a NanoDrop® ND1000 UV/Vis spectrophotometer
and RNA solutions were stored at -80°C (Section 3.5).
3.3 Reverse Transcription To reverse transcribe RNA into cDNA, 1µg total RNA (Sections 3.2.1, 3.2.2) was added
to a 0.5mL microcentrifuge tube along with 1µL (0.5μg) Oligo(dT), 1µL 10mM dNTP23
and ddH2O to a final volume of 12µL, the tube was incubated at 65ºC for 5 minutes to
denature the RNA, then immediately placed on ice for 2-5 minutes. Following
incubation, 4µL 5X First Strand Buffer and 2µL 0.1M DTT were added to each tube
and the tubes were incubated at 42°C for 2 minutes. One µL (200U) Superscript II
Reverse Transcriptase® was added and the tubes were incubated at 42°C for 50 minutes,
then heated at 70°C for 15 minutes to degrade the reverse transcriptase enzyme. cDNA
was stored at -20ºC.
3.4 Polymerase Chain Reaction (PCR)
3.4.1 PCR
PCRs were performed in BioRad C1000TM Thermal Cyclers. Each reaction contained
1µL (~50ng) cDNA (Section 3.3) or 15ng plasmid DNA (Section 3.10), PCR buffer
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(containing dNTPs), MgCl2, 15 pmol sense and antisense primers (Appendix II), DNA
Polymerase and ddH2O to 25μL, with the PCR buffer used dependent upon the type of
DNA Polymerase utilised (Table 3.3). Reactions were carried out for 30-35 cycles, with
the annealing temperature and extension times optimised for each primer pair (Table
3.4, Appendix II) and amplified PCR products were stored at -20°C or electrophoresed
in 1-2% agarose gels2 (Section 3.6).
Table 3.3 – Addition of DNA polymerases to PCRs
Platinum® Taq DNA Polymerase
Phusion®
High-Fidelity DNA Polymerase
Combination Platinum® Taq DNA Polymerase + Phusion
High-Fidelity DNA Polymerase
AmpliTaq® Gold 360 DNA Polymerase
Amount DNA Polymerase per Reaction (Units)
0.5 0.4 0.5/0.2 0.625
PCR Buffer 5x PCR Buffer54
5x Phusion HF Buffer (1.5 mM MgCl2)
5x Phusion HF Buffer (1.5 mM MgCl2)
12.5µL AmpliTaq Gold Mastermix
Table 3.4 – Primer pair PCR conditions
Primersa MgCl2 (mM)
DNA Denaturation/ Time
Annealing Temperature (°C)/Time
Extension Time (72°C)
Number of Cycles
RMND5B BamHI 2 95°C/1 min 55°C /1 min 100 sec 35 Twa1 1.5 95°C/1 min 60°C / 1 min 1 min 35 EMP 1.5 95°C/1 min 57°C /1 min 1 min 35 ARMC8 2 95°C/1 min 55°C /1 min 1 min 35 Muskelin 1.5 95°C/1 min 55°C /1 min 1 min 35 C17orf39 1.5 95°C/1 min 55°C /1 min 1 min 35 RMND5A700S/RMND5A AS
1.5 95°C/1 min 55°C /1 min 1 min 35
RMND5B789-S/RMND5BTOP0-AS
1.5 95°C/1 min 55°C /1 min 1 min 35
RMND5A RING 2 95°C/1 min 55°C /1 min 1 min 35 RMND5B RING 2 95°C/1 min 55°C /1 min 1 min 35 CBL RING 2 95°C/1 min 55°C /1 min 1 min 35 RanBPM687-S/RanBPM2190-AS
1.5 98°C/10 sec 60°C /30 sec 90 sec 35
RMND5A BamHI1-S / RMND5A1176-AS
1.5 98°C/10 sec 64°C /30sec 90 sec 35
a = Appendix II
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3.4.2 “A” Tailing of PCR Products
For TA cloning of PCR amplified products where Phusion® High Fidelity Polymerase
was used, ‘A’ tailing of the PCR products was carried out using Platinum® Taq DNA
Polymerase. For each reaction, 7µL purified PCR product, 1µL 10X Taq PCR Buffer,
0.5µL 10mM dATP14, 5U Platinum® Taq DNA Polymerase and 0.75mM MgCl2 were
combined and the reaction was incubated at 70ºC for 30 minutes. ‘A’-tailed products (1-
2µL) were used in ligation reactions (Section 3.8.4).
3.4.3 Site Directed Mutagenesis
Site directed mutagenesis was performed, based on the Stratagene QuikChange®
Protocol. Overlapping forward and reverse mutagenesis primers of 25-45 bases with 3’
overhangs were designed, with estimated melting temperatures of between 60ºC and
70ºC and the mutation located in the middle of both sequences (Appendix II).
3.4.3.1 Mutagenesis PCR
Mutagenesis PCRs were performed in a BioRad C1000TM Thermal Cycler using 15ng
template plasmid DNA (Sections 3.9, 3.10), 0.5U Phusion® High Fidelity Polymerase
polymerase, 4µL 5x Phusion® HF Buffer, 200µM dNTPs23, 0.5µM forward and reverse
mutagenesis primers (Appendix II), 3% DMSO and ddH2O to 20µL (Table 3.5). To
digest methylated wild-type plasmid DNA, 0.5 µL (10U) DpnI was added to each
reaction and the tubes were incubated for 60 minutes at 37ºC, then heated at 80ºC for 20
minutes to inactivate the DpnI and the PCR products transformed into competent E. coli
DH5α cells (Section 3.8.6).
Table 3.5 – Site directed mutagenesis PCR conditions
Primers (15 pmol/μL)
RMND5A (C356S)
FWD/ RVSE
RMND5A (C356A/H358A)
FWD/RVSE pair 1 and 2
RMND5B (C358S)
FWD/ RVSE
RMND5B (C358A/H360A)
FWD/RVSE
MgCl2 (mM) 1.5mM 1.5mM – 2.5mM 1.5mM 1.5mM – 3mM DNA
denaturation time (98ºC)
30s 40s 30s 40s
Annealing Temperature (°C)
65ºC - 72ºC 64°C-70°C 58ºC - 68ºC 65°C
Annealing Time (seconds)
30s 50s 30s 50s
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Extension Time (72ºC)
3 min 4min 3 min 4min
Number of Cycles 26 26 26 26
3.5 Spectrophotometric Quantitation of RNA/DNA A NanoDrop® ND-1000 spectrophotometer was used to determine the concentration of
both RNA and DNA while a Qubit fluorometer with Quant-iTTMdsDNA BR Assay Kit
was used to determine the concentration of double-stranded (plasmid) DNA. For
measurements using the Nanodrop® ND-1000, the spectrophotometer was blanked with
the appropriate solution (DEPC-treated ddH2O15 or TE buffer) and the optical density
(OD) of the RNA/DNA solutions at 260nm and 280nm was determined. An OD of 1.0
represented 40µg/mL RNA or 50µg/mL DNA, while OD260/OD280 ratios of between 1.8
and 2.0 indicated relatively pure RNA/DNA solutions. For DNA measurements using
the Qubit fluorometer, a Quant-iTTM working solution was prepared by the addition of
1μL Quant-iTTM Reagent to 199μL Quanti-iTTM Buffer. For the standards, 190μL
Quanti-iTTM working solution was aliquoted into two tubes into which 10μL standard
solutions S1 and S2 were added, respectively. Between 1-20μL samples were added to
180-199μL Quant-iTTM working solution to obtain a final volume of 200μL in all tubes.
Tubes were vortexed for 3 seconds and allowed to stand for >2 minutes before reading
the samples using the QubitTM fluorometer. DNA concentrations in μg/mL determined
by the fluorometer were adjusted according to the dilutions used for each sample.
3.6 Agarose Gel Electrophoresis DNA was electrophoresed in 1% or 2% (w/v) agarose gels2 in 1X TAE buffer100. The
appropriate volume of 6X DNA loading dye22 was added to each sample, with 5-15μL
sample loaded into each well and a lane containing 5μL (250ng) 1Kb PlusTM DNA
ladder61 included in each gel. Gels were electrophoresed at 100V for 20-40 minutes and
visualised under UV transillumination using a Gel Doc 2000, with images analysed
using BioRad Quantity One® software.
3.7 DNA Purification
3.7.1 Purification of DNA
PCR products and plasmids were purified using a QIAquick® PCR purification kit and
according to the manufacturer’s instructions. Briefly, 5 volumes of PBI buffer was
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added to each sample, the solution was mixed then added to a QIAquick® column,
which was centrifuged at 13000rpm for 1 minute at room temperature and the flow
through discarded. PE buffer (750µL) was added to each column, columns were
centrifuged at room temperature for 1 minute at 13000rpm at room temperature, the
flow through discarded and the column centrifuged for a further 1 minute at 13000rpm
and room temperature to dry the column matrix. Columns were placed into fresh 1.5mL
microcentrifuge tubes and the DNA eluted by the addition of 50μL EB buffer to the
centre of the QIAquick® column membrane. Columns were incubated at room
temperature for 1 minute then centrifuged for 1 minute at room temperature and
13000rpm. Purified DNA was stored at -20ºC.
3.7.2 Gel Purification of DNA
To gel purify DNA using a Qiagen QIAquick® Gel Purification Kit, samples
electrophoresed in agarose gels (Section 3.6), were visualised under UV
transillumination, the appropriate bands were excised from the gel using sterile scalpel
blades and placed into preweighed 1.5mL microcentrifuge tubes. Tubes were
reweighed, the weight of the agarose calculated and 100μL Buffer QG per 100mg gel
slice was added to each tube. Tubes were incubated at 50ºC for 10 minutes or until the
agarose had melted, 1 gel volume of isopropanol was added to the tube and the solution
mixed by pipetting. The solution was then transferred to a QIAquick® spin column and
centrifuged at 13000rpm for 1 minute at room temperature. The flow through was
discarded, 750μL Buffer PE was added to each column, which was centrifuged at room
temperature for 1 minute at 13000rpm and the flow through discarded. Columns were
centrifuged for a further 1 minute at 13000rpm and room temperature to dry the column
matrix and the columns were transferred to fresh 1.5mL microcentrifuge tubes. To elute
the DNA, 20-50μL Buffer EB was added to each column, columns were incubated for 1
minute at room temperature then centrifuged at 13000rpm for 1 minute. Purified DNA
was stored at -20ºC.
3.8 Cloning of PCR Products
3.8.1 Plasmids
The pGEM®-T Easy cloning vector (Promega) was used for cloning of PCR-amplified
fragments using the A overhangs generated by Platinum® Taq DNA polymerase (Figure
3.1, Section 3.4). The pEGFP-C2 and pmCherry-C1 mammalian expression vectors
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(Clontech) were used to enable the expression of EGFP- and Cherry-tagged proteins in
mammalian cells (Figures 3.2, 3.3). The pGEX-2TK bacterial expression vector (GE
Healthcare) was used to allow the expression of GST-fusion proteins in bacterial cells
(Figure 3.4).
3.8.2 Restriction Enzyme Digestion of Plasmids
For 15μL restriction enzyme digests, purified plasmid (Section 3.7.1, 3.9, 3.10), 1μL
(5U) appropriate restriction enzyme and 1.5μL 10X buffer (appropriate for restriction
enzyme) were added to 0.5mL microcentrifuge tubes and the volume made up to 15μL
using ddH2O. Tubes were incubated for 3 hours at the optimum temperature for each
restriction enzyme in BioRad PTC-100TM Programmable Thermal Cyclers and the
digested plasmids were stored at -20°C.
3.8.3 Shrimp Alkaline Phosphatase Digestion
In order to prevent recircularisation of restriction enzyme digested plasmids (Section
3.8.2), the 5’ phosphate groups were removed using shrimp alkaline phosphatase (SAP).
For 40µL digests, digested plasmid (Section 3.8.2), 4μL 10X SAP dephosphatase buffer
and 1.5μL (1.5U) SAP were made up to 40µL with ddH2O, digests were incubated at
37ºC for 30 minutes, then at 65ºC for 15 minutes to inactivate the SAP. Plasmids were
purified using a QIAquick® Purification kit (Section 3.7.1) and stored at -20ºC or
electrophoresed in agarose gels (Section 3.6).
3.8.4 Ligation Reactions
For 10μL ligation reactions, 50ng linear vector (restriction enzyme digested, SAP
treated (Section 3.8.2, 3.8.3)), 1μL 10X T4 DNA Ligase Reaction Buffer, the
appropriate amount of insert and 1μL T4 DNA Ligase were made up to 10μL with
ddH2O. Ligation reactions were incubated at 16°C overnight in BioRad PTC-100TM
Programmable Thermal Cyclers and stored at -20°C. The amount of insert required for
1:1 molar ratios of insert:vector was calculated using the following equation:
x(ng)insert = 50ng vector x y(kb)insert z(kb)vector
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Figure 3.1: Map of the pGEM®-T Easy cloning vector showing the multiple cloning site (MCS) and the insert position (*). Ligation of inserts into the MCS results in the disruption of the lacZ gene thus enabling the selection of transformed bacterial colonies containing plasmids with insert s (white colonies) and those containing recirularised vector (blue colonies) on LB agar/Ampicillin39 plates containing X-gal (Adapted from www.promega.com/vectors).
*
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Figure 3.2: Map of the pEGFP-C2 expression vector showing the multiple cloning site (MCS) and the EcoRI restriction digest site where RMND5A or RMND5B was inserted (*). The enhanced green fluorescent protein (EGFP) coding region is located upstream of the MCS, therefore in-frame ligation of RMND5A into the plasmid results in the expression of RMND5A with an amino-terminal EGFP tag (EGFP-RMND5A) following transfection of the plasmid into mammalian cells (adapted from www.clontech.com/images/pt/dis_vectors).
*
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Figure 3.3: Map of the pmCherry C1 expression vector showing the multiple cloning site (MCS) and the EcoRI/BamHI restriction enzyme digest sites where RMND5B and RanBPM were inserted. The Cherry fluorescent tag was inserted into a pEGFP-C1 expression vector with the EGFP tag removed. The Cherry fluorescent tag is amino-terminal to the MCS, therefore in-frame ligation of RMND5B and RanBPM into the MCS resulted in the expression of amino-terminally Cherry-tagged RMND5B and RanBPM (adapted from www.staff.ncl.ac.uk/p.dean/pEGFP_C1_map.pdf)
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Figure 3.4: Map of the pGEX-2TK bacterial expression vector showing the multiple cloning site (MCS) and the BamHI/EcoRI restriction enzyme insert position. The GST tag is upstream from the MCS thus allowing the expression of amino-terminal GST tagged fusion proteins in bacterial systems (adapted from http://www.gelifesciences.com/aptrix/upp00919.nsf/Content).
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3.8.5 Preparation of Competent Bacterial Cells
3.8.5.1 Preparation of Competent Escherichia coli DH5α
To prepare competent bacterial cells, an aliquot of E. coli DH5α glycerol stocks was
streaked onto a Luria-Bertani (LB) Agar38 plate and incubated inverted at 37ºC. The
following day, one colony from the overnight plate was inoculated into 10mL LB
Broth43 and incubated overnight at 37ºC with shaking at 225rpm. The next morning,
1mL of the culture was inoculated into 200mL LB Broth43 and the culture incubated at
37ºC with shaking at 225rpm for ~3 hours until the OD600 was between 0.4-0.5. The
culture was divided into four 50mL centrifuge tubes and centrifuged at 4000rpm for 10
minutes at 4ºC to sediment the bacterial cells, the supernatant was discarded and the
bacterial pellets were resuspended in 5mL ice cold Glycerol/PIPES33 buffer until
homogeneous. The cell suspensions were pooled, incubated on ice for 30 minutes and
the bacterial cells repelleted by centrifugation at 4000rpm for 10 minutes at 4ºC. The
supernatant was discarded and the bacterial pellet was resuspended in 2mL
Glycerol/PIPES33 buffer until homogeneous, divided into 100μL aliquots in sterile
1.5mL tubes and stored at -80ºC.
3.8.5.2 Preparation of Competent Escherichia coli BL21
To prepare competent E. coli BL21 bacterial cells, BL21 cells from glycerol stocks
stored at -80ºC were streaked onto an LB Agar38 plate and incubated inverted overnight
at 37ºC. The following day, a single colony from the plate was inoculated into 5mL LB
Broth43 and incubated overnight at 37ºC and 225rpm. The next morning, 0.5mL of
overnight culture was transferred to 7.5mL fresh LB Broth43 and incubated at 37ºC,
225rpm for 1-1.5 hours until the OD600 was 0.4-0.5. At this time, the bacterial cells were
collected by centrifugation at 2500rpm for 10 minutes at 4ºC, the supernatant decanted
and the pellet resuspended in 0.5mL 50mM CaCl27. Another 2mL 50mM CaCl2
7 was
added to the tube and the cell suspension was mixed then stored on ice for 30 minutes.
The bacterial cells were again collected by centrifugation at 2500rpm for 10 minutes at
4ºC, resuspended in 0.5mL 50mM CaCl27 and 50μL aliquots were used immediately in
transformation reactions (Section 3.8.6).
3.8.6 Transformation of Bacterial Cells
Aliquots of 100 µL competent E. coli DH5α cells (Section 3.8.5.1) stored at -80°C, or
50μL freshly prepared competent BL21 cells (Section 3.8.5.2) were thawed on wet ice,
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2-10µL ligation reaction (Section 3.8.4) was added to each tube, the solution was stirred
to mix then incubated on ice for 30 minutes, heated at 42°C for 90 seconds then placed
on ice for 2 minutes. Eight-hundred µL LB broth43 was added to each tube and the tubes
were incubated at 37°C with shaking at 225rpm for 1 hour. Aliquots of 50µL, 100µL
and 150µL of the transformed cells were spread onto LB agar/ampicillin39 or LB
agar/kanamycin41 plates, or for blue/white colony selection, LB/ampicillin/IPTG/X-
gal40 or LB/kanamycin/IPTG/X-gal42 plates and the plates were incubated inverted at
37°C overnight then stored at 4°C.
3.8.7 Preparation of Bacterial/Glycerol Stocks
Five mL bacterial cultures in LB broth/ampicillin44 or LB broth/kanamycin46 were
prepared by overnight incubation at 37°C and 225rpm. The following day, the cultures
were centrifuged at 5000rpm for 8 minutes at 4°C and the supernatants discarded. A
1mL aliquot of prechilled LB broth/10% glycerol45 was added to each pellet, the cells
were evenly suspended, transferred to 2mL cryotubes and stored at -80°C.
3.9 Small Scale Plasmid Purification Bacterial cultures were inoculated into 5mL LB broth43 containing the appropriate
antibiotic and incubated overnight at 37°C and 225rpm. The following day, 1.5mL from
each culture was transferred to a 1.5mL microcentrifuge tube, which was centrifuged at
14000rpm for 30 seconds at room temperature. Supernatants were removed from the
tubes, bacterial pellets were resuspended in 100µL ice cold Solution I92 by vortexing,
then cells were lysed by the addition of 200µL freshly prepared Solution II93 to each
tube. Tubes were immediately inverted 6 times, 150µL ice cold Solution III94 was added
to each tube, and the tubes were gently vortexed three times, incubated on ice for 5
minutes and then centrifuged for 5 minutes and 14000rpm at room temperature.
Supernatants were transferred into fresh 1.5mL microcentrifuge tubes, 2 volumes of
100% ethanol (~900µL) was added, tubes were briefly vortexed, incubated for 2
minutes at room temperature then centrifuged at 12000rpm for 5 minutes at room
temperature. Supernatants were decanted, 600µL 70% ethanol25 was added to each tube,
the tubes were inverted to mix, centrifuged at room temperature for 5 minutes at
12000rpm, the supernatants drained and the tubes inverted to air dry for 20 minutes.
Plasmid pellets were resuspended in ~30µL ddH2O, 1µL RNase A (10mg/mL)70 was
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added to each tube, tubes were incubated at 37°C for 1 hour and purified plasmids were
stored at -20°C.
3.10 Large Scale Plasmid Purification Large scale plasmid preparation was carried out using an Invitrogen PureLinkTM HiPure
Plasmid Midiprep Kit. For this procedure, 5mL bacterial cultures were incubated for 6
hours at 37ºC and 225rpm in LB Broth43 containing the appropriate antibiotic, then
inoculated into 100mL LB broth43 containing the appropriate antibiotic and incubated
overnight at 37°C and 225rpm. The following day the culture was centrifuged at
4000rpm for 10 minutes at room temperature. Supernatants were removed, the pellets
were collected in 4mL Resuspension Buffer containing RNase A, the mixture was
pipetted until homogeneous and transferred to a sterile 15mL tube. Four mL Lysis
Buffer was added, the mixture was inverted to mix, incubated at room temperature for 5
minutes, then 4mL Precipitation Buffer was added and the tube was inverted to mix.
Tubes were centrifuged at 12000rpm for 20 minutes at 4°C and supernatants were
transferred to columns which had been pre-equilibrated by the addition of 10mL
Equilibration Buffer that had been allowed to flow through by gravity flow.
Supernatants were passed through each column by gravity flow, columns were washed
twice with 10mL Wash Buffer and the flow throughs discarded. A sterile 15mL tube
was placed under each column and the DNA was eluted from the column by adding
5mL Elution Buffer and allowing the buffer to pass through the column by gravity flow.
To precipitate the DNA, 3.5mL isopropanol was added to each elution tube, the solution
was inverted to mix and the tubes were centrifuged at 15000rpm for 30 minutes at 4°C.
Pellets were washed with 3mL 70% ethanol25, centrifuged at 15000rpm for 20 minutes
at 4°C, the ethanol removed and the pellets were air dried for 10 minutes then
resuspended in 100µL TE buffer. Purified plasmid DNA was stored at -20°C.
3.11 GST Fusion Protein Production and Purification
3.11.1 Small Scale Production of GST Fusion Proteins
For small scale production of GST fusion proteins, BL21 cells that had been
transformed with pGEX-2TK containing the insert of interest were spread onto LB
Agar/Ampicillin42 plates and incubated overnight at 37ºC (Section 3.8.6). The following
morning, one colony was inoculated into 5mL LB Broth/Ampicillin44 and incubated at
37ºC with shaking at 225rpm for ~2-3 hours until the OD600 was ~0.5. For cultures
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where GST-RING domain proteins were induced, 250µM ZnCl2 was added to the
medium to facilitate RING domain folding. Cultures were then divided in two and 25μL
100mM IPTG35 was added to one culture in order to induce GST fusion protein
production. The cultures were incubated for an additional 2.5 hours at 30ºC and 225rpm
and the OD600 recorded. One mL of each culture was transferred to a sterile 1.5mL tube
and the cells were centrifuged at 14000rpm for 1 minute at 4°C, the supernatants
discarded and the cell pellets resuspended in the required volume of Whole Cell Lysis
Buffer112 according to the formula:
Lysates were drawn through a 23G needle 20-30 times to decrease their viscocity and
stored at -20°C.
For small scale GST Fusion protein production where both the soluble and insoluble
protein fractions were to be collected, LB Agar/Ampicillin42 plates were streaked with
frozen glycerol stocks of BL21 transformed with the pGEX-2TK expression vector
containing the insert of interest (Section 3.8.6, 3.8.7). The plates were incubated
overnight at 37ºC and the following day one colony was inoculated into 5mL LB
Broth/Ampicillin44 and incubated overnight at 37ºC and 225rpm. The next morning,
0.5mL culture was transferred into a fresh 50mL tube containing 10mL LB
Broth/Ampicillin44 and incubated at 37ºC and 225rpm for ~1.5 hours until the OD600
was ~0.5. A 1mL aliquot was taken (uninduced control sample) and 95µL 100mM
IPTG35 was added to the remaining ~9.5mL culture, which was incubated for 3 hours at
30ºC and 225rpm to induce GST fusion protein production. The uninduced cells were
centrifuged at 14000rpm for 1 minute, the pellet resuspended in 200μL Whole Cell
Lysis Buffer112 and the lysate drawn through a 23G needle to decrease the viscosity.
GST fusion proteins were prepared by centrifuging the IPTG-treated cultures at
4000rpm for 5 minutes, discarding the supernatants and resuspending the pellets in
10mL Sonication Buffer95. One hundred μL 10mg/mL Lysozyme48 was added to the
resuspended cells and the tubes were inverted to mix, then incubated on ice for 30
minutes. One mL 10% Triton X-100111 was then added to each tube, the tubes were
inverted to mix and the solution sonicated on ice at 3 x 30sec (Setting 7, 25 watt output)
to lyse the bacterial cells. The solution was centrifuged at 14000rpm for 15 minutes and
the supernatant collected as the soluble fraction whilst the sedimented material
(insoluble fraction) was resuspended in 200μL Whole Cell Lysis Buffer112, as described
Volume (µL) = 1000 x OD600 8
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above. Ten μL of each sample was electrophoresed in 12% separating SDS
polyacrylamide gels78,96 (Section 3.15.2), which were stained with Coomassie blue11
(Section 3.15.5) and destained with Coomassie blue destaining solution10 to visualise
the proteins.
3.11.2 Large Scale GST Fusion Protein Production
To produce larger quantities of GST fusion proteins, BL21 bacterial cells transformed
with pGEX-2TK expression vectors containing the inserts of interest were inoculated
into 5mL LB Broth/Ampicillin44 and incubated overnight at 37ºC and 225rpm. The
following day, the 5mL culture was inoculated into 100mL LB Broth/Ampicillin44 and
incubated at 37ºC and 225rpm for ~2 hours until the OD600 was 0.6. For cultures where
GST-RING domain proteins were induced, 250µM ZnCl2 was added to the medium to
facilitate RING domain folding. GST fusion protein production was induced by the
addition of 1mL 100mM IPTG35 and the culture was incubated at 30ºC and 225rpm for
3 hours. Following incubation, the cultures were centrifuged at 3000rpm for 15 minutes
at 4ºC, the supernatants discarded and the cell pellets resuspended in 20mL Sonication
Buffer95. 200μL 10mg/mL Lysozyme48 was added to each suspension, the tubes were
inverted to mix then incubated on ice for 15 minutes prior to the addition of 144μL
200mM PMSF60, 50μL 0.5M EDTA28, 20μL 1M DTT20, 200μL 1M MgCl249 and 2mL
10% Triton X-100111 to each tube. Tubes were mixed by inversion and the cell
suspensions were lysed by sonication at 3 x 30 seconds (setting 7, 25 watt output).
Lysates were centrifuged at 27000rpm for 15 minutes at 4ºC and to isolate GST fusion
proteins, the supernatants were transferred to fresh 50mL tubes and incubated with
300μL 50% Glutathione agarose beads30 for 15 minutes at 4ºC with rotation. The beads
were collected by centrifugation at 3000rpm/4ºC for 2 minutes, the supernatants
removed and the beads resuspended in 10mL NETN Buffer53 then incubated for 10
minutes with rotation at 4ºC. The beads were again collected by centrifugation for 2
minutes at 3000rpm and 4ºC and washed in NETN Buffer53 as described above. To
elute the GST fusion proteins, the beads were pelleted by centrifugation at 3000rpm and
4ºC for 2 minutes, the supernatants removed and the beads resuspended in 300μL GST
Elution Buffer32 and incubated for 10 minutes at 4ºC with rotation. Beads were
collected by centrifugation at 3000rpm for 2 minutes at 4ºC and the supernatants
transferred to sterile 0.5mL tubes and stored at -80ºC. To confirm expression of the
GST fusion protein, an aliquot was electrophoresed in a 12% SDS polyacrylamide
gel78, 96 (Section 3.15.2) along with BSA standards (1μg - 10μg) and the gel stained with
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Coomassie blue11 and destained with Coomassie blue destaining solution10 (Section
3.15.5).
3.11.3 Total Protein Extraction from E. coli BL21 Cells
To obtain total protein from E. coli BL21 cells, BL21 cells were inoculated from
glycerol stocks into 5mL LB Broth43 and incubated overnight at 225rpm. Two mL
overnight culture was inoculated into 10mL LB Broth43 and the culture was incubated
for 4 hours at 37°C/225rpm. Bacterial cells were harvested by centrifugation, the
supernatant discarded and the cells were resuspended in 5mL Sonication Buffer95 with
the addition of 5µL 10mg/mL Lysozyme48. Cells were lysed by sonication at 3 x 30
seconds on ice (setting 7, 25 watt output), the debris sedimented by centrifugation for 5
minutes at 5000rpm and the supernatant collected and stored at -80°C in 0.5mL
aliquots.
3.12 DNA Sequencing Sequencing reactions were carried out using the dideoxy chain termination method
with Big DyeTM Terminator and Big DyeTM Terminator sequencing buffer (Sanger et
al., 1977). Sequencing reactions were prepared in 0.5mL microcentrifuge tubes and
contained 8μL 2.5x Big DyeTM Terminator sequencing buffer, 3pmol primer (Appendix
II), 250-500ng plasmid DNA, 0.5μL Big DyeTM terminator (v3.1) and ddH2O to a final
volume of 20µL. The sequencing reactions were carried out in PTC-100TM BioRad
Programmable Thermal Cyclers with 25 cycles of DNA denaturation at 96ºC for 15
seconds, primer annealing at 50ºC for 10 seconds, and primer extension at 60ºC for 4
minutes. Reactions were precipitated by adding 2μL 3M sodium acetate81 (pH4.6) and
50μL 95% ethanol25 to each tube, the tubes were vortexed then incubated on ice for 10
minutes. Tubes were centrifuged at 12000rpm for 30 minutes at 4°C, supernatants were
removed, pellets were rinsed with 70% ethanol25, centrifuged at 12000rpm for 5
minutes at 4°C and the supernatants removed. Tubes were air dried for 10 minutes at
room temperature, stored at -20ºC and reactions were sequenced by the The Lotterywest
State Biomedical Facility at Royal Perth Hospital using an ABI Prism 3730 capillary
sequencer. Sequencing chromatograms were viewed using Chromas Lite version 2.23
and analysed using Basic Local Alignment Search Tool (BLAST)
(http://blast.ncbi.nlm.nih.gov/Blast).
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3.13 Immunoprecipitation Immunoprecipitation reactions were carried out using Protein A or Protein G Sepharose
beads using an in-house laboratory protocol and buffers, or alternatively
immunoprecipitations using Protein A/G or anti-GFP microbeads performed using a
µMACSTM GFP purification kit and supplied buffers. Immunoprecipitation reactions
were carried out at 4°C using cells growing in 10cm petri dishes that had been
transfected with expression plasmids and cultured for 48 hours following transfection
(Section 3.1.4). To harvest cells, culture plates were placed on ice, the medium was
aspirated and cells were rinsed 3 times with 1mL ice cold PBS56. The PBS56 was
aspirated, 1mL ice cold RIPA buffer69 or Lysis Buffer47 (anti-GFP microbeads) was
added to each plate, cells were scraped into the buffer then transferred to pre-cooled
1.5mL microcentrifuge tubes on ice. The suspensions were pipetted to disperse, tubes
were rotated for 30 minutes at 4°C to lyse the cells, centrifuged at 10000rpm for 10
minutes to collect the cell debris and the ~1mL supernatant placed in a fresh pre-cooled
1.5mL microcentrifuge tube. A 50µL aliquot of the lysate (total input) was transferred
to a 0.5mL microcentrifuge tube and stored at -20°C. Where microbeads were utilised
for immunoprecipitation reactions, 2-4μg antibody (Table 3.6) and 100μL Protein A/G
microbeads (Miltenyi Biotec) were added to the remaining ~950μL lysate and the tubes
were incubated for 30 minutes on ice. For immunoprecipitation of GFP-tagged proteins,
50μL anti-GFP microbeads (Miltenyi Biotec) was added to the ~950μL cell lysate and
the tubes incubated on ice for 30 minutes.
To prepare μ columns (Miltenyi Biotec), 200μL RIPA Buffer69 or Lysis Buffer47 was
applied to each column and allowed to drain by gravity flow. Cell lysates were added to
the columns and allowed to drain through by gravity flow, the columns were washed 5x
with 200µL RIPA Buffer69 or 4x with 200µL Wash Buffer 1 and once with 100µL
Wash buffer 2 (anti-GFP microbeads). To elute bound proteins, 20μL pre-heated 1X
SDS-PAGE gel loading buffer77 or 20μL Elution buffer (anti-GFP microbeads) was
added to each column, the columns were incubated for 5 minutes at room temperature, a
further 50μL buffer was added to the column and the eluate was collected and analysed
by SDS-PAGE and western blotting (Section 3.15).
Where Protein A64 or Protein G66 Sepharose beads were used, 300µL Sepharose bead
slurry per sample was prepared by washing the beads in 1.5mL PBS56 five times and
centrifugating the slurry at 4000rpm for 2 minutes at 4ºC between washes. The beads
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were then resuspended in 150µL PBS56 to create a 50% slurry and stored in three 100µL
aliquots at 4°C. Cell lysates were prepared as above, then pre-cleared by the addition of
100µL 50% Protein A65/G67 slurry and incubation of the lysates for 1 hour at 4°C with
rotation. The beads were collected by centrifugation at 4000rpm for 2 minutes at 4ºC
and each supernatant divided equally into two fresh 1.5mL tubes, one containing 5µg
antibody (Table 3.6), and the other containing no antibody as a control (mock
immunoprecipitation). For the untransfected control, untransfected cell lysates were
prepared as above and incubated with 5μg antibody (Table 3.6). Tubes were incubated
overnight at 4°C with rotation and the next morning, 100µL 50% Protein A65 or Protein
G67 slurry was added to each tube, the tubes were incubated for 2 hours with rotation at
4°C to allow immunocomplex binding to Protein A or G and the beads collected by
centrifugation at 4000rpm for 2 minutes at 4°C. Supernatants were discarded, the beads
were washed with 1mL ice cold PBS56, then collected by centrifugation at 4000rpm and
4°C for 2 minutes and the supernatants discarded. This washing procedure was carried
out a further 4 times. After the last wash was removed, the proteins were eluted by
heating the beads in 50µL 2X SDS PAGE gel loading buffer77 at 95°C for 5 minutes.
The beads were collected by centrifugation at 4000rpm for 2 minutes at 4°C and the
supernatants were collected and analysed by SDS-PAGE and western blotting (Section
3.15).
Table 3.6 – Beads and buffers utilised for immunoprecipitation reactions
Buffer Utilised/ Microbeads Utilised
Protein A or G Microbeads
Anti-Tag Microbeads
Protein A or G Sepharose
Cell Lysis Buffer RIPA Buffer69 Lysis Buffer47 RIPA Buffer69
Column/Bead Preparation Buffer
RIPA Buffer69 Lysis Buffer47 PBS56
Wash Buffer RIPA Buffer69 4x Wash Buffer 1*
1x Wash Buffer 2* RIPA Buffer69
Column Elution Buffer 1X SDS PAGE gel loading buffer77
Elution Buffer* 2X SDS PAGE gel loading buffer77
* = µMACSTM GFP purification kit
3.14 Ubiquitin Assays
3.14.1 In Vitro Ubiquitin Assay
In vitro ubiquitin assays were carried out using a Ubiquitinylation kit (Enzo Life
Sciences). Each reaction required the addition of E1, E2, E3 enzymes, ATP, ubiquitin
Chapter 3 Methods
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and ubiquitinylation buffer in a 0.5mL tube (Table 3.7) and the reactions were carried
out at 37ºC for 60 minutes. To stop the reactions, 50µL 2x SDS-PAGE loading buffer79
was added to each tube, samples were heated at 95ºC for 8 minutes and the reaction
products separated in 12% polyacrylamide gels78, 96 or in 4-12% gradient
polyacrylamide gels. Biotinylated-ubiquitin was detected by western blotting using a
Vectastain ABC Elite Streptavidin-HRP Kit97 (Section 3.15).
Table 3.7 – In vitro auto-ubiquitination assay components
Auto-ubiquitination Assay (μL)
Minus ATP Control (μL)
Minus E3 enzyme Control (μL)
ddH2O To 50μL To 50μL To 50μL 10X Ubiquitinylation Buffer
5 5 5
100U/mL IPP68 10 10 10 50mM DTT21 1 1 1 0.1M Mg-ATP 2.5 - 2.5 20x E1 5 5 5 10x E2 2.5 2.5 2.5 E3 To 4µM To 4µM - 20x Bt-Ubiquitin 2.5 2.5 2.5
3.14.2 In Vivo Ubiquitin Assay
In vivo ubiquitination assays were performed by transfecting LNCaP cells growing in
10cm petri dishes with the appropriate plasmid combinations (Section 3.1.4, Table
3.8). Transfected cells were incubated at 37°C with 5% CO2 for 42-45 hours at which
time proteasome inhibitors (10µM MG13250 or 10µM Lactacystin37) were added to
allow the accumulation of ubiquitinated proteins, then the cultures were incubated for a
further 3-6 hours (Section 3.1.4). Cells were lysed 48 hours post-transfection and the
protein of interest, either GFP-RMND5 proteins or NKX3.1-V5, was
immunoprecipitated (Section 3.13) using anti-GFP microbeads or Protein A microbeads
with anti-V5 antibody, and the eluate analysed by 4-12% gradient SDS-PAGE and
western blotting (Section 3.15).
Table adapted from Ubiquitinylation Product Data Sheet UW9920 (Enzo Life
Sciences)
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Table 3.8 – In vivo ubiquitination assay plasmid combinations
Immunoprecipitated Protein
Transfected Plasmids (total=30µg/10cm plate)
Protein Tag Immunoprecipitated
RMND5A pEGFP-RMND5A, pCMV-HA-Ubiquitin
GFP
RMND5B pEGFP-RMND5B, pCMV-HA-Ubiquitin
GFP
NKX3.1 pcDNA3.1-NKX3.1-V5, pCMV-HA-Ubiquitin, pEGFP-RMND5A or pEGFP-RMND5B
V5
3.15 Western Blotting
3.15.1 Preparation of Whole Cell Lysates
To prepare whole cell lysates, medium was removed from cells growing in 6 or 12 well
plates (Section 3.1.4, 3.1.5), wells were rinsed with PBS56 and 100-250µL Whole Cell
Lysis Buffer112 was added to each well. Cells were scraped into the buffer using a
spatula and the lysates were transferred to 1.5mL microcentrifuge tubes. Lysates were
drawn through 23G needles into 1mL syringes until no longer viscous and stored at -
20ºC.
3.15.2 Polyacrylamide Gel Electrophoresis
Polyacrylamide gel electrophoresis was performed using a BioRad Protean® 3 Cell or
XCell SureLockTM Mini Cell Electrophoresis system. To prepare polyacrylamide gels,
plates were assembled according to the manufacturer’s instructions, 12% SDS-PAGE
separating gels78 were prepared and pipetted between the glass plates to ~0.5cm below
the level of the wells. The solution was overlayed with ddH2O and gels were
polymerised for 45 minutes at room temperature. The ddH2O was removed and the gel
space rinsed and then filled with 4% stacking gel96 solution. Combs were inserted
between the plates and the stacking gels were polymerised for 45 minutes at room
temperature.
Protein samples were prepared in 0.5mL microcentrifuge tubes and contained 10-20µL
sample (Sections 3.13, 3.14, 3.15.1) and 1-2µL 10X SDS PAGE Loading Buffer77.
Samples were denatured at 95°C for 8 minutes then allowed to cool to room
temperature. The electrophoresis apparatus was assembled according to the
manufacturer’s instructions, filled with 1X Running Buffer76 and samples were added to
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the wells in a fumehood. Where NUPAGE® Bis-Tris 4-12% gradient gels were used, the
XCell SureLockTM Mini Cell electrophoresis system was assembled according to the
manufacturer’s instructions and filled with 1X MOPS buffer51. Each gel included a well
containing 5μL ColorPlus Prestained Protein Marker. Gels were electrophoresed for ~45
minutes at 200V.
3.15.3 Western Transfer
Following electrophoresis, the apparatus was disassembled and gels were transferred to
containers of ice cold Transfer Buffer105. Transfer cassettes were assembled from
“black” to “white” and contained two sponges, two pieces of Whatman filter paper, the
gel, a piece of HybondTM-C Extra nitrocellulose membrane cut to the size of the gel,
two pieces of Whatman filter paper and two sponges. Each component was wetted with
ice cold Transfer Buffer105 before being added to the cassette and air bubbles were
removed by rolling a tube over the surface as each component was added to the cassette.
Cassettes were closed and inserted into a Mini-PROTEAN III cell transfer apparatus
that contained an ice block and magnetic stirrer. Tanks were filled with Transfer
Buffer105, placed on a magnetic stirrer and proteins were transferred overnight at 30V
with gentle stirring. Following overnight transfer, the transfer apparatus was
disassembled and filters were either stored at 4ºC between Whatman filter paper and
wrapped in aluminium foil or used immediately for immunoblotting (western blotting)
(Section 3.15.4).
3.15.4 Immunoblotting (Western Blotting)
Immunoblotting was performed at room temperature with horizontal rotation unless
otherwise indicated. Prior to immunoblotting, nitrocellulose membranes (Section
3.15.3) were cut to size and incubated for 90 minutes in TBS/3% Blotto103 blocking
solution. Filters were then sequentially incubated with primary antibody diluted in
TBST/1% Blotto103 for 90 minutes (Table 3.9), TBST102 for 3 x 10 minute washes,
secondary antibody diluted in TBST/1% Blotto103 for 90 minutes and washed 3 x 10
minutes with TBST102 (Table 3.9). For membranes blotted for biotinylated-ubiquitin,
the membrane was blocked for 1 hour in TBST/1% BSA104, washed 3x 10 minutes with
TBST102, incubated for 1 hour with streptavidin-HRP99 solution diluted in
TBST/1%BSA104 followed by 6 x 10 minute washes with TBST102. Filters were drained
and incubated with Enhanced Chemiluminescence (ECLTM) Western Blotting Detection
Reagent24 for 1 minute, the filters again drained, wrapped in plastic wrap and exposed to
Chapter 3 Methods
86
X-ray film for 10 seconds – 30 minutes as required. Exposed filters were developed in
an AGFA developer, scanned using an HP Photosmart 2710 scanner and analysed using
BioRad Quantity One® software. Using densitometry of the protein bands (BioRad
Quantity One®) proteins of interest were normalised to the housekeeping gene β-actin.
Table 3.9 – Primary and secondary antibodies and their respective dilutions
Primary Antibody Secondary Antibody Antibody Dilution Antibody Dilution
Goat anti-actin IgG 1:2000 Donkey anti-goat HRP conjugate
1:2000
Goat anti-NKX3.1 IgG 1:1000 Donkey anti-goat HRP conjugate
1:2000
Mouse anti-V5 IgG 1:2000 Sheep anti-mouse HRP conjugate
1:2000
Mouse anti-GFP IgG 1:2000 Sheep anti-mouse HRP conjugate
1:2000
Mouse anti-androgen receptor IgG
1:2000 Sheep anti-mouse HRP conjugate
1:2000
Rat anti-RFP IgG* 1:2000 Anti-rat HRP Conjugate
1:5000
Mouse anti-RFP IgG* 1:2000 Sheep anti-mouse HRP conjugate
1:2000
Mouse anti-HA (HRP labelled) 1:1000 - - Streptavidin-HRP97 33.3:1000 - -
* Red fluorescent antibodies show immunogenicity towards Cherry fluorescent tag which is a DsRed derivative
3.15.5 Coomassie Blue Staining
Following polyacrylamide gel electrophoresis (Section 3.15.2), gels were incubated in
Coomassie Blue staining solution11 for 2-16 hours with gentle rotation then destained
using Coomassie Blue Destaining10 solution for 40-60 minutes. Stained gels were dried
at room temperature between cellophane wrap and stored at room temperature. For mass
spectrometric analysis, 12% acrylamide gels were stained with a colloidal Coomassie
Blue solution12 and destained using 1% Acetic Acid1, both for 16-24 hours (Section
3.17).
3.16 Microscopic Imaging of Cells
3.16.1 Preparation of Slides for Fluorescence Microscopy
To prepare cells growing on coverslips in 6 well plates, 0.5μL Hœchst 33258 dye
(10mg/mL)34 was added to each well at 46 hours after transfection (Section 3.1.3, 3.1.4)
Chapter 3 Methods
87
and the cells were incubated for 2 hours at 37º/5% CO2 to allow uptake of the dye.
Medium was removed from the wells and cells were washed 3 x 5 minutes with PBS56,
the PBS56 was aspirated and cells were fixed in 4% formaldehyde29 for 15 minutes at
room temperature. The cells were again washed 3 x 5 minutes with PBS56, the PBS56
was aspirated and the cells were permeabilised by the addition of 200µL 0.1% Triton-X
100111 for 5 minutes at room temperature. Cells were washed for 3 x 5 minutes with
PBS56, the PBS56 was removed then 50µL TRITC-phalloidin dye55 was added to each
coverslip and the coverslips incubated at room temperature for 40 minutes. Phalloidin is
a compound extracted from Amanita phalloides that binds filamentous actin allowing
cytoplasmic staining of cells. Coverslips were washed for 3 x 5 minutes with PBS56 then
mounted onto glass microscope slides using 10µL mounting medium52 and stored in the
dark at 4ºC.
3.16.2 Preparation of Slides for Immunofluorescence Microscopy
For immunofluorescence microscopy, coverslips were initially processed as described
in Section 3.16.1, and following permeabilisation with 0.1% Triton-X100110, coverslips
were washed for 3 x 5 minutes with PBS56, the PBS56 aspirated, 400µL Confocal
Blocking Buffer9 was added to each coverslip and the coverslips incubated for 30
minutes at room temperature. The coverslips were washed 3 x 5 minutes with PBS56 and
incubated with primary antibody diluted 1:2000 in PBS/1% BSA57 overnight at 4ºC,
protected from light. The following morning the coverslips were washed 3 x 5 minutes
with PBS56, 100µL AlexaFluor®546 secondary antibody diluted 1:400 in PBS/1%
BSA57 was added to the coverslips and the coverslips were incubated at room
temperature for 1 hour. Coverslips were washed 5 x 5 minutes with PBS56, the PBS56
aspirated, 50µL phalloidin dye55 was added to each coverslip and the coverslips were
incubated at room temperature for 40 minutes. Coverslips were washed 5 x 5 minutes
with PBS56, mounted onto microscope slides using 10µL mounting medium52 then
stored at 4ºC protected from light.
3.16.3 Fluorescence Microscopy
An Olympus IX71 Inverted Microscope or a Nikon Ti-E Inverted Fluorescence
Microscope were used to visualise the prepared slides (Section 3.16.1, 3.16.2). The
Olympus IX71 microscope uses 6V30W Halogen illumination, whilst the Nikon Ti-E
microscopy uses TI-DS Diascopic illumination pillar 30W, and both microscopes use a
Chapter 3 Methods
88
range of filters to excite fluorophores using specific excitation wavelengths (Table
3.10). UV excitation wavelengths (10-400nm) were used to visualise blue fluorophores
such as Hœchst 3325834, blue excitation wavelengths (400-500nm) were used to
visualise green fluorophores such as EGFP, and green excitation wavelengths (500-
580nm) were used for the visualisation of red fluorophores such as
Tetramethylisothiocyanate (TRITC) phalloidin55 (Table 3.10). Photographs were taken
of the images using Image-Pro® Plus or Nikon Elements software and the images were
overlayed using Adobe Photoshop (Adobe Systems, Inc., San Jose, Calif.) and Confocal
Assistant 4.02 (Todd Clark Brelje) software then saved in .TIFF format.
Table 3.10 – Excitation and emission wavelengths of fluorescent labels
Excitation Wavelength (nm)
Emission Wavelength (nm)
Enhanced Green Fluorescent Protein
488nm 507nm
Hœchst 3325834 360nm-365nm 465nm Phalloidin (TRITC)55 540nm-545nm 570nm-573nm Alexa Fluor 546 (goat anti-mouse antibody)
546nm 570nm
Cherry 587nm 610nm
3.17 Mass Spectrometry To prepare proteins for mass spectrometric analysis, LNCaP cells growing in 4 x 10cm
dishes were transfected with plasmids encoding either GFP-RMND5A or GFP-
RMND5B (Section 3.1.4), the cells lysed and GFP immunoprecipitation of the lysates
performed using anti-GFP microbeads (Section 3.13). Mock immunoprecipitation
reactions using lysates from 3 x 10 cm2 dishes of untransfected cells and anti-GFP
microbeads was also performed as a control (Section 3.13). The immunoprecipitation
products were separated in 12% SDS-PAGE polyacrylamide gels 78, 96 (Section 3.15.2),
with a small aliquot of each sample analysed by GFP western blotting to confirm
efficient immunoprecipitation (Section 3.15.3, 3.15.4). The electrophoresed samples
were stained with Coomassie blue 12 (Section 3.15.5) and the protein bands of interest
were excised, with each added to a 1.5mL tube. 50µL acetonitrile was added to each
tube and the bands were dried by vacuum centrifugation for 1 hour at room temperature
then processed and analysed by the Australian Proteome Analysis Facility, Sydney.
Chapter 3 Methods
89
At APAF, bands were cut into small pieces, destained, dried and trypsin digested in
ammonium bicarbonate (pH8.0) overnight. The peptides were made up to 40µL in ESI
buffer and preconcentrated by injecting the sample onto a Michrome peptide Captrap
then desalted with 0.1% formic acid and 2% acetonitrile at a flow rate of 8µL/min.
Peptides were separated using an Exigent TEMPO nanoflow liquid chromatography
system using an SGE ProteCol C18, 300A, 3µm, 150µm x 10cm analytical column and
eluted from the column using a linear solvent gradient (steps from H2O:CH3CN (100:0,
+0.1% formic acid) to H2O:Ch3CN (10:90, +0.1% formic acid) over 80 minutes at a
flow rate of 500nL/min. The liquid chromatography products were analysed using a Q
Star Elite Mass Spectrometer (AB Sciex), and positive ion nanoflow electrospray, with
the mass spectrometer operated in an information dependent acquisition mode (IDA)
using a TOF mass analyser. The TOF MS survey scan was acquired with the three
largest multiply charged ions (counts >25) in each survey scan subjected to MS/MS
analysis and MS/MS spectra were accumulated for 2s (m/z 100-1600). The resulting
data were submitted to the search program Mascot (Matrix Science Ltd, London, UK)
and the peaklists were searched using the SwissProt database against Homo sapiens.
High scores were confirmed or qualified by operator inspection as a match. A decoy
database was also searched, providing a false positive identification percentage.
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
Chapter 4: Characterisation of RMND5 E3 Ubiquitin Ligase Activity
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
90
4.1 Introduction
Human RMND5 proteins are named after their yeast orthologue, RMD5, and although
the cellular roles of the human orthologues are not well characterised, the functions of
RMD5 are being progressively determined in S.cerevisiae and as such may shed light
on the functions of human RMND5A and RMND5B.
4.1.1 Yeast RMD5/Gid2
Yeast cells growing on non-fermentable carbon sources synthesise glucose via
gluconeogenesis, a pathway that involves the enzyme fructose-1,6-bisphosphatase
(FBPase), which under these conditions has a life-life of approximately 20 hours
(Funayama et al., 1980). Upon exposure to glucose, FBPase gene expression is
repressed and the existing protein undergoes rapid degradation with its half-life reduced
to ~20 minutes in a process known as catabolite inactivation (Gancedo, 1971; Schork et
al., 1995). The route by which FBPase is degraded is dependent on the amount of time
the cells have been starved of glucose prior to its addition to the medium. If cells have
been glucose starved for a short amount of time (<24 hours), FBPase is
polyubiquitinated and degraded by the ubiquitin-proteasome system, while for cells
growing on non-fermentable medium such as acetate for a longer time period (>24
hours), FBPase undergoes vacuolar import and degradation (Schork et al., 1994; Chiang
and Chiang, 1998; Hammerle et al., 1998).
Proteins involved in the degradation of FBPase by the ubiquitin-proteasome system are
known as glucose induced degradation of FBPase (Gid) proteins. Gid1-3, the first of the
Gid proteins identified to be involved in the degradation of FBPase was characterised
following the isolation of three mutants defective in glucose induced degradation
(Hammerle et al., 1998). These mutants were also defective in their ability to degrade
N-end rule proteins which are short lived proteins degraded by the ubiquitin-proteasome
system and recognised due to the presence of defined amino-terminal amino acid
residues (Varshavsky, 1997; Hammerle et al., 1998). Gid2 was also identified in a
screen of a single-gene deletion mutant databank for Saccaromyces cerevisiae genes
required for sporulation and meiosis. Enyenihi and Saunders identified 12 genes that
were not essential for meiotic entry but that were required for meiotic nuclear division
(RMD 1-12), including RMD5/Gid2, although its role in this process has not yet been
elucidated (Enyenihi and Saunders, 2003).
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
91
Gid2 is not present as a monomeric protein in the cell but is associated with a large,
600kDa protein complex, the remaining members of which, Gid4-9, were identified in a
mutational screen (Regelmann et al., 2003). The components of the complex have also
been identified in yeast systematic interaction studies (Ho et al., 2002; Krogan et al.,
2006; Pitre et al., 2006). The complex, named the Vid30/Gid complex, consists of
Gid1/Vid30, RMD5/Gid2, Gid4/Vid24, Gid5/Vid28, Gid7, Gid8 and Gid9/Fyv10, and
includes proteins which function in both of the FBPase degradation pathways
(Regelmann et al., 2003). The Gid proteins that degrade FBPase by the proteasome and
vacuolar import and degradation (Vid) proteins, discussed below, are responsible for
FBPase breakdown by the vacuole (Regelmann et al., 2003). The Vid30 complex is an
E3 ubiquitin ligase complex, with RMD5/Gid2 contributing its enzymatic activity to the
complex, a function mediated by its carboxy-terminal RING domain (Figure 4.1) (Santt
et al., 2008). In RMD5/Gid2 mutant cells, and in in vitro and in vivo ubiquitination
assays utilising an RMD5 RING domain mutant, FBPase is no longer ubiquitinated,
further supporting the role of this protein and the Vid30 complex in targeting FBPase
for degradation by the ubiquitin-proteasome system (Regelmann et al., 2003). Recently,
a second protein within the complex, Gid9/Fyv10, which contains a degenerate RING
domain, has been shown to associate with RMD5, contributing to the E3 ubiquitin
ligase activity of the complex (Braun et al., 2011).
Vid30, another member of the complex appears to be the substrate recognition
component as it was shown to interact with FBPase in immunoprecipitation reactions
and, along with Vid28, Vid30 is proposed to function as a core component of the Vid30
complex (Pitre et al., 2006; Santt et al., 2008). An additional associated member,
Gid4/Vid24 is not present in cells growing on ethanol but its expression is rapidly
induced upon growth of cells on glucose, however as Gid4/Vid24 mRNA levels are
similar under both conditions, Gid4/Vid24 regulation appears to occur at the
translational level (Pitre et al., 2006; Santt et al., 2008). Gid4/Vid24 is hypothesised to
activate the Vid30 complex by altering its conformation following glucose shift,
resulting in the ubiquitination and degradation of FBPase, which is dependent on RMD5
(Santt et al., 2008). Following FBPase degradation, Gid4/Vid24 is itself ubiquitinated
by RMD5 resulting in its proteasomal degradation in a regulatory feedback mechanism
(Figure 4.1) (Braun et al. 2011). The ubiquitin conjugating enzyme, Gid3/Ubc8 is
associated with the Vid30 complex and is involved in the ubiquitination of FBPase and
Gid4/Vid24.
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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Another protein associated with the complex, Gid6/Ubp14 is a deubiquitinating enzyme
that cleaves ubiquitin chains on FBPase, thereby reducing the competition for the
proteasome between polyubiquitin chains attached to the substrate and the substrate
itself (Amerik et al., 1997; Regelmann et al., 2003). Supporting this, cells lacking
Gid6/Ubp14 exhibit a three-fold reduction in FBPase degradation (Regelmann et al.,
2003). In addition to its regulation of FBPase, the Vid30 complex, including
RMD5/Gid2 and Gid9/Fyv10, are implicated in the regulation of other enzymes
involved in the irreversible steps of glucose synthesis, including the gluconeogenic
enzyme phosphoenolpyruvate decarboxykinase (PEPCK) and the TCA cycle enzyme
cytoplasmic malate dehydrogenase (c-MDH) (Figure 4.1) (Santt et al., 2008; Braun et
al., 2011). Interestingly, all three enzymes contain amino terminal proline residues, and
RMD5/Gid2 mutants are associated with defective degradation of N end rule pathway
proteins, while mutation of the amino terminal proline residue of FBPase renders it
incapable of degradation by the proteasome (Hammerle et al., 1998). These findings
suggest that the Vid30 complex plays a broader role in the regulation of responses of the
yeast cells to changing nutrient conditions by recognising substrates with amino
terminal proline residues.
Three members of the Vid30 complex, Gid1/Vid30, Gid5/Vid28 and RMD5/Gid2 have
also been implicated in the degradation of the high affinity hexose transporter 7 (Hxt7)
in response to rapamycin or nitrogen-starvation (Snowdon et al., 2008). Gid1/Vid30 and
Figure 4.1: Proposed mechanism of action of the Vid30 complex. Upon exposure to glucose, the Vid30 complex is activated by Gid4/Vid24, which enables RMD5/Gid2 and Gid9/Fyv10 to ubiquitinate FBPase, PEPCK and c-MDH, thereby targeting them for degradation by the proteasome (Braun et al., 2011).
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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Gid5/Vid28 play overlapping roles in Hxt7 internalisation and degradation as only
Vid30/Vid28 double mutants display substantial Hxt7 stabilisation (Snowdon et al.,
2008). Although RMD5/Gid2 mutant cells also exhibited delayed internalisation and
degradation of Hxt7, the transporter was eventually internalised and degraded in all
conditions implicating the involvement of other proteins in this process (Snowdon et al.,
2008). Vid30 has been shown to play a role in nitrogen catabolite repression by shifting
the cells towards glutamate production, particularly under low ammonia conditions (van
der Merwe et al., 2001). Therefore, Gid1/Vid30, Gid5/Vid28 and RMD5/Gid2 and the
Vid30 complex were hypothesised to function closely with the target of rapamycin
(TOR) pathway (Snowdon et al., 2008). A human orthologue of the Vid30 complex has
been reported and this complex, named the CTLH complex will be discussed in detail in
Chapter 6 (Kobayashi et al., 2007).
As described above, FBPase can also be degraded by the vacuole. FBPase is recognised
by Vid proteins and packaged into Vid vesicles, which fuse with the vacuole, resulting
in its degradation (Hoffman and Chiang, 1996; Chiang and Chiang, 1998). One protein
involved in this process is Gid4/Vid24, which is synthesised in the presence of glucose,
is associated with Vid vesicles and is required for the transport of FBPase in Vid
vesicles to the vacuole. Consistent with this function, Gid4/Vid24 mutants show an
accumulation of FBPase in Vid vesicles which do not move to the vacuole (Chiang and
Chiang, 1998). Coatomer proteins such as Sec28 which play roles in the endocytic
trafficking of proteins in yeast and mammalian cells, form part of Vid vesicles and
associate with Gid4/Vid24, leading to the hypothesis that Vid vesicles merge with the
endocytic pathway in the transport of FBPase to the vacuole for degradation (Brown et
al., 2008). Recently it has been shown that the Vid pathway does indeed merge with the
endocytic pathway at actin patches at the plasma membrane where early endocytosis
takes place, thereby utilising the endocytic pathway to deliver Vid vesicles and their
cargo including FBPase and MDH2 to the vacuole for degradation (Brown et al., 2010).
When glucose is added to starved cells, FBPase, MDH2 and the Vid vesicle associated
proteins Gid4/Vid24 and Sec28 are present at actin patches where Gid1/Vid30 attached
to Vid vesicles associates with the actin patches, thereby merging the two pathways
(Brown et al., 2010; Alibhoy et al., 2012).
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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4.1.2 Human RMND5 Proteins
Human RMND5A (Gene ID 64795) and RMND5B (Gene ID 64777) are the
uncharacterised human orthologues of yeast RMD5. The RMND5 proteins share 70%
amino acid homology and are highly conserved between mammalian species, indicating
that they perform both similar and important cellular roles.
4.1.2.1 RMND5A
RMND5A (p44CTLH/FLJ13910), which is located at the chromosomal locus 2p11.2,
spans approximately 54.7 kilobase pairs (kbp) and contains an open reading frame of 9
exons, producing an mRNA of 6201bp. Translation results in a 391 amino acid protein
that contains four protein-protein interaction domains, a Lissencephaly 1 homology
motif (LisH), a C-terminal to LisH motif (CTLH), a CT11-RanBPM (CRA) motif and a
Really Interesting New Gene (RING) domain. The RMND5A gene is conserved in dog,
cow, chimpanzee, mouse, rat, fruit fly, mosquito, Arabidopsis plants, nematode, mould
and rice (Homologene 5668).
4.1.2.2 RMND5B
RMND5B (FLJ22318), which has provisional protein coding status, is located at 5q35.3
and spans ~17.4kbp with an open reading frame of 11 exons. The transcribed mRNA is
1825bp and encodes a protein of 393 amino acid residues which, like RMND5A,
consists of four protein-protein interaction domains, a LisH, CTLH, CRA and RING
domains. RMND5B orthologues are found in dog, cow, chimpanzee, mouse, rat,
zebrafish and Arabidopsis plants (Homologene 100777).
4.1.3 Protein Domains
4.1.3.1 Lissencephaly 1 Homology Motif (LisH)
There are 2382 LisH domains present in 2364 proteins in the SMART non-redundant
database, with 1.09% (26) of these found in human proteins including RanBPM,
RanBP10 and OFD (SMART SM00667) (Schultz et al., 1998). In all 26 of the human
proteins, the LisH domain is found at the N-terminal except for one protein, DDB1 and
CUL4 Associated Factor 1 (DCAF1) in which the domain is found midway through the
protein (Table 4.1). Six of these 26 LisH-containing proteins also contain a CTLH and a
CRA domain, with a further two containing a LisH and CTLH domain. In addition to a
LisH domain seven proteins contain WD40 repeats. A further seven contain no other
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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identified protein domains, but may contain coiled coil repeats (Table 4.1) (Schultz et
al., 1998).
Table 4.1 – Human LisH domain containing proteins
Human LisH Domain Containing Proteins
Full Name Other protein Domains or Regions
Cellular Role
OFD1 Oral-facial-digital syndrome type 1
Coiled coil regions
Neural cell migration
NOLC1 Nucleolar and coiled-body phosphoprotein
SRP40 Transcription
SSBP2 Single-stranded DNA-binding protein 2
SSDP Regulator of haematopoietic growth/differentiation
SSBP3 Single-stranded DNA-binding protein 3
SSDP Regulation of development
SSBP4 Single-stranded DNA-binding protein 4
SSDP Unknown
DCAF1 DDB1 and Cul4 associated factor 1
Coiled coil region
SCF E3 ubiquitin ligase copmplex member
RMND5A Required for Meiotic Nuclear Division 5A
CTLH, CRA, RING
Unknown
RMND5B Required for Meiotic Nuclear Division 5B
CTLH, CRA, RING
Unknown
FR1OP FGFR1 oncogene partner - Erythroid proliferation/differentiation
MKLN1 Muskelin 1 CTLH, Discoidin-like domain, Kelch Repeat
Mediates cell spreading by interacting with TSP-1
TBL1X Transducin β Like Protein 1 X (F-Box like protein)
WD40, F Box Transcription
TBL1Y Transducin β Like Protein 1 Y
WD40 Transcription
ARMC9 Armadillo repeat containing protein 9 isoform 2
Coiled coil region
Downregulates α-catenin
LIS1 Lissencephaly 1/ Platelet-activating factor acetylhydrolase IB subunit alpha
WD40, coiled coil region
Neural cell migration
CP063/RHOT2 C16orf63/Ras homologue family member T2
- Rho GTPase
NPAT Nuclear protein of the ataxia telangiectasia mutated locus
- Transcription/histone gene expression
TWA1/CT011 C20orf11/Twa1 CTLH, CRA Unknown MAEA Macrophage erythroblast
attacher/Erythroblast macrophage protein
CTLH, CRA, putative RING
Erythroblast maturation
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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A8MX09 Putative uncharacterised protein WDR47
CTLH, WD40, coiled coil
Unknown
RanBP9/RanBPM Ran binding protein 9 SPRY, CTLH, CRA
Adaptor protein/cell signalling
RanBP10 Ran binding protein 10 SPRY, CTLH, CRA
Guanine nucleotide exchange factor
TBL1XR1 Transducin β-like 1 X-linked receptor 1
WD40 Transcription
SMU1 Suppressor of mec-8 and unc-52 homologue
CTLH, WD40 RNA splicing
Treacle Treacle Treacle domains
Nuclear trafficking phosphoprotein
TAF5 Transcription initiation factor TFIID subunit 5
TFIID, WD40 Transcription
KIAA1468 LisH domain and HEAT repeat-containing protein KIAA1468
Coiled coil regions
Unknown
The LisH domain was originally identified in the Lissencephaly 1 (LIS1) protein to
mediate LIS1 dimerisation and oligomerisation, along with the LIS1 coiled-coil domain
(Tai et al., 2002; Kim et al., 2004). By associating with Cytoplasmic Dynein Heavy
Chain (CDHC), which plays an integral role in the microtubule based transport of
organelles and cytoskeletal components, LIS1 modulates CDHC activity during neural
cell migration, axon growth and retrograde transport and is thereby proposed to regulate
microtubule dynamics (Sasaki et al., 2000; Smith et al., 2000). Mutations in the LIS1
protein, including those in the LisH domain, lead to Miller-Dieker lissencephaly, a
disease caused by defective neural cell migration, leading to mental retardation,
epilepsy and premature death (Cardoso et al., 2000; Emes and Ponting, 2001).
Similarly, mice that are heterozygous for a truncated Lis1 gene in which residues
encoding the LisH domain and a coiled coil region (1-63) are missing, display abnormal
cortex morphology hypothesised to be due to defective neuronal migration (Cahana et
al., 2001).
The LisH domain is also present in other proteins which are involved in neural cell
migration, including Oral-facial-digital syndrome type 1 (OFD1), Transducing β like 1
(TBL1) and Treacle, abnormalities in which give rise to neurological or craniofacial
disorders, suggesting that the LisH domain plays a similar role in each of these proteins
(Emes and Ponting, 2001; Kim et al., 2004). OFD1 is an X-linked dominant disorder
caused by mutations in the CXorf5 gene. The condition is lethal in males, while affected
females exhibit facial and oral malformations, and malformation of their digits,
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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suggesting the involvement of neural cell migration (Towfighi et al., 1985; Gerlitz et
al., 2005). Treacher Collins Syndrome arises from mutations in the Treacher Collins-
Franceschetti syndrome 1 (TCOF1) gene which encodes Treacle and results in the
abnormal migration of cells of the neural crest, leading to craniofacial abnormalities and
hearing impairment (Towfighi et al., 1985; Emes and Ponting, 2001; Marszalek et al.,
2002). TBL1, an F-box containing protein that functions as a substrate recognition
component in SCF E3 ubiquitin ligase complexes, has been implicated in the
pathogenesis of ocular albinism (Dimitrova et al., 2010). Mutations of key amino acids
in the LisH domains of LIS1, OFD1 and TBL1 reduce their half-life and lead to their
abnormal cellular localisation suggesting that the LisH domain is involved in the
regulation of these aspects of protein function (Gerlitz et al., 2005). Recently, the LisH
domain of muskelin has been shown to direct the nuclear localisation of this protein and
in a muskelin LisH domain–vinculin chimera, mutation of the LisH domain resulted in
the cytoplasmic mislocalisation of the vinculin chimera (Valiyaveettil et al., 2008).
Thus, the LisH domain appears to be an important determinant of intracellular
localisation.
The LisH domains of TBL1 and LIS1 are also essential for their dimerisation and
resolution of the crystal structure of the fibroblast growth factor receptor 1 (FGFR1)
oncogene partner (FOP), including its LisH domain identified that the LisH domain is
important for FOP dimerisation and centrosomal localisation (Gerlitz et al., 2005;
Mikolajka et al., 2006). Oligomerisation of the TBL1 and TBLR1 proteins is reliant on
the LisH domain. TBL1 and its receptor TBLR1 are associated with nuclear receptor
corepressor (N-CoR) and silencing mediator for retinoid and thyroid receptors (SMRT)
in large protein complexes which are associated with HDAC3. TBL1-TBLR1 stabilise
the structure of the corepressor complexes by forming interactions with HDAC3 and
histones H2B and H4, thereby aiding in chromatin-substrate recognition (Li et al., 2000;
Zhang et al., 2002; Yoon et al., 2003). Deletion of the TBL1 and TBLR1 LisH domains
results in their failure to interact with histone H4 and the inability of the N-CoR
complex to associate with chromatin, thereby inhibiting the function of the complex to
act as a transcriptional repressor (Choi et al., 2008). Similarly, in the yeast orthologue
of TBL1, Sif2, the LisH domain mediates protein tetramerisation and Sif2 interaction
with the Set3 complex, which shows homology to the human N-CoR and SMRT
corepressor complexes (Cerna and Wilson, 2005). The LisH domain of DCAF1 is
required for the formation of Cullin4A-RING E3 Ubiquitin Ligase (CRL4)
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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supramolecular complexes (Ahn et al., 2011). Thus, in multiple proteins, the LisH
domain is required for the formation of large protein complexes, including those
involved in the ubiquitin-proteasome system such as TBL1 and DCAF1.
Although its function is incompletely characterised, based on the functions of LisH
domain containing proteins, the LisH motif is proposed to be involved in determining
protein half-life, facilitating the formation of protein complexes and regulating
microtubule function by mediating protein dimerisation or by binding microtubules or
CDHC directly (Emes and Ponting, 2001). In addition, the LisH domain has been
proposed to be involved in cell migration, nucleokinesis and chromosome segregation
(Emes and Ponting, 2001).
4.1.3.2 C-Terminal to LisH (CTLH) Domain
The CTLH domain is an ill-conserved 58 amino acid residue motif with 5
characteristically arranged leucine residues (residues 12, 21, 31, 42, 50) which form a
“U” shaped domain structure (Zeng et al., 2006). According to the SMART database,
there are 708 CTLH domains in 679 proteins, however only nine of these (1.03%) are
found in humans (SMART SM0068) (Schultz et al., 1998). Eight of these proteins also
contain an N-terminal LisH domain and many also contain C-terminal WD40 repeats
(Table 4.1). A single protein, WD repeat containing protein 26 (WDR26), contains only
a CTLH domain and WD40 repeats and is therefore similar in domain structure to other
LisH and CTLH domain containing proteins. WDR26, originally described in 2004, is a
G-beta-like protein which is involved in the regulation of MAPK signalling pathways
and mediates MEKK1 repression of the transcriptional activity of the ETS transcription
factor ELK-1 (Zhu et al., 2004). WDR26 is also implicated in mediating the
transcriptional activity of other proteins. For example, WDR26 opposes H2O2 induced
cell death by down regulating AP-1 transcriptional activity (Zhao et al., 2009; Feng et
al., 2012). These findings therefore implicate the CTLH domain, in part, in
transcriptional regulation. Interestingly, the LisH and CTLH domains of yeast Vid30 are
important for its function in merging the Vid and endocytic pathways for FBPase and
MDH2 degradation as deletion of these domains results in the impairment of FBPase
association with actin patches (Section 4.1.1) (Alibhoy et al., 2012).
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
99
4.1.3.3 CT11-RanBPM (CRA) Domain
The CRA domain was initially identified in the C-terminus of RanBPM, and according
to the SMART database there are six human proteins, including RanBPM, which
contain this domain, all of which also contain the LisH and CTLH domains (Table 4.1)
(Schultz et al., 1998; Menon et al., 2004). RanBPM uses its CRA domain to interact
with the C-terminus of the Fragile X Mental Retardation Protein (FMRP), a region of
FMRP that contains an RGG box with which it interacts with RNA. For this reason it
was proposed that by using its CRA domain to interact with FMRP, RanBPM is able to
modulate the RNA binding-properties of FMRP (Menon et al., 2004). Additional
functions of the CRA domain of either RanBPM or of other CRA domain containing
proteins (Table 4.1) have not been reported.
4.1.3.4 Really Interesting New Gene (RING) Domain
The RING domain, typically found in E3 ubiquitin ligases contains the characteristic
consensus sequence, CX2X(9-39)CX(1-3)HX(2-3)C/HX2CX(4-48)CX2C (Section
1.7.2). In addition, RING variants such as RBQ1 and RBX1 (Section 1.7.2.1) are able to
function as E3 ubiquitin ligases with aspartate and asparagine residues in place of
conserved cysteine or histidine residues (Deshaies and Joazeiro, 2009). Yeast RMD5 is
another example of a functional E3 ubiquitin ligase that does not possess all eight
cysteine/histidine amino acids and analysis of the RING domains of RMND5A and
RMND5B indicated that they are similar in amino acid composition to yeast RMD5
(Section 4.2.1) (Santt et al., 2008).
4.1.4 RMND5 Proteins and Cancer
Disruption of RMND5A and RMND5B has been detected in a number of cancers. Using
differential display, quantitative RT-PCR and RNA in situ hybridisation, Li et al. (2008)
identified that RMND5A is overexpressed in ovarian cancer (Li et al., 2008). The
RMND5A chromosomal locus, 2p11.2 is amplified in pilocytic astrocytomas and
atypical lobular hyperplasia of the breast, whilst this region undergoes loss in mantle
cell lymphoma cell lines (Camps et al., 2006; Mastracci et al., 2006; Belirgen et al.,
2012). The chromosomal locus of RMND5B, 5q35.3 has been found to be deleted in a
number of cancers including neuroblastoma and non-small cell lung carcinomas
(NSLC) (Mendes-da-Silva et al., 2000; Mosse et al., 2005). Additionally, amplification
of this chromosomal locus has been identified in patients with asbestos related lung
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
100
tumours, clear cell renal carcinoma and uterine leiomyosarcoma (Nymark et al., 2006;
Chen et al., 2009; Raish et al., 2012). The 5q35.3 chromosomal locus undergoes loss of
heterozygosity (LOH) in 82% of breast tumours that also display mutations of the breast
cancer 1 (BRCA1) gene and in 44% of BRCA2 mutated breast tumours (Johannsdottir et
al., 2006). The region is also located within an uncharacterised prostate cancer
heritability locus identified in a genome-wide linkage analysis of 1233 prostate cancer
families, however the genes with positive linkage within the 5q35 locus have not been
further investigated (Xu et al., 2005; Christensen et al., 2010). As such, although the
chromosomal regions continuing the RMND5A and RMND5B genes are disrupted in a
number of cancers, characterisation of specific genes in the two loci, 5q35.3 and 2p11.2
which are responsible for tumour initiation and/or progression has not been reported.
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
101
4.2 Results
4.2.1 Bioinformatics Analyses of RMND5 Protein Architecture
In order to identify potential cellular functions of RMND5 proteins, the protein domain
architecture of RMND5A and RMND5B was predicted using online protein domain
prediction tools including the National Centre for Biotechnology Information (NCBI)
(http://www.ncbi.nlm.nih.gov/gene/64795), SBASE (http://www.icgeb.trieste.it/sbase)
and Simple Modular Architecture Research Tool (SMART) (http://smart.embl-
heidelberg.de/) databases (Vlahovicek et al., 2005, Schultz et al., 1998). These
databases identified four protein-protein interaction domains present in each of
RMND5A and RMND5B. RMND5A contained a Lissencephaly 1 homology (LisH)
(amino acids 114-146), a C-Terminal to LisH (CTLH) (amino acids 153-210), a CT-11
RanBPM (CRA) (amino acids 209-302) and a Really Interesting New Gene (RING)
(amino acids 336-377) domain, while RMND5B contained a LisH (amino acids 116-
148), a CTLH (amino acids 155-212), a CRA (amino acids 211-305) and a RING
(amino acids 338-379) domain (Figure 4.2A). Additionally, SBASE database identified
loosely conserved putative protein domains in the amino-terminal regions of RMND5A
and RMND5B, with RMND5A containing a ribulose phosphate 3-epimerase like
domain (amino acids 11-40) and a GAT-like domain (amino acids 42-94), and
RMND5B containing a putative myosin tail like domain (amino acids 26-91) (not
shown) (Vlahovicek et al., 2005). The functions of LisH, CTLH and CRA domain have
not been well characterised, however RING domains are well characterised motifs
commonly found in E3 ubiquitin ligases, suggesting that RMND5 proteins are able to
function as E3 ubiquitin ligases. Alignment of the RING domains of RMND5A,
RMND5B and yeast RMD5 identified that all eight zinc coordinating residues required
for RING domain folding and function are identical between the three proteins,
supporting the hypothesis that RMND5 proteins possess this enzymatic activity (Figure
4.2B).
4.2.2 Cloning of Full Length RMND5A into pGEX-2TK and Expression of
GST-RMND5 Proteins for In Vitro Ubiquitination Assays
4.2.2.1 Cloning of RMND5A into pGEX-2TK
To determine whether RMND5 proteins possess E3 ubiquitin ligase activity, full length
RMND5A was cloned into the pGEX-2TK expression vector to allow expression of
GST-RMND5A in bacteria. The pGEX-RMND5B construct was already available in
Chapter 4 Characterising RMND5 E3 Ubiquitin Ligase Activity
102
Figure 4.2: Protein domain architecture of RMND5 proteins. (A) Bioinformatics analyses identified four protein-protein interaction domains in RMND5 proteins with (i) RMND5A containing a Lissencephaly 1 homology (LisH) (aa 114-146), a C-Terminal to LisH (CTLH) (aa 153-210), a CT-11 RanBPM (CRA) (aa 209-302) and finally a Really Interesting New Gene (RING) (aa 336-377) domain. (ii) RMND5B was identified to contain a LisH (aa 116-148), a CTLH (155-212aa), a CRA (aa 211-305) and a RING (aa 338-379) domain. The protein domains of RMND5A and RMND5B exhibit a high degree of amino acid homology. (B) Alignment of the RING domains of RMND5A, RMND5B and yeast RMD5 identified that all zinc co-ordinating residues required for RING domain folding and function (red) were identical between the three proteins.
A (i)
(ii)
B
Amino acid position
1
1
80%
391
Lissencephaly 1 Homology Domain C-Terminal to LisH Domain CT-11 RanBPM Domain Really Interesting New Gene Domain
114 146 153 210 209 302 336 377
393
116 148 155 212 211 305 338 379
RMND5A
RMND5B
76% 60% 68% 74% Amino acid homology 84%
CPILRQQTTDNNPPMKLVCGHIISRDALNKMFNGS--KLKCPYC
CPILRQQTSDSNPPIKLICGHVISRDALNKLINGG--KLKCPYC
CPVLKEETTTENPPYSLACHHIISKKALDRLSKNGTITFKCPYC
RMND5A
RMND5B
RMD5 361 404
336 377
338 379
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
103
the laboratory (Dawson, 2006). To prepare RMND5A insert for ligation, the RMND5A
coding region was amplified by PCR using pEGFP-RMND5A as a template, the
RMND5ABamHI1-S and RMND5A1176-AS primers and a range of annealing
temperatures from 64°C - 68°C for 35 cycles (Sections 3.4.1, 3.8, Appendix II). The
PCR products were electrophoresed in a 1% agarose gel, identifying a product at the
expected size of ~1.2kb in PCRs using all annealing temperatures tested (Section 3.4,
Figure 4.3A). Five PCRs were performed using the optimised annealing temperature of
64°C, the PCR products were combined, “A” tails added and a 5µL aliquot of the
purified products electrophoresed in a 1% agarose gel, verifying a single band at the
expected size of ~1.2kb (Sections 3.4.1, 3.4.2, Figure 4.3B).
To obtain pGEMT-RMND5A, 100ng (2µL) of the RMND5A insert was ligated into
50ng (1µL) pGEM®T-Easy by TA cloning and the ligation products were transformed
into competent E. coli DH5α (Sections 3.8.4, 3.8.5.1, 3.8.6). Transformed bacteria were
spread on LB Agar/Ampicillin plates containing X-gal for blue/white colony selection
and after overnight incubation, 4 white colonies were picked, inoculated into LB
Broth/Ampicillin and incubated overnight (Sections 3.8.6, 3.9). The following day,
plasmids were isolated from the bacteria, RNase treated then digested with EcoRI to
identify clones containing an RMND5A insert (Sections 3.8.2, 3.9). The products were
electrophoresed in a 1% agarose gel which identified the correctly sized ~1.2kb insert in
all pGEMT-RMND5A clones 1-8 (Section 3.6, Figure 4.3C). To confirm the correct
sequence of RMND5A inserts, pGEMT-RMND5A clones 1 to 4 were purified, 5µL of
each was electrophoresed in a 1% agarose gel to estimate plasmid DNA concentration
and 2µL of pGEMT-RMND5A clones 1 to 4 was sequenced using M13-S and M13-AS
primers (Sections 3.6, 3.7.1, 3.12, Appendix II, not shown). Analysis of chromatograms
using BLASTTM verified the correct RMND5A sequence in all pGEMT-RMND5A
clones (Section 3.12, not shown).
To prepare RMND5A insert for ligation into pGEX-2TK, the RMND5A insert from
pGEMT-RMND5A clone 1 was released by EcoRI digestion, the products were
electrophoresed in a 1% agarose gel and the ~1.2kb RMND5A insert gel purified
(Sections 3.6, 3.8.2). Five µL of the purified RMND5A insert was electrophoresed in a
1% agarose gel, with 50ng (5µL) ligated into 50ng (2µL) pGEX-2TK that had been
EcoRI digested, SAP treated and purified (Sections 3.8.2, 3.8.3, Figure 4.3D, E). The
ligation products were transformed into E. coli DH5α and selected by growth on
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
104
Lane 1: MW Marker Lane 2: 64°C Lane 3: 65°C Lane 4: 66°C Lane 5: 67°C Lane 6: 68°C Lane 7: Negative Control
Lane 1: MW Marker Lane 2: Purified RMND5A Lane 3: Negative Control
A
~1.2kb
~1.2kb
Lane 1: MW Marker Lane 2, 4, 6, 8, 10, 12, 14, 16: Undigested pGEMT-RMND5A clones 1, 2, 3, 4, 5, 6, 7, 8 Lane 3, 5, 7, 9, 11, 13, 15, 17: EcoRI digested pGEMT- RMND5A clones 1, 2, 3, 4, 5, 6, 7, 8
C
D
~1.2kb ~4.2kb
~5kb
B
Lane 1: MW Marker Lane 2: pGEX-2TK Lane 3: BamHI/EcoRI/SAP digested pGEX-2TK
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
105
Figure 4.3: Cloning of full length RMND5A into pGEX-2TK. (A) pEGFP-RMND5A (template) and an annealing temperature gradient (64°C to 68°C) were used to amplify the RMND5A coding region. The products were electrophoresed in a 1% agarose gel, identifying the ~1.2kb insert in all reactions. (B) Following optimisation, the RMND5A coding region was amplified in quadruplicate reactions, the reactions were combined and purified. 5µL product was electrophoresed in a 1% agarose gel from which the DNA concentration was estimated to be ~50ng/µL. (C) pGEMT-RMND5A clones 1 to 8 were EcoRI digested to release the ~1.2kb RMND5A insert and the products were electrophoresed in a 1% agarose gel identifying inserts in all clones. (D) 5µL BamHI/EcoRI digested and purified pGEX-2TK plasmid was electrophoresed in a 1% agarose gel, from which the concentration of DNA was estimated to be 25ng/µL. (E) pGEMT-RMND5A was EcoRI digested, releasing the ~1.2kb RMND5A insert and 5µL gel purified product was electrophoresed in a 1% agarose gel. The DNA concentration was estimated from the gel to be ~10ng/µL. (F) pGEX-RMND5A clones 1-6 were EcoRI digested and the products were electrophoresed in a 1% agarose gel, identifying the RMND5A insert in all clones.
~1.2kb ~5kb
~1.2kb
E
F
Lane 1: MW Marker Lane 2: Purified RMND5A
Lanes 1, 14: MW Marker Lane 2, 4, 6, 8, 10, 12: Undigested pGEX-RMND5A clones 1, 2, 3, 4, 5, 6 Lane 3, 5, 7, 9, 11, 13: EcoRI digested pGEX- RMND5A clones 1, 2, 3, 4, 5, 6
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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LB Agar/Ampicillin plates (Section 3.8.6). Six colonies of pGEX-RMND5A were
inoculated into LB Broth/Ampicillin and small scale plasmid isolation was performed
followed by RNase treatment then EcoRI digestion to release the insert (Sections 3.8.2,
3.9). The reaction products were electrophoresed in a 1% agarose gel, identifying that
all six clones contained a product of the expected insert size of ~1.2kb (Section 3.6,
Figure 4.3F). pGEX-RMND5A clones 1 to 4 were purified and 5µL aliquots were
electrophoresed in a 1% agarose gel (Sections 3.6, 3.7.1, not shown). Based on the gel
image, 2µL each of pGEX-RMND5A clones 1 to 3 were sequenced using the pGEX-S
and pGEX-AS primers, with no mutations identified in pGEX-RMND5A clone 2 using
BLASTTM analysis (Section 3.12, Appendix II, Appendix III).
4.2.2.2 Small Scale Production of GST, GST-RMND5A and GST-RMND5B
pGEX-RMND5A clone 2 was transformed into E. coli BL21 cells and a glycerol stock
was prepared (Section 3.8.7, 4.2.2.1). To verify GST-fusion protein expression in
bacteria, pGEX-RMND5A and pGEX-RMND5B transformed E. coli BL21 cells were
selected on LB Agar/Ampicillin plates along with E. coli BL21 cells transformed with
pGEX-2TK (without insert) as a control. Expression of the GST-fusion proteins was
induced by the addition of 1mM IPTG (final concentration) and lysates of the cells were
electrophoresed in 12% polyacrylamide gels then stained with Coomassie blue to
visualise cellular proteins (Sections 3.11.1, 3.15.5, 3.15.1, 3.15.2). The presence in
lysates from IPTG-induced cultures of bands of ~25kDa (GST) and ~60kDa (GST-
RMND5A, GST-RMND5B) indicated that the transformed bacteria expressed the GST
fusion proteins, although the apparent molecular size of the GST-RMND5A and GST-
RMND5B was smaller than the expected molecular size of ~69kDa (Figure 4.4A).
4.2.2.3 Large Scale Production of GST-RMND5A
Following confirmation of bacterial production of GST-RMND5A (Section 4.2.2.2),
large scale purification of GST-RMND5A (Section 3.11.2) was performed and the
purified GST-RMND5A protein, along with aliquots collected from lysates of the
uninduced and IPTG induced cells, and the insoluble and unbound fractions were
electrophoresed in a 12% polyacrylamide gel, which was stained with Coomassie blue
(Sections 3.15.2, 3.15.5, Figure 4.5B). Although a prominent ~60kDa band was evident
in lysates from IPTG-induced cells, a ~60kDa band of purified GST-RMND5A was
only barely visible (Figure 4.4B). The prominent ~60kDa band was not evident in the
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
107
unbound fraction, which would usually indicate that the majority of the GST-RMND5A
had been bound to the glutathione sepharose beads, however, electrophoresis of the
insoluble fraction, which included a prominent ~60kDa band, showed that the majority
of the GST-RMND5A fusion protein was present in insoluble aggregates (Figure 4.4B).
To optimise the extraction of soluble GST-RMND5A, culture and purification
conditions were adjusted including IPTG induction of the cultures at room temperature,
28°, 30°C and 37°C, and from 30 minutes to 4 hours to capture the induced protein prior
to aggregation (not shown). In order to extract GST-RMND5A from the insoluble
aggregates, the bacterial lysates were sonicated for 4-6 x 30 second pulses on ice, and
up to seven elutions of the GST-RMND5A fusion proteins from the glutathione
sepharose was carried out at both room temperature or 4°C, however, similar low yields
of purified GST-RMND5A were obtained (Figure 4.4B). Finally, the pGEX-RMND5A
plasmid was transformed into Rosetta BL21 cells, which are codon-optimised bacterial
cells, and the production of GST-RMND5A was performed to determine whether this
strain also packaged the GST-RMND5A fusion protein into insoluble aggregates,
however, again the GST-RMND5A protein was present in the inclusion bodies with
very little in the soluble fraction (not shown). Thus, full length GST-RMND5A and
GST-RMND5B could not be purified from bacterial cells and an alternative approach
was taken whereby the RING domains of RMND5A and RMND5B would be cloned
into pGEX-2TK to generate GST-RMND5A RING and GST-RMND5B RING for use
in in vitro ubiquitination assays.
4.2.3 Cloning of RMND5 RING Domains for In Vitro Ubiquitination Assays
4.2.3.1 Cloning of RMND5 RING Domains into pGEX-2TK
To investigate the E3 ubiquitin ligase activity of RMND5A and RMND5B using the
RING domains of each of the proteins, the RMND5A (126bp) and RMND5B (126bp)
RING domains were PCR amplified from pEGFP-RMND5A and pEGFP-RMND5B,
respectively in PCRs utilising 1-2mM MgCl2 and a 55°C annealing temperature for 35
cycles (Section 3.4.1). The 117bp RING domain of CBL, a well-characterised E3
ubiquitin ligase was similarly PCR amplified from pCMV-CBL and electrophoresis of
each of the RING domains resulted in identification of bands of the expected sizes of
117bp (CBL) or 126bp (RMND5A, RMND5B) (Section 3.6, Figure 4.5A). The optimum
PCR conditions of 1.5mM MgCl2 and a 55°C annealing temperature were utilised to
amplify in quadruplicate the RING domains of each of RMND5A, RMND5B and CBL,
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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Figure 4.4: Expression and purification of full length GST-RMND5A and GST-RMND5B. (A) Expression of full length GST-RMND5A and GST-RMND5B was verified by electrophoresis and Coomassie blue staining of lysates from IPTG induced bacterial cells transformed with pGEX-RMND5A and pGEX-RMND5B. Protein bands at ~60kDa corresponding in size to GST-RMND5A and GST-RMND5A were identified in lysates from the IPTG-induced cultures. (B) GST-RMND5A was purified from IPTG induced pGEX-RMND5A transformed bacterial cells and the products electrophoresed in a 12% polyacrylamide gel which was stained with Coomassie blue. A ~60kDa band corresponding in size to GST-RMND5A was identified in lysates and purified products from IPTG induced cultures.
1 2 4 5 7
Lane 1: MW Marker Lane 2: GST: no IPTG Lane 3: GST: IPTG Lane 4: GST-RMND5A: no IPTG Lane 5: GST-RMND5A: IPTG Lane 6: GST-RMND5B: no IPTG Lane 7: GST-RMND5B: IPTG
~25kDa
~60kDa
1 2 3 4 5 6 7
Lane 1: MW Marker Lane 2: GST-RMND5A: no IPTG Lane 3: GST-RMND5A: IPTG, whole lysate Lane 4: GST-RMND5A: IPTG, soluble fraction Lane 5: GST-RMND5A: IPTG, unbound fraction Lane 6: GST-RMND5A: IPTG, purified fraction Lane 7: GST-RMND5A: IPTG, insoluble fraction
~60kDa
A
B
6 3
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
109
the four reactions for each RING domain were combined, purified and 5µL aliquots
were electrophoresed in a 2% agarose gel to verify product amplification (Sections
3.4.1, 3.6, Figure 4.5B).
The purified RING domains of RMND5A, RMND5B and CBL were ligated into the
pGEM®T-Easy cloning vector by ligating 30ng (1µL) of each PCR product into 50ng
(1µL) pGEM®T-Easy by TA cloning (Section 3.8.4). The ligation products were
transformed into competent E. coli DH5α and the transformed bacteria spread onto LB
Agar/Ampicillin plates containing X-gal to identify plasmids containing inserts using
blue/white colony selection (Section 3.8.6). Four white colonies for each of pGEMT-
RMND5A RING, pGEMT-RMND5B RING and pGEMT-CBL RING were picked,
inoculated into LB Broth/Ampicillin and incubated overnight. Plasmids were isolated
by small scale plasmid isolation, RNase treated, digested with BamHI and EcoRI and
the products were electrophoresed in 2% agarose gels (Section 3.6, 3.8.2, 3.9). Faint
insert bands of the appropriate size were identified in all of the plasmids and glycerol
stocks were prepared for each clone (Section 3.8.7, not shown).
To confirm that the correct sequences of the RMND5A, RMND5B and CBL RING
domains were present, plasmids from two clones of each of pGEMT-RMND5A RING,
pGEMT-RMND5B RING and pGEMT-CBL RING were purified, 2µL of each purified
plasmid was electrophoresed in a 1% agarose gel and based on the gels, 2µL of each
plasmid was sequenced using M13-S and M13-AS primers (Section 3.6, 3.7.1, 3.12, not
shown). Using BLASTTM, the correct RING domain sequences in pGEMT-RMND5A
clone 1, pGEMT-RMND5B clone 1 and pGEMT-CBL clone 1 were verified (Section
3.12, Appendix II, not shown).
To prepare the RING domains of RMND5A, RMND5B and CBL for subcloning into
pGEX-2TK, the RING domains of pGEMT-RMND5A RING clone 1, pGEMT-
RMND5B RING clone 1 and pGEMT-CBL RING clone 1 were released by digestion
with BamHI and EcoRI, the products were electrophoresed in 2% agarose gels, the
RING domain inserts were gel purified and 5µL of each electrophoresed in a 2%
agarose gel (Section 3.6, 3.7.1, 3.8.2, Figure 4.5C). Based on the gel, 7µL (30ng) of
each RING domain insert was ligated into 2µL (20ng) pGEX-2TK which had been
digested with BamHI and EcoRI and SAP treated (Section 3.8.2, 3.8.3, 3.8.4, Figure
4.5D). The ligation products were transformed into E. coli DH5α, selected by growth on
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
110
C Lane 1: MW Marker Lane 2: Purified RING RMND5A Lane 3: Purified RING RMND5B Lane 4: Purified RING CBL
~120bp
~120bp
Lane 1: MW Marker Lane 2: RMND5A RING (1mM MgCl2) Lane 3: RMND5A RING (1.5mM MgCl2) Lane 4: RMND5A RING (2mM MgCl2) Lane 5: RMND5B RING (1mM MgCl2) Lane 6: RMND5B RING (1.5mM MgCl2) Lane 7: RMND5B RING (2mM MgCl2) Lane 8: Negative Control Lane 9: MW Marker Lane 10: CBL RING (1mM MgCl2) Lane 11: CBL RING (1.5mM MgCl2) Lane 12: CBL RING (2mM MgCl2) Lane 13: Negative Control
A
~120bp
B Lane 1: MW Marker Lane 2: Purified RMND5A RING Lane 3: Purified RMND5B RING Lane 4: Purified CBL RING Lane 5: Negative Control
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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Figure 4.5: Cloning of sequences encoding the RING domains of RMND5A, RMND5B and CBL into pGEX-2TK. (A) PCR conditions for the amplification of the RING domains of RMND5A, RMND5B and CBL were optimised using LNCaP cDNA, an annealing temperature of 55°C and 1mM – 2mM MgCl2. (B) Following PCR optimisation, each RING domain was amplified in quadruplicate reactions, the reactions for each RING domain were combined and purified and 5µL of each purified RING domain was electrophoresed in a 2% agarose gel, from which the DNA concentrations were estimated to be ~30ng/µL. (C) pGEMT-RMND5A RING, pGEMT-RMND5B RING and pGEMT-CBL RING were digested with BamHI and EcoRI to release the RING inserts, the inserts were gel purified and 5µL of each purified insert was electrophoresed in a 2% agarose gel from which the concentration of each was estimated to be ~5ng/µL. (D) 5µL purified BamHI/EcoRI digested and SAP treated pGEX-2TK plasmid was electrophoresed in a 1% agarose gel from which the DNA concentration was estimated to be ~10ng/µL. (E) pGEX-RMND5A RING, pGEX-RMND5B RING and pGEX-CBL RING were purified and 5µL each purified product was electrophoresed in a 1% agarose gel.
Figure 4.9: BamHI and EcoRI digests and purification of pGEX-RING domains of RMND5A, RMND5B and CBL. (B) pGEX-RMND5A RING, pGEX-RMND5B RING and pGEX-CBL RING (i) clone 3 and (ii) clone 4 were purified and 5µL of each plasmid was electrophoresed in 1% agarose gels.
Lane 1: MW Marker Lane 2: Undigested pGEX-2TK Lane 3: Purified BamHI/EcoRI SAP digested pGEX-2TK
D
~5kb
Lane 1: MW Marker Lane 2: Purified pGEX-RING RMND5A clone 4 Lane 3: Purified pGEX-RING RMND5B clone 4 Lane 4: Purified pGEX-RING CBL clone 4
E
~5kb
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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LB Agar/Ampicillin plates and 4 colonies of bacteria transformed with each of pGEX-
RMND5A RING, pGEX-RMND5B RING and pGEX-CBL RING were inoculated into
LB Broth/Ampicillin for small scale plasmid isolation (Section 3.8.6, 3.9). Purified
plasmids were digested with RNase and following BamHI and EcoRI digestion to
release the inserts, plasmids were electrophoresed in 2% agarose gels, however no
inserts were visualised in the gels, potentially due to poor visibility of the small (117bp
and 126bp) sizes of the RING domain inserts (not shown). pGEX-RMND5A RING
clone 4, pGEX-RMND5B RING clone 4 and pGEX-CBL RING clone 4 were purified,
5µL aliquots of each were electrophoresed in a 1% agarose gel and 2µL of each plasmid
was sequenced using the pGEX-S and pGEX-AS primers (Section 3.6, 3.7.1, 3.12,
Appendix II, Figure 4.5E). Sequencing results indicated the presence of RING domain
inserts containing no mutations in all plasmids (Appendix III).
4.2.3.2 pGEX-RING Domain Protein Expression
Since each RING domain chelates two zinc ions, the medium in which bacterial
production of the GST fusion proteins was induced was supplemented with ZnCl2, as
was the buffer in which the eluted GST-fusion proteins was stored. This addition was
included in order to aid in the correct conformation and therefore functioning of the
RING domains. A range of ZnCl2 concentrations from 50µM - 250µM was tested to
determine whether the growth of E. coli BL21 cells was inhibited by the addition of
ZnCl2. For these experiments, the production of the GST-RMND5A RING domain
fusion protein was induced by the addition of 1mM IPTG to 5mL cultures, with 15µL
aliquots of the cell lysates electrophoresed in 12% polyacrylamide gels. Coomassie blue
staining of the gels identified that the amounts of bacterial proteins and the expression
of the GST-RMND5A RING domain were similar under all ZnCl2 concentrations
tested, indicating that the growth of E. coli BL21 cells and the production of GST-
fusion proteins was not affected by the addition of ZnCl2 to the culture medium (Section
3.11.1, 3.11.2). As such, 250µM ZnCl2 was added to the LB broth and 25µM ZnCl2 was
added to the elution buffer for subsequent experiments (Figure 4.6A).
For small scale production of GST-RING domains, the pGEX-RMND5A RING clone
4, pGEX-RMND5B RING clone 4 and pGEX-CBL RING clone 4 plasmids (Section
4.2.3.1) were transformed into E. coli BL21 cells and glycerol stocks were generated
(Section 3.8.7). To verify GST-fusion protein expression in bacteria, E. coli BL21 cells
transformed with pGEX-RMND5A RING clone 4, pGEX-RMND5B RING clone 4 and
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
113
pGEX-CBL RING clone 4 were streaked onto LB Agar/Ampicillin plates, individual
colonies were inoculated into LB broth/Ampicillin and expression of GST or the GST-
fusion proteins was induced by the addition of 1mM IPTG for 2.5 hours. Cells were
lysed, 15µL aliquots were electrophoresed in 12% polyacrylamide gels and the gels
were stained with Coomassie blue to visualise cellular proteins (Sections 3.11.1, 3.15.2,
3.15.5, Figure 4.6B). A prominent band of ~25kDa was evident in lysates of IPTG
treated cells transformed with pGEX-2TK (GST), while in those extracts of cells
transformed with pGEX-RMND5A RING, pGEX-RMND5B RING and pGEX-CBL
RING, prominent protein bands of the expected size of ~28kDa were present (Figure
4.6B). In lysates of cells transformed with pGEX-CBL RING, a strong band running at
a slightly higher than expected molecular weight of ~30kDa was evident (Figure 4.6B).
These findings indicated that following IPTG induction, each of the GST fusion
proteins was strongly expressed. For in vitro ubiquitination assays, GST and each of the
GST-RING domains were purified from 100mL bacterial cultures (Section 3.11.2,
4.2.4) and the purified proteins were electrophoresed in 12% polyacrylamide gels along
with BSA standards (Section 3.15.2). Gels were stained with Coomassie blue, indicating
successful production of GST and each of the GST-RING domain fusion proteins
(Section 3.15.5, Figure 4.6C, D).
4.2.4 In Vitro Auto-Ubiquitination Assays
4.2.4.1 Optimisation of In Vitro Auto-ubiquitination Assays using the GST-
CBL RING Domain
To optimise experimental conditions, in vitro ubiquitination assays were performed
using 4µM (final concentration) of the GST-RING domain of CBL and the E2
conjugating enzyme UbcH5b, which has been shown to mediate ubiquitin transfer with
CBL in vitro (Huang et al., 2009). These 50µL reactions were set up using an in vitro
Ubiquitinylation kit (ENZO Biosciences), reactions were terminated by the addition of
50µL 2x non-reducing loading dye and the products were electrophoresed in 12%
polyacrylamide gels (Section 3.14.1). Western blotting for biotinylated ubiquitin
resulted in the identification of a prominent band at >100kDa, as expected for a positive
result, however multiple additional bands at lower molecular weights were also present,
representing proteins bound to ubiquitin via isopeptide and thioester bonds (Figure 4.7).
In order to minimise the thioester-linked ubiquitinated protein bands, subsequent in
vitro ubiquitination assays were terminated by the addition of 2x reducing loading dye
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
114
A
B
1 2 3 4 5 6 7 8 9 10 Lane 1: MW Marker
Lane 2: 1µg BSA
Lane 3: 2.5µg BSA
Lane 4: 5µg BSA
Lane 5: 7.5µg BSA
Lane 6: 10µg BSA
Lane 7: 1µL GST-RMND5A RING
Lane 8: 3µL GST-RMND5A RING
Lane 9: 3µL GST-RMND5B RING
Lane 10: 1µL GST-RMND5B RING
~28kDa
C
Lane 1: MW Marker
Lane 2: GST: no IPTG
Lane 3: GST: IPTG
Lane 4: GST-RMND5A RING: no IPTG
Lane 5: GST-RMND5A RING: IPTG
Lane 6: GST-RMND5B RING: no IPTG
Lane 7: GST-RMND5B RING: IPTG
Lane 8: GST-CBL RING: no IPTG
Lane 9: GST-CBL RING: IPTG
~28kDa
1 2 3 4 5 6 7 8 9
~28kDa
1 2 3 4 5 6 7
Lane 1: MW Marker
Lane 2: GST-RMND5A RING: no IPTG
Lane 3: GST-RMND5A RING: IPTG
Lane 4: GST-RMND5A RING: IPTG and 50µM ZnCl2
Lane 5: GST-RMND5A RING: IPTG and 100µM ZnCl2
Lane 6: GST-RMND5A RING: IPTG 200µM ZnCl2
Lane 7: GST-RMND5A RING: IPTG and 250µM ZnCl2
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
115
Figure 4.6: Expression and purification of the GST-RING domains of
RMND5A, RMND5B and CBL. (A) The production of the GST-RMND5A RING
domain (~28kDa) was assessed by IPTG induction of pGEX-RMND5A RING
transformed E. coli BL21 cells that had been cultured in LB broth containing 50µM-
250µM ZnCl2. 15µL aliquots of each bacterial lysate was electrophoresed in a 12%
polyacrylamide gel and the gel was stained with Coomassie blue. (B) The
production of GST-RMND5A RING, GST-RMND5B RING and GST-CBL RING
domains was induced by the addition of IPTG to E. coli BL21 cultures transformed
with pGEX-RMND5A RING, pGEX-RMND5B RING and pGEX-CBL RING,
respectively. 15µL aliquots of each bacterial lysate was electrophoresed in 12%
polyacrylamide gels which were stained with Coomassie blue to determine GST
fusion protein production. The GST-RMND5A RING, GST-RMND5B RING and
GST-CBL RING domains were purified from large scale IPTG induced bacterial
cultures and the purified products electrophoresed in 12% polyacrylamide gels then
stained with Coomassie blue. The concentration of each GST-RING domain was
estimated in comparison to BSA standards, with the concentration of (C) GST-
RMND5A RING domain and GST-RMND5B RING domain estimated to be
~0.7µg/µL and ~0.9µg/µL, respectively, whilst (D) the concentration of GST-CBL
and GST were estimated to be ~1.9µg/µL and ~2.1µg/µL, respectively.
Lane 1: MW Marker
Lane 2: 1µg BSA
Lane 3: 2.5µg BSA
Lane 4: 5µg BSA
Lane 5: 7.5µg BSA
Lane 6: 10µg BSA
Lane 7: 1µL GST
Lane 8: 3µL GST
Lane 9: 1µL GST-CBL RING
Lane 10: 3µL GST-CBL RING
1 2 3 4 5 6 7 8 9 10
~25kDa ~28kDa
D
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
116
and the samples were boiled at 95°C for 5 minutes (Section 3.14.1). This resulted in the
detection of fewer bands compared to experiments where non-reducing loading dye was
used and the samples were not boiled (Figure 4.7).
4.2.4.2 In Vitro Auto-ubiquitination Assays Using the GST-RMND5A and
GST-RMND5B RING Domains
In vitro ubiquitination assays were performed using 4µM (final concentration) each of
the GST-RMND5A RING and GST-RMND5B RING domains along with the GST-
CBL RING domain as a positive control (Section 4.2.3.2). A negative control reaction
that lacked ATP was also included as the E1 enzyme requires ATP for the activation of
ubiquitin. Again the reactions were terminated using 2x reducing loading dye and the
products boiled at 95°C for 5 minutes prior to electrophoresis in 12% polyacrylamide
gels and western blotting for biotinylated ubiquitin (Section 3.14.1, 3.15). Under these
conditions, the negative control reaction resulted in a single band at ~10kDa
corresponding to free ubiquitin, as expected, whilst reactions where the GST-RING
domains of RMND5A and RMND5B were utilised resulted in a prominent band at
>100kDa, similar to that of the CBL RING domain (Figure 4.7). Lower molecular
weight bands were also present, corresponding to ubiquitinated RING domains and
ubiquitin isopeptide and thioester linked to the E2 conjugating enzyme UbcH5b. Initial
experiments therefore indicated that these experimental conditions would be suitable for
evaluation of RMND5A and RMND5B auto-ubiquitination activity.
4.2.4.3 Screening of E2 Conjugating Enzymes in In Vitro Ubiquitination
Assays
In order to determine the E2 enzymes with which RMND5A and RMND5B could
interact to mediate the transfer of ubiquitin, a panel of eleven E2 conjugating enzymes
including UbcH5b was utilised in in vitro ubiquitination assays (Section 3.14.1). The E2
enzymes UbcH1, UbcH2, UbcH5a, UbcH5b, UbcH5c, UbcH6, UbcH7, UbcH8,
UbcH10 and UbcH13 were tested with the GST-RING domains of RMND5A and
RMND5B in these experiments. Control reactions included UbcH5b and GST-CBL
RING domain (positive control), a reaction lacking ATP (negative control) and a
reaction lacking a GST-RING domain (negative control). Following electrophoresis of
reaction products in 12% polyacrylamide gels and western blotting for ubiquitin, high
molecular weight bands were present when the GST-RMND5A RING domain was
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
117
Figure 4.7: Optimisation of in vitro ubiquitination assays. The GST-CBL RING domain and UbcH5b were used to optimise in vitro ubiquitination assays. Reactions were initially terminated by the addition of 2x non-reducing loading dye (left panel) and 10µL of each reaction product was electrophoresed in a 12% polyacrylamide gel then analysed by western blotting for biotinylated ubiquitin. To reduce the presence of thioester-ubiquitinated proteins, subsequent reactions were terminated by the addition of 2x reducing loading dye and heating at 95°C for 5 minutes (middle panel). Using this method, in vitro ubiquitination assays containing the GST-CBL, GST-RMND5A or GST-RMND5B RING domains resulted in high molecular weight protein bands corresponding to polyubiquitinated proteins (right panel). Experiment was performed twice.
Chapter 4
Characterisation of RM
ND5 E3 U
biquitin Ligase Activity
Figure 4.8: RMND5 RING domains mediate ubiquitin transfer with specific E2 conjugating enzymes. In vitro ubiquitination assays were carried out using the GST-RMND5A or GST-RMND5B RING domains along with a panel of 11 E2 conjugating enzymes and 10µL each reaction product was electrophoresed in 4-12% gradient gels. Control reactions were also performed lacking ATP, E3 enzyme (negative controls) or containing the functional E3 ubiquitin ligase CBL (positive control). Following western blotting for biotinylated ubiquitin, high molecular weight bands corresponding to polyubiquitinated proteins were visible in assays containing the GST-RMND5A RING domain with UbcH2, UbcH5b and UbcH5c and the GST-RMND5B RING domain with UbcH5b and UbcH5c. Experiment was performed once.
118
RMND5A RING domain RMND5B RING domain
Ub- RING(>100kDa)
UbProteins
(~15-50kDa)
Free Ubiquitin (~10kDa)
Controls
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
119
utilised in in vitro reactions with the E2 enzymes UbcH2, UbcH5b and UbcH5c, whilst
similar high molecular weight bands were present when the GST-RMND5B RING
domain was utilised in in vitro ubiquitination assays with UbcH5b and UbcH5c (Section
3.14.1, 3.15, Figure 4.8). These results indicated that RMND5A and RMND5B both
exhibit E3 ubiquitin ligase activity in vitro and that RMND5A interacts with UbcH2,
UbcH5b and UbcH5c, while RMND5B interacts with UbcH5b and UbcH5c to mediate
ubiquitin transfer.
4.2.4.4 Control In Vitro Auto-Ubiquitination Assays
In order to determine whether the results obtained in in vitro ubiquitination assays
(Section 4.2.4.2, 4.2.4.3) were specific to the inclusion of GST-RMND5A RING or
GST-RMND5B RING domains, the assays were repeated using the E2 enzyme UbcH5b
and each time omitting a reaction ingredient (Section 3.14.1). In addition, GST alone
was used in a control reaction. Where GST alone was utilised, lower molecular weight
bands were present, corresponding to free ubiquitin, polyubiquitin or ubiquitinated E2
enzyme (Figure 4.9). In negative control reactions where ubiquitin, ATP, E1 or E2 were
omitted, only free ubiquitin or polyubiquitin bands were present, whilst reactions where
GST-RMND5A RING or GST-RMND5B RING domains were omitted resulted in the
same bands as well as additional products corresponding to ubiquitinated E2 enzyme
(Figure 4.9). In reactions where GST-RMND5A RING and GST-RMND5B RING
domains were added, bands corresponding to free ubiquitin, polyubiquitin and
ubiquitinated E2 enzymes at lower molecular weights were evident, in addition to a
band of ~38kDa that was not present in other reactions, which corresponded in size to
the monoubiquitinated RING domains (Figure 4.9). These results indicated that the
positive results in in vitro ubiquitination assays (Section 4.2.4.2, 4.2.4.3) were specific
for the GST-RMND5A RING and GST-RMND5B RING domains and that RMND5
proteins were able to auto-monoubiquitinate their RING domains.
4.2.5 In Vivo Ubiquitination Assays
In order to determine whether full length RMND5 proteins possess E3 ubiquitin ligase
activity in mammalian cells, in vivo ubiquitination assays were performed by
transfecting LNCaP cells with plasmids encoding GFP-RMND5A or GFP-RMND5B
and HA-Ubiquitin, and following 6 hours of proteasome inhibition with 10µM
lactacystin the cells were lysed at 48 hours post-transfection (Section 3.1.4, 3.1.5, 3.10).
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
120
-
RMND5BRING-Ub~38kDa
Free Ub(~10kDa)
RMND5ARING-Ub~38kDa
Free Ub(~10kDa)
ATPUbiquitin
E1UbcH5bGST-RINGGST
++++
+-
++++-
-+
+++-
-
++
++-
-
+++
+-
-
++++
-
++++
-+
WB: Bt-Ubiquitin
Figure 4.9: Control reactions for in vitro ubiquitination assays. In vitro
ubiquitination assays were performed using UbcH5b and a full set of
controls for each of RMND5A and RMND5B. Controls included the
omission of one ingredient per reaction and inclusion of GST instead of
GST-RMND5A RING domain or GST-RMND5B RING domain. The
presence of a band at ~38kDa corresponding in size to the
monoubiquitinated RING domain was only present in reactions containing
all assay ingredients and either GST-RMND5A or GST-RMND5B. The
experiment was performed twice and representative results are shown.
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
121
GFP immunoprecipitation was used to isolate GFP-tagged RMND5A and RMND5B
proteins, which were electrophoresed in 4-12% gradient polyacrylamide gels to allow
separation of high molecular weight ubiquitinated proteins. Increased expression of high
molecular weight protein bands were detected by HA western blotting (for HA-
ubiquitin) in lysates of cells that overexpressed GFP-RMND5A or GFP-RMND5B,
indicating that RMND5A and RMND5B were associated with ubiquitinated proteins in
LNCaP cells (Section 3.13, 3.15, Figure 4.10). These results were enhanced when
cultures were treated with the proteasome inhibitor, lactacystin which allowed
accumulation of ubiquitinated proteins, producing intense smears of high molecular
weight proteins of >80kDa (Figure 4.10). Western blotting of the total cell lysates of
these samples identified the presence of GFP, GFP-RMND5A and GFP-RMND5B and
the similar expression of HA-ubiquitin in all samples, indicating that the results were
due to differences in levels of GFP-RMND5A/GFP-RMND5B associated ubiquitinated
proteins (not shown). These experiments provided evidence that RMND5 proteins are
associated with ubiquitinated proteins in LNCaP cells, which is consistent with the in
vitro ubiquitination assay results.
4.2.6 Investigation of the E3 Ubiquitin Ligase Activity of the RMND5A and
RMND5B RING Domains using RMND5A (C356S) and RMND5B (C358S)
RING Domain Mutants
In order to demonstrate that the RING domains of RMND5 proteins were responsible
for their E3 ubiquitin ligase activity in in vitro and in vivo ubiquitination assays, a single
amino acid change was introduced into the RING domains of RMND5A (C356S) and
RMND5B (C358S) using site-directed mutagenesis (Section 3.4.3, Figure 4.11). The
corresponding mutation in the yeast RMD5 (C379S) was previously shown to inhibit its
ability to ubiquitinate its substrate protein, fructose-1, 6-bisphosphatase (Santt et al.,
2008). Once the full length pEGFP-RMND5A (C356S) and pEGFP-RMND5B (C358S)
mutants were generated, the RMND5A (C356S) and RMND5B (C358S) RING domains
were cloned into the pGEX-2TK expression vector to allow the expression of GST-
RMND5A (C356S) RING and GST-RMND5B (C358S) RING.
4.2.6.1. Introduction of C356S into RMND5A RING Domain
Site-directed mutagenesis was performed using a PCR based method and initial
optimisation of PCR conditions was carried out in 26 cycle reactions using a gradient of
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
122
Figure 4.10: In vivo ubiquitination activity of RMND5 proteins. LNCaP cells were transfected with plasmids encoding HA-Ubiquitin, GFP-RMND5A or GFP-RMND5B or empty vector and cultured for 48 hours post-transfection, with 10µM Lactacystin added for the final 6 hours of culture. Following GFP immunoprecipitation of cell lysates, HA western blotting was performed on the immunoprecipitated proteins, identifying increased association of GFP-RMND5A and GFP-RMND5B with ubiquitinated proteins that was enhanced by proteasome inhibition. The experiment was performed twice and representative results are shown.
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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Figure 4.11: Site directed mutagenesis of the RING domains of RMND5A and RMND5B. Alignment of the RING domains of RMND5A, RMND5B and yeast RMD5 showing all eight amino acid residues required for RING domain activity (red/orange). These include the conserved cysteine residues (bold, orange) to be mutated to serine (green) in order to reduce or inactivate the RING domain activity of RMND5A and RMND5B. Numbers indicate amino acid number.
CPILRQQTTDNNPPMKLVCGHIISRDALNKMFNGS--KLKCPYC
CPILRQQTSDSNPPIKLICGHVISRDALNKLINGG--KLKCPYC
CPVLKEETTTENPPYSLACHHIISKKALDRLSKNGTITFKCPYC
RMND5A (C356S)
RMND5B (C358S)
RMD5 (C379S)
S
336 377
338 379
361 404
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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annealing temperatures ranging from 65°C-72°C, 1.5mM MgCl2 and the pEGFP-
RMND5A plasmid as the DNA template (Section 3.4.3). Electrophoresis of 10µL of the
amplified products identified a single band at the expected size of ~5.9Kb, which was
present at all annealing temperatures tested but was more prominent at lower annealing
temperatures (Figure 4.12A). The reaction products from PCRs performed with
annealing temperatures of 65°C and 65.5°C were digested with DpnI to degrade the
parental methylated plasmid DNA and the reaction products were transformed into E.
coli DH5α and selected by plating on LB Agar/Kanamycin (Section 3.4.3.1, 3.8.6). Six
of the resulting colonies were picked, cultured in LB Broth/Kanamycin and plasmids
isolated by small scale plasmid purification (Section 3.9). Plasmids were treated with
RNase then digested with EcoRI to release the RMND5A insert and 10µL of the reaction
products were electrophoresed in a 1% agarose gel (Section 3.6, 3.8.2, Figure 4.12B).
Bands corresponding to the ~1.2kb RMND5A (C356S) insert were identified in all
clones and the pEGFP-RMND5A (C356S) clones 1 to 4 were purified, 5µL of each
purified clone was electrophoresed in a 1% agarose gel to verify the presence of the
~5.9kb insert and based on the gel, 2µL of each clone was sequenced using the
RMND5A1176-AS primer (Section 3.6, 3.7.1, 3.12, Appendix II, not shown).
BLASTTM analysis of the sequencing chromatograms verified the presence of the
C356S mutation in clones 1 and 4, whilst clones 2 and 3 each had a region of inserted
DNA, therefore, the entire coding region of the pEGFP-RMND5A (C356S) clones 1
and 4 was sequenced using the RMND5A603-S, RMND5A1-S and RMND5A490-AS
primers (Section 3.12, Appendix II). Sequencing analysis detected no mutations aside
from the G1061C base substitution that encoded the C356S amino acid change (not
shown).
To ensure that no further mutations were present in the pEGFP-C2 vector that contained
the RMND5A (C356S) RING domain mutation, the RMND5A coding region was
excised from the pEGFP-C2 plasmid by digestion with EcoRI, gel purified and the
purified insert electrophoresed in a 1% agarose gel, verifying the presence of the ~1.2kb
product (Sections 3.6, 3.7.2, 3.8.2, Figure 4.12C). Fresh pEGFP-C2 vector was digested
with EcoRI, SAP treated, purified and a 2µL aliquot electrophoresed in a 1% agarose
gel (Section 3.8.2, 3.8.3, 3.6, Figure 4.12D). Sixty ng (4µL) RMND5A (C356S) was
ligated with fifty ng (0.5µL) pEGFP-C2 and the products were transformed into
competent E. coli DH5α (Sections 3.8.2, 3.8.4). Transformed bacteria were grown on
LB Agar/Kanamycin plates, 4 colonies were inoculated into LB Broth/Kanamycin
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
125
~1.2kb
~4.7kb
Lane 1: MW Marker Lane 2: Undigested pEGFP-RMND5A (C356S) Clone 1 Lane 3-6: EcoRI digested pEGFP-RMND5A (C356S) Clone 1-4
E
A Lane 1: MW Marker Lane 2: 72°C Lane 3: 71.6°C Lane 4: 70.9°C Lane 5: 69.5°C Lane 6: 67.8°C Lane 7: 66.4°C Lane 8: 65.5°C Lane 9: 65°C Lane 10: Negative Control
~5.9kb
Lane 1: MW Marker Lane 2: Undigested pEGFP-RMND5A (C356S) Clone 1 Lane 3-8: EcoRI digested pEGFP-RMND5A (C356S) Clones 1-6
B
~1.2kb
~4.7kb
Lane 1: MW Marker Lane 2: RMND5A (C356S) Clone 1 Lane 3: RMND5A (C356S) Clone 4
~1.2kb
Lane 1: MW Marker Lane 2: Undigested pEGFP-C2 Lane 3: EcoRI digested pEGFP-C2
~4.7kb
C D
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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Figure 4.12: Preparation of pEGFP-RMND5A (C356S). (A) Wild type pEGFP-RMND5A and an annealing temperature gradient (65°C to 72°C) were used to optimise site directed mutagenesis PCR conditions. 10µL of each DpnI digested PCR product was electrophoresed in a 1% agarose gel, visualising the mutagenesis products at ~5.9kb in all reactions. (B) Plasmids isolated from pEGFP-RMND5A (C356S) clones 1 to 6 were digested with EcoRI to liberate the ~1.2kb insert then electrophoresed in a 1% agarose gel confirming the presence of insert in all clones. (C) pEGFP-RMND5A (C356S) clones 1 and 4 were digested with EcoRI to release the ~1.2kb RMND5A (C356S) coding region which was gel purified. 5µL of each purified product was electrophoresed in a 1% agarose gel from which the DNA concentration was estimated to be ~15ng/µL. (D) 2µL of purified EcoRI/SAP digested pEGFP-C2 plasmid was electrophoresed in a 1% agarose gel from which the concentration of DNA was estimated to be ~100ng/µL. (E) Plasmids isolated from pEGFP-RMND5A (C356S) clones 1 to 4 were digested with EcoRI to liberate the ~1.2kb insert, then electrophoresed in a 1% agarose gel, confirming that all clones contained an insert. (F) To determine the insert orientation, pEGFP-RMND5A (C356S) mutant clones 1 to 4 were digested with SacI, then electrophoresed in a 1% agarose gel. The presence of a band at ~300bp in the digested plasmids of clones 1-4 indicated that the RMND5A (C356S) insert was in the sense orientation in all plasmids.
~300bp
~5.6kb
Lane 1: MW Marker Lane 2: SacI digested wild type pEGFP-RMND5A Lane 3: Undigested pEGFP-RMND5A (C356S) Clone 1 Lane 4-7: SacI digested pEGFP-RMND5A (C356S) Clone 1-4
F
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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cultures for small scale plasmid purification and the purified plasmids RNase treated
then digested with EcoRI to release the insert (Section 3.6, 3.8.2, 3.9). Following
electrophoresis in a 1% agarose gel, a band at the expected size of ~1.2kb was present in
all four clones, and to determine insert orientation, the plasmids were digested with SacI
then electrophoresed in a 1% agarose gel (Sections 3.6, 3.8.2, Figure 4.12E). The
presence of a band at ~300bp indicated that all clones were inserted in the pEGFP-C2
vector in the sense orientation (Figure 4.12F). Each of the pEGFP-RMND5A (C356S)
clones was purified and 5µL product electrophoresed in a 1% agarose gel to estimate
the DNA concentration (Sections 3.6, 3.7.1, not shown). Based on the gel, 2µL of each
of clones 2 and 3 was utilised in sequencing reactions using the RMND5A1-S,
RMND5A1176-AS, RMND5A603-S and RMND5A490-AS primers, the products of
which were analysed using BLASTTM (Section 3.12, Appendix II). From this analysis,
pEGFP-RMND5A (C356S) clone 2 was verified as mutation free, the sense orientation
of the insert was confirmed and glycerol stocks of this clone were prepared (Section
3.8.7, 3.12, Appendix III).
4.2.6.2 Introduction of C358S into the RMND5B RING Domain
Initial site-directed mutagenesis PCRs using RMND5B (C358S) Primer set 1 (Appendix
II) to incorporate the RMND5B (C358S) RING domain mutation did not amplify
products and optimisation of PCR conditions including the use of a range of annealing
temperatures from 50°C - 72°C, 1.5mM and 2mM MgCl2, high fidelity and high GC
content buffers and increased initial denaturation, annealing and extension times were
not successful (not shown). The mutagenesis PCR primers were redesigned and
shortened (RMND5B (C358S) Primer set 2) and the PCR conditions re-optimised using
a range of annealing temperatures from 65°C - 72°C, which resulted in amplification of
similar levels of a single product at the expected size of ~5.9kb at all annealing
temperatures (Section 3.4.3, 3.6, Figure 4.13A). The negative control contained a faint
smear of DNA which was attributed to primer self-annealing (Figure 4.13A). PCR
products from reactions amplified with an annealing temperature of 65°C and 65.5°C
were digested with DpnI to degrade the wild-type parental DNA, the products were
transformed into competent E. coli DH5α cells and selected by plating onto LB
Agar/Kanamycin (Sections 3.4.3, 3.8.6). Four of the resulting colonies were picked,
incubated overnight in LB Broth/Kanamycin and plasmids isolated by small scale
plasmid purification then treated with RNase and SalI digested to liberate the insert
(Section 3.8.2, 3.9). Electrophoresis of 10µL of each product identified a band at the
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
128
expected size of ~1.2kb and each of the plasmids was purified then electrophoresed in
preparation for sequencing reactions (Section 3.6, 3.7.1, Figure 4.13B). Based on the
gel, 3µL of each of clones 1 to 4 was sequenced using the RMND5B790-S primer, and
the sequencing chromatograms analysed using BLASTTM, verifying the presence of the
RMND5B C358S mutation in each of the four clones (not shown). To confirm the entire
RMND5B sequence, pEGFP-RMND5B (C358S) clones 1 and 2 were sequenced using
the RMND5BSalI1-S, RMND5BSalI1182-AS and pEGFP1266-S primers, verifying the
absence of mutations in the remainder of the RMND5B coding region (Appendix II, not
shown).
To subclone RMND5B (C358S) into pEGFP-C2, pEGFP-RMND5B (C358S) clone 1
was digested with SalI to release the RMND5B (C358S) insert, the insert was gel
purified then 5µL was electrophoresed in a 1% agarose gel (Section 3.6, 3.7.2, 3.8.2,
Figure 4.13C). pEGFP-C2 was prepared by digestion with SalI, SAP treatment,
purification, then 2µL product was electrophoresed in a 1% agarose gel (Section 3.6,
3.8.2, 3.8.3, Figure 4.13C). The ligation reaction contained 50ng (5µL) RMND5B
(C358S) insert and 50ng pEGFP-C2, the ligation products were transformed into
competent E. coli DH5α and transformed bacteria were selected by growth overnight on
LB Agar/Kanamycin (Section 3.8.4, 3.8.6). Ten colonies were picked, cultured in LB
Broth/Kanamycin for small scale plasmid purification, the resulting plasmids treated
with RNase, digested with SalI to liberate the insert and 10µL of each product was
electrophoresed in a 1% agarose gel (Section 3.6, 3.8.2, 3.9, Figure 4.13D). A band at
the expected size of ~1.2kb was present in clones 5, 6 and 10, and to determine the
insert orientation, these were digested with PstI, with clone 6 the only plasmid
producing a single band at ~315bp, indicating that this clone contained the insert in the
sense orientation (Section 3.6, 3.8.2, Figure 4.13E). pEGFP-RMND5B (C358S) clone 6
was purified, sequenced using the RMND5BSalI1-S, RMND5BSalI1182-AS,
RMND5B790-S and pEGFP1266-S primers and the sequences verified to be mutation
free (apart from the G1067C base change resulting in the C358S mutation) and in the
sense orientation (Section 3.12, Appendix II, Appendix III). A glycerol stock was
prepared of this mutant (Section 3.8.7).
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
129
Lane 1: MW Marker Lane 2: 72°C Lane 3: 71.6°C Lane 4: 70.9°C Lane 5: 69.5°C Lane 6: 67.8°C Lane 7: 66.4°C Lane 8: 65.5°C Lane 9: 65°C Lane 10: Negative Control
~5.9kb
Lane 1: MW Marker Lane 2: Undigested pEGFP-RMND5B (C358S) Clone 1 Lane 3-6: SalI digested pEGFP-RMND5B (C358S) Clone 1-4
~1.2kb ~4.7kb
B
~1.2kb ~4.7kb
Lane 1: MW Marker Lane 2: RMND5B (C358S) Clone 1 Lane 3: Undigested pEGFP-C2 Lane 4: SalI digested pEGFP-C2
C
A
~1.2kb ~4.7kb
Lane 1: MW Marker Lane 2: Undigested pEGFP-RMND5B (C358S) Clone 1 Lane 3 - 10: SalI digested pEGFP-RMND5B (C358S) Clone 1 - 10
D
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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Figure 4.13: Preparation of pEGFP-RMND5B (C358S). (A) Wild type pEGFP-RMND5B and an annealing temperature gradient ranging from 65°C to 72°C were used to optimise RMND5B site-directed mutagenesis PCR conditions. 10µL of each DpnI digested PCR product was electrophoresed in a 1% agarose gel, and the mutagenesis products visualised at ~5.9kb. (B) Plasmids isolated from pEGFP-RMND5B (C358S) clones 1 to 4 were digested with SalI to liberate the ~1.2kb insert then electrophoresed in a 1% agarose gel, confirming the presence of insert in all clones. (C) pEGFP-RMND5B (C358S) mutant clone 1 was SalI digested to liberate the RMND5B (C358S) coding region and 5µL of the gel purified insert was electrophoresed in a 1% agarose gel from which the concentration was estimated to be 10ng/µL. 2µL of purified SalI/SAP digested pEGFP-C2 plasmid was electrophoresed in a 1% agarose gel from which the DNA concentration was estimated to be 300ng/µL. (D) Plasmids isolated from pEGFP-RMND5B (C358S) clones 1 to 10 were digested with SalI then 5µL of each digested product was electrophoresed in a 1% agarose gel. Clones 5, 6 and 10 contained the RMND5B (C358S) insert at ~1.2kb. (E) To determine insert orientation, pEGFP-RMND5B (C358S) clones 4, 5, 6, 10 were digested with PstI then electrophoresed in a 1% agarose gel. The presence of two bands at ~1kb and ~300bp indicated that the RMND5B (C358S) inserts in clones 5 and 6 were in the antisense orientation whilst a single band at ~300bp indicated that the RMND5B (C358S) insert was present in the sense orientation.
~1 kb
Lane 1: MW Marker Lane 2: Undigested pEGFP-RMND5B (C358S) Clone 4 Lane 3: PstI digested pEGFP-RMND5B (C358S) Clone 4 Lane 4: PstI digested pEGFP-RMND5B (C358S) Clone 5 Lane 5: PstI digested pEGFP-RMND5B (C358S) Clone 10 Lane 6: MW Marker Lane 7: Undigested pEGFP-RMND5B (C358S) Clone 6 Lane 8: PstI digested pEGFP-RMND5B (C358S) Clone 6
~5.6kb
~300bp
E ~5.6kb ~300bp
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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4.2.6.3 Cloning of the RMND5A (C356S) and RMND5B (C358S) RING
Domains into pGEX-2TK
Following development of RMND5A (C356S) (Section 4.2.6.1) and RMND5B (C358S)
(Section 4.2.6.2), the mutant RING domains were PCR amplified from these plasmids
using the previously optimised PCR conditions, the amplified products purified,
electrophoresed in a 2% agarose gel, then 60ng (2µL) of each PCR product was ligated
into 50ng (1µL) pGEM®T-Easy cloning vector (Section 3.4, 3.6, 3.7.1, Figure 4.14A).
The ligation products were transformed into competent E. coli DH5α cells and selected
by growth of the transformed bacteria on LB Agar/Ampicillin plates with blue/white
colony selection, then 4 colonies each of the pGEMT-RMND5 RING domain mutants
were picked and incubated in LB Broth/Ampicillin for small scale plasmid purification
(Section 3.8.6). Purified plasmids were treated with RNase, digested with BamHI and
EcoRI to release the insert, and the digestion products electrophoresed in a 2% agarose
gel (Section 3.6, 3.8.2). A band of the expected molecular weight of ~126bp was
present in all digested plasmids of each of pGEMT-RMND5A (C356S) RING and
pGEMT-RMND5B (C358S) RING, therefore clones 1 and 2 were purified then
electrophoresed in a 1% agarose gel in preparation for sequencing (Section 3.6, 3.7.1,
Figure 4.14B). Based on the gel, 4µL pGEMT-RMND5A (C356S) RING clones 1 and 2
and 2µL pGEMT-RMND5B (C358S) RING clones 1 and 2 were sequenced using the
M13-S and M13-AS primers, with BLASTTM analysis of the sequencing products
verifying the presence of the RMND5A (C356S) and RMND5B (C358S) RING domain
mutations and no additional base changes (Section 3.12, Appendix II, not shown).
To prepare the RMND5A (C356S) RING and RMND5B (C358S) RING domains for
ligation, pGEMT-RMND5A (C356S) RING clone 1 and pGEMT-RMND5B (C358S)
RING clone 1 were each digested with BamHI and EcoRI to release the ~126bp inserts,
the products gel purified and 5µL of each electrophoresed in a 2% agarose gel in
preparation for ligation reactions (Sections 3.6, 3.7.2, 3.8.2, Figure 4.14C). The pGEX-
2TK vector was prepared by small scale plasmid purification, RNase treatment, BamHI
and EcoRI digestion and SAP treatment, prior to purification and electrophoresis in a
1% agarose gel (Sections 3.6, 3.7.1, 3.8.2, 3.9, Figure 4.14D). Based on the gels, 40ng
(8µL) RMND5A (C356S) RING and 60ng (4µL) RMND5B (C358S) RING were ligated
with 50ng (2.5µL) pGEX-2TK, the resulting products were transformed into competent
E. coli DH5α cells and selected by plating cultures on LB Agar/Ampicillin (Sections
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
132
Lane 1: MW Marker Lane 2: RMND5A (C356A) RING Lane 3: RMND5B (C358S) RING Lane 4: Negative Control
Lane 1: MW Marker Lane 2: Undigested pGEMT-RMND5A (C356S) RING clone 1 Lane 3-6: EcoRI digested RMND5A (C356S) RING clone 1-4 Lane 7-10: EcoRI digested RMND5B (C358S) RING clone 1-4 Lane 11: MW Marker
~126bp
~126bp
~3kb
A
B
Lane 1: MW Marker Lane 2: RMND5A (C356S) RING Lane 3: RMND5B (C358S) RING Lane 4: Negative Control
~126bp
~5kb
Lane 1: MW Marker Lane 2: Undigested pGEX-2TK Lane 3: BamHI/EcoRI SAP digested pGEX-2TK
C
D
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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Figure 4.14: Cloning of sequences encoding the RMND5A (C356S) and RMND5B (C358S) RING domains into pGEX-2TK. (A) The RING domains of pEGFP-RMND5A (C356S) and pEGFP-RMND5B (C358S) were PCR amplified and 5µL of each purified product was electrophoresed in a 2% agarose gel from which the DNA concentration of each was estimated to be 30ng/µL. (B) Plasmids isolated from pGEMT-RMND5A (C356S) RING and pGEMT-RMND5B (C358S) RING clones 1 to 4 were BamHI and EcoRI digested and the products were electrophoresed in a 2% agarose gel confirming that each clone contained an insert. (C) pGEMT-RMND5A (C356S) RING and pGEMT-RMND5B (C358S) RING were BamHI/EcoRI digested, gel purified and 5µL product was electrophoresed in a 2% agarose gel from which the concentration of RMND5A (C356S) and RMND5B (C358S) were estimated to be ~5ng/µL and 15ng/µL, respectively. (D) 5µL of purified BamHI/EcoRI digested pGEX-2TK plasmid was electrophoresed in a 1% agarose gel and the plasmid concentration estimated from the gel to be ~20ng/µL. (E) pGEX-RMNDA (C356S) RING and pGEX-RMND5B (C358S) RING clones 1 and 2 were purified and 5μL product electrophoresed in a 1% agarose gel.
Lane 1: MW Marker Lane 2: Purified pGEX-RMND5A (C356S) RING clone 1 Lane 3: Purified pGEX-RMND5A (C356S) RING clone 2 Lane 4: Purified pGEX-RMND5B (C358S) RING clone 1 Lane 5: Purified pGEX-RMND5B (C358S) RING clone 2
~5kb
E
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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3.8.4, 3.8.6). Four colonies of each of the pGEX-RMND5 RING domain mutants were
inoculated into LB Broth/Ampicillin for small scale plasmid purification, plasmids were
treated with RNase, digested with BamHI and EcoRI to liberate the RING domain
inserts and 10µL of each product was electrophoresed in a 2% agarose gel (Sections 3.6,
3.8.2, 3.9). A faint ~126bp band was evident in all pGEX-RMND5A (C356S) RING
clones 1-4 and pGEX-RMND5B (C358S) RING clones 1-4 (not shown), and pGEX-
RMND5A (C356S) RING and pGEX-RMND5B (C358S) RING clones 1 and 2 were
purified and electrophoresed in a 1% agarose gel in preparation for sequencing
(Sections 3.6, 3.7.1, Figures 4.14E). Based on the gel, 4µL of each of pGEX-RMND5A
(C356S) RING clones 1 and 2 and 7µL pGEX-RMND5B (C358S) RING clones 1 and 2
were sequenced using the pGEX-S and pGEX-AS primers, with BLASTTM analysis
identifying that pGEX-RMND5A (C356S) RING clone 2 and pGEX-RMND5B
(C358S) RING clone 2 each contained no additional mutations (Section 3.12, Appendix
II, not shown). These plasmids were transformed into E. coli BL21 cells and glycerol
stocks were prepared (Section 3.8.6, 3.8.7).
4.2.6.4 Expression and Intracellular Localisation of RMND5A (C356S) and
RMND5B (C358S)
In preparation for transfection into mammalian cells, large scale plasmid preparations of
pEGFP-RMND5A (C356S) and pEGFP-RMND5B (C358S) were performed and the
plasmid DNA concentrations determined to be 1.93µg/µL for pEGFP-RMND5A
(C356S) and 2.2µg/µL for pEGFP-RMND5B (C358S) (Section 3.10, 4.2.6.1, 4.2.6.2).
To verify the expression of GFP-RMND5A (C356S) and GFP-RMND5B (C358S),
LNCaP cells growing in 6 well plates were transfected with 4µg plasmids encoding
either wild-type or mutant GFP tagged-RMND5A or RMND5B (Section 3.1.4).
Cultures were harvested 48 hours following transfection and GFP western blotting
identified the presence of a ~70kDa band in the lysates of cells transfected with each of
the constructs (Section 3.15, Figure 4.15A). These findings indicated that mutation of
the RING domains of RMND5A and RMND5B did not inhibit the expression or levels
of expression of the proteins.
The intracellular localisation of the GFP-RMND5A (C356S) and GFP-RMND5B
(C358S) was investigated by transfection of LNCaP cells growing on coverslips with
2µg plasmids encoding GFP-tagged wild-type or mutant RMND5 proteins, with the
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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A
B GFP Nucleus Cytoplasm Overlay
GFP-RMND5A
Wild Type
C356S
GFP-RMND5B
Wild Type
C358S
Untransfected Negative Control
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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Figure 4.15: Expression and cellular localisation of GFP-RMND5A (C356S) and GFP-RMND5B (C358S) proteins. LNCaP cells were transfected with plasmids encoding either GPF-RMND5A, GFP-RMND5B, GFP-RMND5A (C356S) or GFP-RMND5B (C358S) and at 48 hours post-transfection (A) the cells were harvested for GFP western blotting, which identified bands corresponding to GFP-RMND5A, GFP-RMND5A (C356S), GFP-RMND5B and GFP-RMND5B (C358S) at ~70kDa. (B) Alternatively, the cells were prepared for fluorescence microscopy and stained with Hœchst 33258 and tetramethylisothiocyanate (TRITC)-Phalloidin to image the nucleus and cytoplasm, respectively. GFP-RMND5A, GFP-RMND5B, GFP-RMND5A (C356S) and GFP-RMND5B (C358S) displayed diffuse nuclear and cytoplasmic localisation, with GFP-RMND5B and GFP-RMND5B (C358S) also exhibiting a punctate cytoplasmic distribution (Magnification x1000,). Experiments were performed twice and representative results are shown. (WT= wild-type).
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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cells fixed at 48 hours post transfection and viewed by fluorescence microscopy
(Section 3.1.3, 3.1.4, 3.16.1). Both GFP-RMND5A and GFP-RMND5A (C356S)
proteins exhibited a diffuse appearance and were localised in the nucleus and
cytoplasm, with occasional cells transfected with wild-type RMND5A exhibiting
punctate cytoplasmic staining (Figure 4.15B). The GFP-RMND5B and GFP-RMND5B
(C358S) proteins similarly displayed a diffuse appearance and both nuclear and
cytoplasmic localisation, with most cells also exhibiting a punctate cytoplasmic
distribution (Figure 4.15B). These findings indicated that the RING domain mutations
did not markedly alter the cellular intracellular localisation of RMND5 proteins in
comparison to the wild-type RMND5A and RMND5B.
4.2.6.5 In Vivo Ubiquitination Activity of RMND5A (C356S) and RMND5B
(C358S)
The activity of RMND5A (C356S) and RMND5B (C358S) was evaluated in in vivo
ubiquitination assays in comparison to the activity of wild-type RMND5 proteins. For
these experiments, LNCaP cells were cotransfected with 15µg of plasmids encoding
HA-Ubiquitin and GFP-RMND5A, GFP-RMND5A (C356S), GFP-RMND5B or GFP-
RMND5B (C358S), cultured for 42 hours, then treated for the final 6 hours with the
proteasome inhibitor, MG132 to allow the accumulation of ubiquitinated proteins within
the cells (Section 3.1.4, 3.1.5, 3.14.2). The cells were harvested at 48 hours post-
transfection, GFP-tagged RMND5 proteins were immunoprecipitated, and the
immunoprecipitated proteins analysed by HA western blotting (for HA-Ubiquitin)
(Section 3.13, 3.15, Figures 4.18, 4.16). All GFP immunoprecipitated products
contained high molecular weight HA (ubiquitin) bands, corresponding to ubiquitinated
proteins which were more prominent in immunoprecipitates from the MG132 treated
cells (Figure 4.16). Both of the (wild-type) GFP-RMND5A and GFP-RMND5B
immunoprecipitates were associated with increased levels of high molecular weight
ubiquitinated proteins in comparison to cells overexpressing GFP alone, and the levels
of ubiquitinated proteins were further enhanced when cells were treated with MG132
(Figure 4.16). However, GFP-RMND5A, GFP-RMND5A (C356S), GFP-RMND5B and
GFP-RMND5B (C358S) immunoprecipitated proteins were each associated with
similar amounts of high molecular weight proteins corresponding to ubiquitinated
proteins, indicating that the RMND5A (C356S) and RMND5B (C358S) RING domain
mutations did not markedly reduce the in vivo ubiquitination activity of the GFP-
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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Figure 4.16: In vivo ubiquitination activity of RMND5A (C356S) and RMND5B
(C358S). LNCaP cells were cotransfected with plasmids encoding HA-ubiquitin and
GFP-RMND5A, GFP-RMND5B, GFP-RMND5A (C356S), GFP-RMND5B
(C358S) or GFP (empty vector). Cells were cultured for 48 hours, with 10μM
MG132 added for the final 6 hours of culture. Following GFP immunoprecipitation
of the cell lysates, HA western blotting was performed on the immunoprecipitated
proteins, identifying low levels of ubiquitinated proteins associated with GFP, (A)
GFP-RMND5A and GFP-RMND5A (C356S), or (B) GFP-RMND5B and GFP-
RMND5B (C358S). The levels of ubiquitinated proteins were increased following
MG132 treatment of cultures and in comparison to GFP-expressing cells, the levels
of ubiquitinated proteins were evaluated in cells that overexpressed wild-type or
mutant RMND5A and RMND5B. The experiment was performed twice and
representative blots are shown (WT= wild-type).
A
B
WT (C356S)
- - - + + +
Empty Vector
10µM MG132
GFP (~70kDa)
HA (Ubiquitin)
Ubn-Proteins
(~80kDa – 175kDa)
GFP-RMND5A
- - - + + +
Empty Vector WT (C358S)
10µM MG132
HA-Ubiquitin
GFP (~70kDa)
Ubn-Proteins (~80
– 175kDa)
GFP-RMND5B
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
139
RMND5 proteins. Alternatively, the E3 ubiquitin ligase activity of endogenous protein
may be masking the reduced activity of the mutant proteins. Western blotting of the
total cellular inputs for HA-Ubiquitin detected similar expression in all lysates of cells
transfected with pCMV-HA-Ubiquitin, and similarly GFP western blotting of the total
cellular inputs showed that GFP, and wild-type or mutant GFP-RMND5A and GFP-
RMND5B were each expressed (not shown), indicating that the above result was not
due to markedly altered expression from the transfected plasmids.
4.2.6.6 In Vitro Auto-Ubiquitination Activity of RMND5A (C356S) and
RMND5B (C358S) RING Domains
To verify that the GST-RMND5A (C356S) and GST-RMND5B (C358S) RING
domains (Section 4.2.6.3) could be expressed in bacterial cells, each plasmid was
transformed into competent E. coli BL21 cells, glycerol stocks were prepared and cells
from each stock were streaked onto LB Agar/Ampicillin plates (Section 3.8.7).
Individual colonies were inoculated into LB Broth/Ampicillin, expression of the GST-
fusion proteins was induced by the addition of 1mM IPTG, then aliquots of the cells
were lysed and electrophoresed in 12% acrylamide gels, which were stained with
Coomassie blue (Sections 3.11.1, 3.15.2, 3.15.5). The remaining aliquots of bacterial
cells were sonicated, the soluble and insoluble fractions collected and 15µL aliquots
electrophoresed in 12% polyacrylamide gels, which were similarly stained with
Coomassie blue to determine the localisation of the mutant GST-RING domains in the
bacterial cells (Section 3.11.1, 3.15, Figure 4.17A). Only cellular proteins were present
in lysates from untreated cells, whilst a prominent band at ~28kDa was visible in
extracts of cells where expression of GST-RMND5A (C356S) RING or GST-RMND5B
(C358S) RING domains had been induced by IPTG treatment. A prominent band at
~28kDa was present in both the soluble and insoluble fractions, indicating that the GST-
RMND5A (C356S) RING and GST-RMND5B (C358S) RING proteins were packaged
into insoluble inclusion bodies but were also present in the soluble fraction in sufficient
quantities to be purified for in vitro auto-ubiquitination assays. To obtain purified GST-
RMND5A (C356S) RING and GST-RMND5B (C358S) RING domains for in vitro
ubiquitination assays, the proteins were extracted from 100mL bacterial cultures and
electrophoresed along with BSA standards in 12% polyacrylamide gels, which were
stained with Coomassie blue (Section 3.11.2, 3.15.2, 3.15.5, Figure 4.17B, C). In vitro
ubiquitination assays were also performed using bacterial proteins as the substrate, and
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
140
Lane 1: MW Marker
Lane 2: GST-RMND5A (C356S) RING: no IPTG
Lane 3: GST-RMND5A (C356S) RING: IPTG
Lane 4: GST-RMND5A (C356S) RING: IPTG, soluble fraction
Lane 5: GST-RMND5A (C356S) RING: IPTG, insoluble fraction
Lane 6: GST-RMND5B (C358S) RING: IPTG
Lane 7: GST-RMND5B (C358S) RING: IPTG, soluble fraction
Lane 8: GST-RMND5B (C358S) RING: IPTG, insoluble fraction
1 2 3 4 5 6 7 8
~28kDa
1 2 3 4 5 6 7 8
Lane 1: MW Marker
Lane 2: 1µg BSA
Lane 3: 2.5µg BSA
Lane 4: 5µg BSA
Lane 5: 7.5µg BSA
Lane 6: 10µg BSA
Lane 7: 1µL GST-RMND5B (C358S) RING
Lane 8: 3µL GST-RMND5B (C358S) RING
~28kDa
1 2 3 4 5 6 7 8 Lane 1: MW Marker
Lane 2: 1µg BSA
Lane 3: 2.5µg BSA
Lane 4: 5µg BSA
Lane 5: 7.5µg BSA
Lane 6: 10µg BSA
Lane 7: 1µL GST-RMND5A (C356S) RING
Lane 8: 3µL GST-RMND5A (C356S) RING
~28kDa
B
C
A
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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Figure 4.17: Expression and purification of GST-RMND5A (C356S) RING
and GST-RMND5B (C358S) RING domains. E. coli BL21 cells transformed
with pGEX-RMND5A (C356S) RING or pGEX-RMND5B (C358S) RING were
induced with IPTG to produce GST-RMND5A (C356S) RING or GST-RMND5B
(C358S) RING domains (~28kDa). (A) The bacterial cells were separated into
soluble and insoluble fractions and a 15µL aliquot of each sample was
electrophoresed in a 12% polyacrylamide gel followed by Coomassie blue
staining to visualise the compartmentalisation of the induced GST fusion proteins.
(B) GST-RMND5A (C356S) RING and (C) GST-RMND5B (C358S) RING
domains were purified from the bacterial cells, an aliquot electrophoresed in
polyacrylamide gels and stained with Coomassie blue, from which the
concentration of the protein was estimated in comparison to BSA standards. The
concentrations of the GST-RMND5A (C356S) RING and the GST-RMND5B
(C358S) RING domain was estimated to be ~2.3µg/µL and ~2.8µg/µL,
respectively. (D) Proteins were extracted from E. coli BL21 cells and 1µL, 5µL
and 8µL aliquots were electrophoresed in a 12% polyacrylamide gel to visualise
the bacterial proteins.
1 2 3 4
Lane 1: MW Marker
Lane 2: 1µL extracted bacterial proteins
Lane 3: 5µL extracted bacterial proteins
Lane 4: 8µL extracted bacterial proteins
D
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
142
to generate proteins for these assays, total proteins were isolated from E. coli BL21
bacterial cells (as bacteria do not ubiquitinate proteins) (Section 3.11.3). Electrophoresis
and Coomassie blue staining of the bacterial proteins indicated that a range of low-high
molecular weight proteins were present, which was suitable for these analyses (Sections
3.15.2, 3.15.5, Figure 4.17D).
In vitro ubiquitination assays using the E2 enzyme UbcH5b were carried out to compare
the activity of wild-type GST-RMND5A and GST-RMND5B RING domains to that of
the GST-RMND5A (C356S) and GST-RMND5B (C358S) RING domains. In vitro
ubiquitination reactions were carried out at 37°C for 1 hour and the reaction products
were electrophoresed in 12% polyacrylamide gels followed by western blotting for
biotinylated ubiquitin (Section 3.14.1, 3.15). The negative control reaction lacking ATP
yielded a single protein band at ~10kDa corresponding to free ubiquitin, whilst the
negative control reactions lacking GST-RING domains (E3) yielded protein bands
corresponding to ubiquitinated E2 enzyme, as observed previously (Section 4.2.4.3).
Biotinylated-ubiquitin western blotting of reactions containing wild-type GST-RMND5
RING domains yielded prominent protein bands at >100kDa corresponding to
polyubiquitinated proteins, and the appearance or intensity of the protein bands in
reactions containing wild-type GST-RMND5A RING and GST-RMND5A (C356S)
RING domain proteins was similar (Figure 4.18). These results indicated that the
RMND5A (C356S) RING domain mutation did not reduce RMND5A RING domain E3
ubiquitin ligase activity. Biotinylated ubiquitin western blotting of in vitro
ubiquitination assays using the GST-RMND5B (C358S) RING domain identified a
small reduction in the intensity of prominent high molecular weight protein bands at
>100kDa, corresponding to polyubiquitinated proteins in comparison to reactions
containing GST-RMND5B RING domain, indicating a reduction in the E3 ubiquitin
ligase activity of the RMND5B (C358S) RING domain (Figure 4.18). In vitro
ubiquitination assays using the GST-RMND5A RING, GST-RMND5A (C356S) RING,
GST-RMND5B RING or GST-RMND5B (C358S) RING domains were repeated with
the addition of 2µL extracted bacterial proteins as substrates (Section 3.14.1, 3.15). In
these assays, the presence of protein bands ranging from 7kDa to >100kDa in western
blots for biotinylated ubiquitin, indicated that all RING domains were able to
ubiquitinate bacterial proteins (Figure 4.18). Evidence of a small reduction in protein
banding present in the reactions using GST-RMND5B (C358S) RING domain
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
143
compared to reactions containing the GST-RMND5B RING domain supported initial
findings that mutation of the RMND5B RING domain had reduced its ubiquitination
activity. However, the appearance and intensity of protein bands corresponding to
ubiquitinated proteins were similar in reactions containing GST-RMND5A RING or
GST-RMND5A (C356S) RING domains indicating that the mutation had not altered the
ubiquitination activity of the RMND5A RING domain (Figure 4.18).
To determine whether the GST-RMND5A (C356S) RING domain exhibited differences
in the rate at which it was able to mediate ubiquitin transfer compared to the wild-type
GSTRMND5A RING domain, in vitro ubiquitination assays were repeated at a reaction
temperature of 30°C (rather than 37°C), and with aliquots of each reaction taken at 10,
30, 60 and 90 minutes (Section 3.14.1, Figure 4.18). Biotinylated ubiquitin western
blotting of reaction products from the in vitro ubiquitination assays identified high
molecular weight protein bands corresponding to polyubiquitinated proteins at >100kDa
of a similar intensity in reactions containing either GST-RMND5A RING or GST-
RMND5A (C356S) under all conditions tested, indicating no alterations in the
ubiquitination activity of the mutant RMND5A RING domain. The only notable
difference in in vitro ubiquitination activity of the GST-RMND5A (C356S) RING
domain was evident in the appearance of a band at ~38kDa corresponding in size to the
monoubiquitinated GST-RING domain (Figure 4.18). This band appeared more intense
in western blots of products from reactions performed using the GST-RMND5A
(C356S) RING domain, suggesting that the mutant RMND5A RING domain favoured
automonoubiquitination in comparison to the wild-type RMND5A RING domain under
these conditions. The intensity of this band was similar for both GST-RMND5A RING
and GST-RMND5A (C356S) RING domains when reactions were carried out at 37°C
(Figure 4.18).
4.2.7 Examination of the E3 Ubiquitin Ligase Activity of the RMND5A and
RMND5B RING Domains by the Introduction of Dual Mutations in the
RMND5 RING Domains
As introduction of a single amino acid change into the RING domains of RMND5A and
RMND5B did not cause a marked reduction in E3 ubiquitin ligase activity, substitution
of two amino acids in each of the RING domains was introduced by site-directed
mutagenesis, generating RMND5A (C356A/H358A) and RMND5B (C358A/H360A)
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
144
WT WTC356S C358S WT WTC356S C358S- - - - + + + + Bacterial Protein Substrate
Ubn Proteins (~8 - >100kDa)
10 10 30 60 90 30 60 90 minutes
Ubn Proteins (~8kDa ->100kDa)
RING-Ub (~38kDa)
Figure 4.18: In vitro ubiquitination activity of GST-RMND5A (C356S) and GST-RMND5B (C358S) RING domains. (A) GST-RMND5A RING, GST-RMND5B RING, GST-RMND5A (C356S) RING or GST-RMND5B (C358S) RING were used in in vitro ubiquitination assays with UbcH5b. Reactions were incubated at 37°C for 60 minutes and 10μL of each product was electrophoresed in 4-12% gradient gels. Western blotting for biotinylated ubiquitin identified high molecular weight bands in reactions containing both wild-type and mutant GST-RMND5 RING domains (left panel). In reactions containing bacterial proteins (right panel) and either wild-type GST-RMND5A RING or mutant GST-RMND5A (C356S) RING, similar levels of ubiquitinated proteins spanning the blot were evident. In vitro ubiquitination assays containing GST-RMND5B (C358S) RING exhibited a slight reduction in banding corresponding to ubiquitinated proteins compared to reactions using wild-type GST-RMND5B RING. (B) Wild-type GST-RMND5A or GST-RMND5A (C356A) RING domains were used in in vitro ubiquitination assays with UbcH5b and the reactions were incubated at 30°C for 10 - 90 minutes. Ten μL of each product was electrophoresed in 4-12% gradient gels followed by western blotting for biotinylated ubiquitin, identifying high molecular weight protein bands of a similar intensity spanning the blot in reactions containing both wild-type GST-RMND5A and GST-RMND5A (C356S) RING domains. Experiments were performed twice and representative blots are shown (WT= wild-type).
A
B B
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
145
(Section 3.4.3, Figure 4.19). The two amino acids were selected for mutation as they are
the third and fourth zinc coordinating residues in the RING domain and mutation of the
homologous residues in other E3 ubiquitin ligases has been reported to reduce their
activity (Zhang et al., 2009). In addition, in RMND5 proteins these two residues are the
canonical RING domain zinc coordinating residues (cysteine and histidine) as opposed
to the first two and fifth and sixth zinc coordinating residues which are not canonical
zinc binding amino acids (Figure 4.19) (Zhang et al., 2009).
4.2.7.1 Mutation of the RMND5A (C356A/H358A) RING Domain
PCR primers were designed to introduce three base changes into the RMND5A RING
domain using RMND5A (C356S) as a template, however although multiple PCR
conditions were utilised to optimise the reaction including a range of annealing
temperatures between 50°C - 72°C, 1.5 – 3mM MgCl2 and high fidelity and GC buffers,
no PCR products were amplified (Section 3.4.3) (not shown). The mutagenesis PCR
primers were therefore redesigned to introduce the RMND5A (C356A/H358A)
mutations in two consecutive PCRs with the C356A mutation introduced via a single
base change in the first round of PCR (RMND5A Primer Set 1) and once confirmed, the
two additional base changes required to introduce the H358A mutation were introduced
using the pEGFP-RMND5A (C356A) plasmid DNA as a template (RMND5A Primer
Set 2). Initial PCRs to introduce the RMND5A (C356A) mutation were carried out using
a gradient of annealing temperatures between 58°C - 66°C for 26 cycles, which
produced a faint band at the expected size of ~5.9kb at all annealing temperatures tested
(Section 3.4.3, 3.6, Figure 4.20A). Amplified products from PCRs performed at
annealing temperatures of 64°C and 66°C were treated with DpnI to digest the parental
methylated plasmid DNA and the reaction products were transformed into E. coli DH5α
then selected by plating on LB Agar/Kanamycin (Sections 3.8.4, 3.8.6). Four of the
colonies were inoculated into LB Broth/Kanamycin for small scale plasmid purification,
the plasmids RNase treated, purified and 5µL electrophoresed in a 1% agarose gel
which resulted in the identification of ~5.9kb bands corresponding in size to pEGFP-
RMND5A (C356A) (Section 3.6, 3.9, not shown). Two µL of each of pEGFP-
RMND5A (C356A) clones 1-4 were sequenced using the RMND5A1176-AS primer,
and BLASTTM analysis of the sequences verified the presence of the C356A mutation in
all clones (Section 3.12, Appendix II, not shown).
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
146
Figure 4.19: Site directed mutagenesis of the RING domains of RMND5A and RMND5B to produce RMND5A (C356A/H358A) RING and RMND5B (C358A/H360A) RING. Alignment of the RING domains of RMND5A, RMND5B and yeast RMD5 showing all eight amino acid residues required for RING domain folding and function (red/orange) including the conserved cysteine residues (bold, orange) to be mutated to alanine (blue) to reduce or inactivate the RING domain activity of RMND5A and RMND5B. Numbers indicate amino acid number.
CPILRQQTTDNNPPMKLVCGHIISRDALNKMFNGS--KLKCPYC
CPILRQQTSDSNPPIKLICGHVISRDALNKLINGG--KLKCPYC
CPVLKEETTTENPPYSLACHHIISKKALDRLSKNGTITFKCPYC
RMND5A (C356A/H358A)
RMND5B (C358A/H360A)
RMD5 (C379S)
A A
336 377
338 379
361 404
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
147
The pEGFP-RMND5A (C356A) plasmid was then used in site-directed mutagenesis
reactions to incorporate the remaining two base changes in the RMND5A RING domain.
These PCRs were carried out using a gradient of annealing temperatures between 64°C
and 72°C for 26 cycles, and electrophoresis of the amplified product identified a faint
band at the expected size of ~5.9kb with more intense bands present in reactions
performed with the lower annealing temperatures of 64°C and 66.4°C (Section 3.4.3,
3.6, Figure 4.20B). The PCR products from reactions with the annealing temperatures
of 64°C and 66.4°C were digested with DpnI then transformed into competent E. coli
DH5α and selected on LB/Kanamycin plates (Section 3.4.3, 3.8.6). Four colonies were
picked and cultured in LB Broth/Kanamycin for small scale plasmid purification, the
plasmids were treated with RNase, purified and 5µL of each of pEGFP-RMND5A
(C356A/H358A) clones 1-4 were electrophoresed in a 1% agarose gel (Section 3.6, 3.9,
Figure 4.20C). Based on the gel, 2µL of each was used in sequencing reactions with the
RMND5A1176-AS primer, and BLASTTM analysis of the chromatograms verified the
presence of the RMND5A (C356A/H358A) mutations in all clones (Section 3.12,
Appendix II, not shown). The entire coding region of pEGFP-RMND5A
(C356A/H358A) clones 1 and 2 was sequenced using the RMND5A603-S, RMND5A1-
S and RMND5A490-AS primers, verifying that no additional mutations had been
incorporated in the site-directed mutagenesis procedure (Appendix II, not shown).
To ensure that no further mutations were present in the pEGFP-C2 vector in which the
RMND5A (C356A/H358A) RING mutations were induced, the RMND5A coding
region was excised from the pEGFP-RMND5A (C356A/H358A) plasmid by digestion
with EcoRI, gel purified and the products electrophoresed in a 1% agarose gel (Sections
3.6, 3.7.2, 3.8.2, Figure 4.20D). Fresh pEGFP-C2 vector was prepared by digestion with
EcoRI, SAP treatment, purification then electrophoresis of the purified products in a 1%
agarose gel (Section 3.6, 3.8.2, 3.8.3, Figure 4.20E). Based on these gels, 60ng (2µL)
RMND5A (C356A/H358A) insert was ligated into 50ng (0.5µL) pEGFP-C2 and the
products were transformed into competent E. coli DH5α cells (Sections 3.8.4, 3.8.6).
Transformed bacteria were selected by growth on LB Agar/Kanamycin plates, three
colonies were picked, cultured in LB Broth/Kanamycin for small scale plasmid
purification, and the plasmids were RNase treated and digested with EcoRI to release
the RMND5A (C356A/H358A) insert (Sections 3.8.2, 3.9). Electrophoresis of the
digested plasmids identified a band at the expected size of ~1.2kb in pEGFP-RMND5A
(C356A/H358A) clones 1 and 2, these clones were purified then 5µL of each was
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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Lane 1: MW Marker Lane 2: Undigested pEGFP-C2 Lane 3: EcoRI and SAP digested pEGFP-C2
~4.7kb
Lane 1: MW Marker Lane 2-6: pEGFP-RMND5A (C356A/H358A) clone 1-4
Lane 1: MW Marker Lane 2: RMND5A (C356A/H358A)
~1.2kb
D E
Lane 1: MW Marker Lane 2: 66ºC Lane 3: 64ºC Lane 4: 60ºC Lane 5: 58ºC Lane 6: Negative Control
~5.9kb
A
B ~5.9kb
~5.9kb
C
Lane 1: MW Marker Lane 2: 70°C Lane 3: 69.5°C Lane 4: 68.4°C Lane 5: 66.4°C Lane 6: 64°C Lane 7: Negative Control
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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Figure 4.20: Generation of RMND5A (C356A/H358A) by site-directed mutagenesis and cloning of pEGFP-RMND5A (C356A/H358A). (A) pEGFP-RMND5A (C356S) and an annealing temperature gradient ranging from 58°C to 66°C were used to optimise RMND5A (C356A) site-directed mutagenesis PCR conditions. PCR products were digested with DpnI and 10µL of each digested product was electrophoresed in a 1% agarose gel, identifying the ~5.9kb mutagenesis products. (B) An annealing temperature gradient from 64°C to 70°C and the pEGFP-RMND5A (C356A) template were used to optimise RMND5A (C356A/H358A) site directed mutagenesis, and 10µL of each of the DpnI digested PCR products were electrophoresed in a 1% agarose gel. The mutagenesis products were identified at ~5.9kb. (C) 5µL of each of purified pEGFP-RMND5A (C356A/H358A) clones 1 to 4 was electrophoresed in a 1% agarose gel. (D) pEGFP-RMND5A (C356A/H358A) was EcoRI digested to release the RMND5A (C356A/H358A) coding region which was gel purified. 5µL purified product was electrophoresed in a 1% agarose gel from which the concentration of the ~1.2kb insert was estimated to be 30ng/µL. (E) 2µL of purified EcoRI/SAP digested pEGFP-C2 plasmid was electrophoresed in a 1% agarose gel from which the concentration of DNA was estimated at ~100ng/µL. (F) Plasmids isolated from pEGFP-RMND5A (C356A/H358A) clones 1 to 3 were EcoRI digested to liberate the ~1.2kb insert and the products were electrophoresed in a 1% agarose gel. Clones 1 and 2 contained the RMND5A (C356A/H358A) insert. (G) pEGFP-RMND5A (C356A/H358A) clones 1 and 2 were purified and 5µL of each purified plasmid was electrophoresed in a 1% agarose gel.
Lane 1: MW Marker Lane 2: Undigested pEGFP-RMND5A (C356A/H358A) Lane 3-5: EcoRI digested pEGFP-RMND5A (C356A/H358A) clones 1-3
~1.2kb
~4.7kb
Lane 1: MW Marker Lane 2: Purified pEGFP-RMND5A (C356A/H358A) clone 1 Lane 3-5: Purified pEGFP-RMND5A (C356A/H358A) clones 2
F
G
~5.9kb
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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electrophoresed in a 1% agarose gel in preparation for sequencing reactions (Sections
3.6, 3.7.1, Figure 4.20E, F). Based on the gel, 1µL of each clone was used for
sequencing reactions with the RMND5A1-S, RMND5A1176-AS, RMND5A603-S and
RMND5A490-AS primers, BLASTTM analysis of which indicated that pEGFP-
RMND5A (C356A/H358A) clone 2 contained no additional mutations (aside from the
T1060G/G1061C/C1066G/A1067C base changes corresponding to the C356A/H358A
amino acids changes) and was inserted in the sense orientation (Appendix II, Appendix
III).
4.2.7.2 Cloning of the RMND5A (C356A/H358A) RING Domain into pGEX-
2TK
Following production of RMND5A (C356A/H358A) (Section 4.2.7.1), the mutant
RMND5A RING domain was PCR amplified in quadruplicate using
RMND5ARING1006-S and RMND5ARING1131-AS primers and the previously
optimised PCR conditions, the products were purified and 5µL was electrophoresed in a
2% agarose gel (Section 3.4.3, 3.6, 4.2.3.1, Figure 4.21A, Appendix II). Based on the
gel, 100ng (2µL) of the RMND5A (C356A/H358A) RING domain PCR product was
ligated into 50ng (1µL) pGEM®T-Easy cloning vector and the products transformed
into competent E. coli DH5α cells and selected by growth on LB Agar/Ampicillin plates
(Section 3.8.4). Four colonies were inoculated into LB Broth/Ampicillin for small scale
plasmid purification, the purified plasmids were RNase treated, digested with BamHI
and EcoRI to release the insert, and 10µL of each product was electrophoresed in a 2%
agarose gel (Section 3.6, 3.8.2, 3.9, Figure 4.21B). A band of the expected molecular
weight of ~126bp was present in pGEMT-RMND5A (C356A/H358A) RING clones 1,
3 and 4, clones 1 and 3 were purified and 5µL of each was electrophoresed in a 1%
agarose gel in preparation for sequencing (Section 3.7.1, not shown). Based on the gel,
3µL of each of clones 1 and 2 were sequenced using M13-S and M13-AS primers, and
the presence of the C356A and H358A mutations in pGEMT-RMND5A
(C356A/H358A) clone 1 were verified using BLASTTM analysis (Section 3.12).
To prepare the RMND5A (C356A/H358A) RING domain for ligation into pGEX-2TK
to generate pGEX-RMND5A (C356A/H358A) RING, the pGEMT-RMND5A
(C356A/H358A) RING clone 1 plasmid was digested with BamHI and EcoRI to release
the ~126bp insert, the product gel purified and 5µL of the purified product
Chapter 4 Characterisation of RMND5 E3 ubiquitin ligase activity
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Figure 4.21: Cloning of sequences encoding the RMND5A (C356A/H358A) RING
domain into pGEX-2TK. (A) The RING domain sequence of pEGFP-RMND5A
(C356A/H358A) was PCR amplified and 5µL purified PCR product was
electrophoresed in a 2% agarose gel from which the DNA concentration was
estimated to be 50ng/µL. (B) Plasmids isolated from pGEMT-RMND5A
(C356A/H358A) RING clones 1 to 4 were BamHI/EcoRI digested to release the
~126bp insert and 10µL of each product was electrophoresed in a 2% agarose gel. (C)
pGEMT-RMND5A (C356A/H358A) RING was digested with BamHI and EcoRI, the
insert was gel purified and 5µL purified product was electrophoresed in a 2% agarose
gel, from which the concentration was estimated to be ~5ng/µL. (D) 5µL of purified
BamHI/EcoRI and SAP digested pGEX-2TK plasmid was electrophoresed in a 1%
agarose gel from which the concentration was estimated to be ~10ng/µL.
B
Lane 1: MW Marker
Lane 2: RMND5A (C356A/H358A) RING
~126bp
Lane 1: RMND5A (C356A/H358A)
RING
Lane 2: Negative Control
Lane 3: MW Marker
~126bp
~3kb
Lane 1: MW Marker
Lane 2: Undigested pGEMT-RMND5A
(C356A/H358A) RING
Lane 3-6: BamHI/EcoRI digested
pGEMT-RMND5A (C356A/H358A)
RING clones 1-4
A
~126bp
p
C
D Lane 1: MW Marker
Lane 2: Undigested pGEX-2TK
Lane 3: BamHI/EcoRI/SAP treated pGEX-2TK ~1.2kb
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
152
electrophoresed in a 2% agarose gel (Section 3.6, 3.8.2, Figure 4.21C). The pGEX-2TK
vector was prepared by small scale plasmid purification, the plasmid RNase treated,
BamHI and EcoRI digested and SAP treated, then purified and 5µL of the product was
electrophoresed in a 1% agarose gel (Section 3.6, 3.8.2, 3.8.3, 3.9, Figure 4.21D). Based
on the gel, 40ng (8µL) RMND5A (C356A/H358A) RING domain was ligated into 50ng
(5µL) pGEX-2TK and the products were transformed into competent E. coli DH5α cells
then selected by plating on LB Agar/Ampicillin (Section 3.8.4, 3.8.6). Four colonies of
pGEX-RMND5A (C356A/H358A) RING were picked and cultured in LB
Broth/Ampicillin for small scale purification. The purified plasmids were RNase treated
then digested with BamHI and EcoRI to liberate the ~126bp RMND5A (C356A/H358A)
RING domain and 10µL of each product was electrophoresed in a 2% agarose gel
(Section 3.6, 3.8.2, 3.9, not shown). Although no insert bands were visible on the gel
(potentially due to their small size of ~126bp), pGEX-RMND5A (C356A/H358A)
RING clones 1 and 3 were purified and 3µL of each of clone 1 and 3 was sequenced
using the pGEX-S and pGEX-AS primers. BLASTTM analysis of the sequencing
products indicated that pGEX-RMND5A (C356A/H358A) RING clone 3 contained no
additional mutations, apart from those encoding the C356A/H358A amino acid changes
(Section 3.7.1, 3.12, Appendix II, not shown). This plasmid was then transformed into
competent E. coli BL21 cells and a glycerol stock was prepared (Section 3.8.7).
4.2.7.3 Mutation of the RMND5B (C358A/H360A) RING Domain
To introduce the C358A and H360A mutations into the RMND5B RING domain, PCR
primers were designed to introduce the three base changes required, with the pEGFP-
RMND5B (C358S) plasmid to be used as the DNA template. Initial PCRs were carried
out using an annealing temperature of 65°C and a range of MgCl2 concentrations from
1.5 – 3mM MgCl2 for 26 cycles, producing a faint band at the expected size of ~5.9kb
in reactions containing 2mM, 2.5mM and 3mM MgCl2 when the PCR products were
electrophoresed in a 1% agarose gel (Sections 3.4.3, Figure 4.22A). The products from
reactions using 2mM and 3mM MgCl2 were treated with DpnI to digest the parental
methylated plasmid DNA, purified, then the digest products were transformed into E.
coli DH5α and selected by plating on LB Agar/Kanamycin (Section 3.8.6). Four
resulting colonies were picked, cultured in LB Broth/Kanamycin for small scale
plasmid purification, the plasmids treated with RNase and purified, with electrophoresis
of the products in a 1% agarose gel identifying bands corresponding to the ~5.9kb
pEGFP-RMND5B (C358A/H360A) (Section 3.6, 3.9, not shown). Based on the gel,
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
153
1µL of each of pEGFP-RMND5B (C358A/H360A) clones 1-4 were sequenced
reactions using the RMND5B790-S primer, which verified the presence of the
C358A/H360A mutations in all clones (Section 3.12). The entire coding region of
pEGFP-RMND5B (C358A/H360A) clones 1 and 2 was sequenced using the
RMND5BSalI1-S, RMND5BSalI1182-AS and pEGFP1266-S primers, and BLASTTM
analysis of the chromatograms confirmed that both inserts contained no additional
mutations (Appendix II, not shown).
To subclone the RMND5B (C358A/H360A) insert into pEGFP-C2, the RMND5B
coding region was excised from the plasmids by digestion with SalI, gel purified and
electrophoresed in a 1% agarose gel (Sections 3.6, 3.7.2, 3.8.2, Figure 4.22B). The
pEGFP-C2 vector was prepared by SalI digestion, SAP treatment, purification then
electrophoresis of the product in a 1% agarose gel (Section 3.7.1, 3.8.2, 3.8.3, Figure
4.22C). Based on the gels, 35ng (7µL) RMND5B (C358A/H360A) insert was ligated
into 50ng (0.5µL) pEGFP-C2 and the products were transformed into competent E. coli
DH5α cells (Section 3.8.4). Three colonies were inoculated into LB Broth/Kanamycin
for small scale plasmid purification, the plasmids RNase treated and digested with SalI
to release the insert then electrophoresed in a 1% agarose gel (Section 3.6, 3.8.2, 3.9,
Figure 4.22D). A band at the expected size of ~1.2kb was present in clone 1, and this
plasmid was purified and 5µL product electrophoresed in a 1% agarose gel (Sections
3.6, 3.7.1, Figure 4.22E). Based on the gel, 1µL of clone 1 was sequenced using the
RMND5BSalI1-S, RMND5BSalI1182-AS and pEGFP1266-S primers and BLASTTM
analysis verified that pEGFP-RMND5B (C358A/H360A) clone 1 contained no
additional mutations (aside from the T1066G/G1067/C1072G/A1073C base changes
corresponding to the C358A/H360A amino acid changes) and was in sense orientation,
therefore glycerol stocks were prepared of this clone (Section 3.8.7, 3.12, Appendix II,
Appendix III).
4.2.7.4 Cloning of the RMND5B (C358A/H360A) RING Domain into pGEX-
2TK
Following the production of pEGFP-RMND5B (C358A/H360A) (Section 4.2.7.3), the
RMND5B (C358A/H360A) RING domain was PCR amplified in quadruplicate using
the RMND5BRING1012-S and RMND5BRING1137-AS primers, the reactions were
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
154
Figure 4.22: Generation of RMND5B (C358A/H360A) by site-directed mutagenesis. (A) pEGFP-RMND5B (C358S) template, an annealing temperature of 65°C and 1.5mM-3mM MgCl2 were used to optimise RMND5B (C358A/H360A) site-directed mutagenesis PCR conditions. PCR products were DpnI digested and 10µL of each product was electrophoresed in a 1% gel to visualise the ~5.9kb mutagenesis products. (B) pEGFP-RMND5B (C358A/H360A) was SalI digested, releasing the RMND5B (C358A/H360A) insert. 5µL gel purified insert was electrophoresed in a 1% agarose gel from which the concentration was estimated to be ~5ng/µL. (C) 2µL purified SalI and SAP digested pEGFP-C2 was electrophoresed in a 1% agarose gel, from which the DNA concentration was estimated to be ~100ng/µL. (D) pEGFP-RMND5B (C358A/H360A) clones 1 to 3 were digested with SalI to liberate the ~1.2kb insert then electrophoresed in a 1% agarose gel. Clone 2 contained an insert. (E) 5µL purified pEGFP-RMND5B (C358A/H360A) clone 2 was electrophoresed in a 1% agarose gel.
Lane 1: MW Marker Lane 2: 1.5mM MgCl2 Lane 3: 2mM MgCl2 Lane 4: 2.5mM MgCl2 Lane 5: 3mM MgCl2 Lane 6: Negative Control
~5.9kb
A
Lane 1: MW Marker Lane 2: Purified pEGFP-RMND5B (C358A/H360A) clone 1 ~5.9kb
E
Lane 1: MW Marker Lane 2: Undigested pEGFP-C2 Lane 3: SalI digested pEGFP-C2
~4.7kb
C
Lane 1: MW Marker Lane 2: RMND5B (C358A/H360A)
~1.2kb
B
D
~1.2kb
~4.7kb
Lane 1: MW Marker Lane 2: Undigested pEGFP-RMND5B (C358A/H360A) Lane 3-5: SalI digested pEGFP-RMND5B (C358A/H360A) clones 1-3
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
155
combined and purified and 5µL of the purified product was electrophoresed in a 2%
agarose gel (Section 3.4, 3.6, 3.7.1, Figure 4.23A). Based on the gels, 60ng (4µL)
RMND5B (C358A/H360A) was ligated with 50ng (1µL) pGEM®T-Easy and the
products were transformed into competent E. coli DH5α then selected by growth on LB
Agar/Ampicillin plates (Section 3.8.4, 3.8.6). Four colonies of pGEMT-RMND5B
(C358A/H360A) RING were inoculated into LB Broth/Ampicillin and incubated
overnight prior to small scale plasmid purification, the plasmids were RNase treated,
BamHI and EcoRI digested to release the insert and electrophoresed in a 2% agarose gel
(Section 3.6, 3.8.2, Figure 4.23B). A band of ~126bp was present in all clones, pGEMT-
RMND5B (C358A/H360A) RING clones 1-4 were purified and 5µL of each product
was electrophoresed in a 1% agarose gel (Section 3.6, 3.7.1, not shown). Based on the
gel, 3µL of each of pGEMT-RMND5B (C358A/H360A) RING clones 1 and 2 were
sequenced using the M13-S and M13-AS primers (Section 3.12, Appendix II).
BLASTTM analysis verified that RMND5B (C358A/H360A) RING clone 1 contained no
additional mutations (not shown).
To subclone the RMND5B (C358A/H360A) RING into pGEX-2TK, pGEMT-RMND5B
(C358A/H360A) RING clone 1 was digested with BamHI and EcoRI, the ~126bp insert
gel purified and 5µL of the product electrophoresed in a 2% agarose gel (Section 3.8.2,
Figure 4.23C). The pGEX-2TK vector was prepared by small scale plasmid
purification, RNase treatment, BamHI and EcoRI digestion and SAP treatment, then
5µL of the purified product was electrophoresed in a 1% agarose gel (Section 3.6, 3.8.2,
3.8.3, 3.9, Figure 4.23D). Based on the gels, 40ng (8µL) RMND5B (C358A/H360A)
RING and 50ng (5µL) pGEX-2TK were ligated and the products transformed into
competent E. coli DH5α then selected by plating on LB Agar/Ampicillin (Section 3.8.4,
3.6). Four colonies of pGEX-RMND5B C358A/H360A RING were picked and cultured
overnight in LB Broth/Ampicillin for small scale purification. Plasmids were RNase
treated, digested with BamHI and EcoRI to liberate the ~126bp RING domain and 10µL
of each product was electrophoresed in a 2% agarose gel (Section 3.6, 3.8.2). Although
no insert bands were visible due to the small size of the products (not shown), pGEX-
RMND5B (C358A/H360A) RING clones 1 and 2 were purified and 2µL of each clone
was sequenced using the pGEX-S and pGEX-AS primers, BLASTTM analysis of which
confirmed that pGEX-RMND5B (C358A/H360A) RING clone 1 was mutation free
apart from the base substitutions encoding the C358A/H360A amino acid changes
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
156
Figure 4.23: Cloning of sequences encoding the RMND5B (C358A/H360A) RING
domain into pGEX-2TK. (A) 5µL purified PCR amplified RMND5B
(C358A/H360A) was electrophoresed in a 2% agarose gel and the DNA concentration
was estimated to be ~15ng/µL. (B) pGEMT-RMND5B (C358A/H360A) RING
clones 1 to 4 were BamHI and EcoRI digested to release the ~126bp insert and the
products were electrophoresed in a 2% agarose gel, identifying inserts in all clones.
(C) 5µL gel purified BamHI and EcoRI digested pGEMT-RMND5B
(C358A/H360A) RING was electrophoresed in a 2% agarose gel from which the
DNA concentration was estimated to be ~5ng/µL. (D) BamHI/EcoRI and SAP
digested pGEX-2TK plasmid was purified and 5µL purified product was
electrophoresed in a 1% agarose gel from which the concentration was estimated to
be ~10ng/µL.
Lane 1: MW Marker
Lane 2: RMND5B RING (C358A/H360A)
Lane 3: Negative Control
~126bp
Lane 1: MW Marker
Lane 2: Undigested pGEMT-RMND5B (C358A/H360A) RING
Lane 3-6: BamHI/EcoRI digested pGEMT-RMND5B (C358A/H360A)
RING clones 1-4
~126bp
~3kb
A
B
~5kb
Lane 1: MW Marker
Lane 2: Undigested pGEX-2TK
Lane 3: BamHI/EcoRI SAP
digested pGEX-2TK
D
Lane 1: MW Marker
Lane 2: RMND5B
(C358A/H360A) RING
C
~126bp
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
157
(Section 3.12, Appendix II). This plasmid was then transformed into E. coli BL21 cells
and a glycerol stock was prepared (Section 3.8.7).
4.2.7.5 Expression and Intracellular Localisation of RMND5A
(C356A/H358A) and RMND5B (C358A/H360A)
To examine the expression and intracellular localisation of the mutant RMND5
proteins, large scale purification of the pEGFP-RMND5A (C356A/H358A) and
pEGFP-RMND5B (C358A/H360A) plasmids was performed and the concentration of
the purified plasmid was determined to be 1.2µg/µL for pEGFP-RMND5A
(C356A/H358A) and 1.3µg/µL for pEGFP-RMND5B (C358A/H360A) (Section 3.10,
4.2.7.1, 4.2.7.3). Expression of pEGFP-RMND5A (C356A/H358A) and pEGFP-
RMND5B (C358A/H360A) was evaluated by western blotting following transient
transfection of LNCaP cells with 4µg plasmids encoding each of wild-type or mutant
GFP-RMND5 proteins (Section 3.1.4, 3.15, Figure 4.24A). GFP western blotting
identified a prominent band at ~70kDa in the lysates of all transfected cells, indicating
expression of each of GFP-RMND5A, GFP-RMND5B, GFP-RMND5A
(C356A/H358A) and GFP-RMND5B (C358A/H360A) (Figure 2.24A).
To determine the cellular localisation of GFP-RMND5A (C356A/H358A) and GFP-
RMND5B (C358A/H360A), LNCaP cells growing on coverslips were transfected with
2µg plasmid encoding either wild-type or mutant GFP-RMND5 proteins (Section 3.1.3,
3.1.4). The cells were fixed 48 hours post transfection and viewed by fluorescence
microscopy, which identified that GFP-RMND5A and GFP-RMND5A
(C356A/H358A) proteins were diffusely distributed in the nucleus and cytoplasm, with
some cells exhibiting a punctate cytoplasmic distribution (Section 3.16, Figure 4.24B).
The GFP-RMND5B and GFP-RMND5B (C358A/H360A) proteins also appeared to be
diffusely distributed in the nucleus and cytoplasm, with most cells also containing
punctate cytoplasmic granules with GFP fluorescence (Figure 4.24B). These results
indicated that the RMND5 RING domain mutations did not alter the cellular
localisation of the RMND5 proteins in comparison to that of the wild-type protein.
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
158
A
B GFP Nucleus Cytoplasm Overlay
GFP-RMND5A
Wild Type
C356A/ H358A
GFP-RMND5B
Wild Type
C358A/ H360A
Untransfected Negative Control
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
159
Figure 4.24: Expression and cellular localisation of GFP-RMND5A (C356A/H358A) and GFP-RMND5B (C358A/H360A). LNCaP cells were transfected with plasmids encoding GFP-RMND5A, GFP-RMND5B, GFP-RMND5A (C356A/H358A) or GFP-RMND5B (C358A/H360A). At 48 hours post-transfection, (A) cells were harvested for GFP western blotting, which identified ~70kDa bands corresponding to GFP-RMND5A, GFP-RMND5A (C356A/H358A), GFP-RMND5B or GFP-RMND5B (C358A/H360A). (B) Alternatively, the cells were prepared for fluorescence microscopy and stained with Hœchst 33258 and tetramethylisothiocyanate (TRITC)-Phalloidin to visualise the nucleus and cytoplasm, respectively. GFP-RMND5A, GFP-RMND5B, GFP-RMND5A (C356A/H358A) and GFP-RMND5B (C358A/H360A) displayed diffuse nuclear and cytoplasmic localisation, with GFP-RMND5B and GFP-RMND5B (C358A/H360A) also exhibiting a punctate distribution in the cytoplasm (Magnification x1000). Experiments were preformed twice and representative results are shown. (WT= wild type).
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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4.2.7.6 In Vivo Ubiquitination Activity of RMND5A (C356A/H358A) and
RMND5B (C358A/C360A)
The activity of RMND5A (C356A/H358A) and RMND5B (C358A/H360A) was
assessed using in vivo ubiquitination assays performed following cotransfection of
LNCaP cells plasmids encoding HA-ubiquitin and either GFP-RMND5A, GFP-
RMND5A (C356A/H358A), GFP-RMND5B or GFP-RMND5B (C358A/H360A). To
promote the accumulation of ubiquitinated proteins, cells were incubated with 10µM
MG132 for the final 2 hours prior to harvest at 48 hours following transfection (Section
3.1.4, 3.1.5, 3.14.2). The shorter time period of proteasome inhibition was used to
facilitate identification of differences in the rate at which the wild-type and mutant
RMND5 proteins were able to associate with ubiquitinated proteins. GFP-tagged
proteins were immunoprecipitated from the cell lysates and electrophoresed in 4-12%
gradient polyacrylamide gels, with HA western blotting of GFP-RMND5A, GFP-
RMND5A (C356A/H358A) and empty vector immunoprecipitates identifying protein
bands corresponding to polyubiquitinated proteins at a range of molecular weights
(~80kDa – 175kDa) (Section 3.15, Figures 4.25A). The levels of ubiquitinated proteins
were markedly increased in the immunoprecipitates of GFP-RMND5A and GFP-
RMND5A (C356A/H358A) expressing cells that had been MG132 treated (Figure
4.25A). GFP-RMND5A and GFP-RMND5A (C356A/H358A) were associated with
similar amounts of ubiquitinated proteins, indicating that either the RMND5A
(C356A/H358A) mutation did not reduce the E3 ubiquitin ligase activity of RMND5A
(C356A/H358A) or that endogenous active proteins were masking alterations in the
activity of the mutant protein.
Similar in vivo ubiquitination assays were performed using GFP-RMND5B and GFP-
RMND5B (C358A/H360A), with HA (ubiquitin) western blotting identifying high
molecular weight proteins (~80kDa – 175kDa) corresponding to ubiquitinated and
polyubiquitinated proteins associated with both wild-type and mutant GFP-RMND5B
(Figure 4.25B). Increased levels of ubiquitinated proteins were detected in
immunoprecipitates from cells overexpressing wild-type GFP-RMND5B that had been
treated with MG132 (Figure 4.25B). In comparison, markedly reduced amounts of
ubiquitinated proteins were detected in association with GFP-RMND5B
(C358A/H360A), suggesting that the RING domain mutations had reduced the E3
ubiquitin ligase activity of RMND5B (Figure 4.25B). Western blotting of the total
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
161
A
B
WT (C356S)
- - - + + +
Empty Vector
10µM MG132
GFP (~70kDa)
HA (Ubiquitin)
Ubn-Proteins (~80kDa – 175kDa)
GFP-RMND5A
- - - + + +
Empty Vector WT (C358S)
10µM MG132 HA-Ubiquitin
GFP (~70kDa)
Ubn-Proteins (~80 – 175kDa)
GFP-RMND5B
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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Figure 4.25: In vivo ubiquitination activity of GFP-RMND5A (C356A/H358A) and GFP-RMND5B (C358A/H360A). LNCaP cells were cotransfected with plasmids encoding HA-ubiquitin and GFP-RMND5A, GFP-RMND5A (C356A/H358A) GFP-RMND5B, GFP-RMND5B (C358A/H360A) or GFP (empty vector). Cells were cultured for 48 hours with 10μM MG132 added for the final 2 hours of culture. Following GFP immunoprecipitation of the cell lysates, HA western blotting was performed on the immunoprecipitated proteins and identified enhanced accumulation of ubiquitinated proteins in lysates from MG132 treated cultures. (A) GFP-RMND5A and GFP-RMND5A (C356A/H358A) were associated with similar levels of ubiquitinated proteins, while (B) in comparison to GFP-RMND5B, GFP-RMND5B (C358A/C360A) was associated with reduced levels of ubiquitinated proteins. Experiments were performed twice and representative results are shown. (WT = wild type)
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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cellular input samples for HA (Ubiquitin), and GFP (wild-type/mutant
RMND5A/RMND5B) detected similar levels in each sample, suggesting that the results
were not due to differences in the transfection efficiency or expression of the fusion
proteins (not shown).
4.2.7.7 In Vitro Auto-Ubiquitination Activity of RMND5A (C356A/H358A)
and RMND5B (C358A/H360A) RING Domains
To verify that the GST-RMND5A (C356A/H358A) RING and GST-RMND5B
(C358A/H360A) RING domains were expressed in E. coli BL21 cells, the pGEX-
RMND5A (C356A/H358A) RING and pGEX-RMND5B (C358A/H360A) RING
plasmids were each transformed into competent E. coli BL21 cells and glycerol stocks
were prepared (Sections 3.8.7, 4.2.7.2, 4.2.7.4). Transformed E. coli BL21 cells were
selected on LB Agar/Ampicillin plates and individual colonies were picked and
inoculated into LB Broth/Ampicillin (Section 3.11.1). Expression of the GST-fusion
proteins was induced by the addition of 1mM IPTG for 2.5 hours, cells were harvested
and 15µL aliquots of lysates from each culture was electrophoresed in 12%
polyacrylamide gels and stained with Coomassie blue (Sections 3.11.1, 3.15.2, 3.15.5,
Figure 4.26A). A prominent band at ~28kDa was present in lysates from the IPTG-
induced cells, with GST-RMND5A (C356A/H358A) RING and GST-RMND5B
(C358A/H360A) RING domains in both the soluble and insoluble fractions, indicating
that RMND5 RING domain mutants were packaged into inclusion bodies, however
sufficient levels of the proteins were present in the soluble fractions for purification and
use in in vitro ubiquitination assays. To obtain purified GST-RMND5A
(C356A/H358A) RING and GST-RMND5B (C358A/H360A) RING domain proteins
for in vitro ubiquitination assays, proteins were induced by the addition of 1mM IPTG
and extracted from 100mL bacterial cultures (Section 3.11.2). Purified GST-RMND5
RING proteins were electrophoresed in 12% polyacrylamide gels with BSA standards
and Coomassie blue stained to estimate the concentration of the purified GST-fusion
proteins (Section 3.25.2, 3.15.5, Figure 4.26B, C).
Prior to the assessment of the GST-RMND5A (C356A/H358A) RING and GST-
RMND5B (C358A/H360A) RING domain activity in comparison to that of the wild-
type GST-RMND5A RING and GST-RMND5B RING domains, in vitro ubiquitination
assays were re-optimised as the enzyme concentrations used in the Ubiquitinylation kit
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
164
B
C 1 2 3 4 5 6 7 8 Lane 1: MW Marker Lane 2: 1µg BSA Lane 3: 2.5µg BSA Lane 4: 5µg BSA Lane 5: 7.5µg BSA Lane 6: 10µg BSA Lane 7: 1µL GST-RMND5B RING Lane 8: 3µL GST-RMND5B RING Lane 9: 1µL GST- RMND5B (C358A/H360A) RING Lane 10:1µL GST- RMND5B (C358A/H360A) RING
~28kDa
9 10
1 2 3 4 5 6 7 8
Lane 1: MW Marker Lane 2: 1µg BSA Lane 3: 2.5µg BSA Lane 4: 5µg BSA Lane 5: 7.5µg BSA Lane 6: 10µg BSA Lane 7: 1µL GST-RMND5A RING Lane 8: 3µL GST-RMND5A RING Lane 9: 1µL GST-RMND5A (C356A/H358A) RING Lane 10: 3µL GST-RMND5A (C356A/H358A) RING
~28kDa
9 10
1 2 3 4 5 6 7
Lane 1: MW Marker Lane 2: GST-RMND5A (C356A/H358A) RING: no IPTG Lane 3: GST-RMND5A (C356A/H358A) RING: IPTG Lane 4: GST-RMND5A (C356A/H358A) RING: IPTG, insoluble fraction Lane 5: GST-RMND5A (C356A/H358A) RING: IPTG, soluble fraction Lane 6: GST-RMND5B (C358A/H360A) RING: no IPTG Lane 7: GST-RMND5B (C358A/H360A) RING: IPTG Lane 8: GST-RMND5B (C358A/H360A) RING: IPTG, insoluble fraction Lane 9: GST-RMND5B (C358A/H360A) RING: IPTG, soluble fraction
~28kDa
8 9 A
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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Figure 4.26: Expression and purification of GST-RMND5A (C356A/H358A) RING and GST-RMND5B (C358A/H360A) RING. (A) E. coli BL21 cells transformed with pGEX-RMND5A (C356A/H358A) RING or pGEX-RMND5B (C358A/H360A) RING were induced with IPTG and the presence of the GST-RMND5A (C356A/H358A) RING or GST-RMND5B (C358A/H360A) RING domains in the soluble and insoluble fractions prepared from the cultures was investigated by electrophoresis of the fractions in 12% polyacrylamide gels, which were stained with Coomassie blue. Following purification, the RING domain proteins were electrophoresed in 12% polyacrylamide gels, and the gels stained with Coomassie blue. In comparison to BSA standards, the concentration of (B) GST-RMND5A RING and GST-RMND5A (C356A/H358A) RING was determined to be ~2.6µg/µL and ~1.26µg/µL, respectively. (C) The concentration of GST-RMND5B RING and GST-RMND5B (C358A/H360A) RING was estimated to be ~4.3µg/µL and ~0.7µg/µL, respectively.
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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(ENZO Life Sciences) were high in comparison to those used in the literature (David et
al., 2011, Yang et al., 2000). Although this was suitable for initial in vitro
ubiquitination assays, in order to determine differences in the activity of wild-type and
mutant RMND5A and RMND5B RING domains, the concentrations of each enzyme
were re-evaluated (Table 4.2). In vitro ubiquitination assays were also carried out for 30
minutes at 37°C (compared to 60 minutes), and the reactions terminated with the
addition of 50µL 2x reducing loading dye and heating for 5 minutes at 95°C (Section
3.14.1).
Table 4.2 – Optimisation of in vitro ubiquitination assay enzyme concentrations
Enzyme Concentration
Utilised in Initial
Screen
Optimised
Concentration 1
Optimised
Concentration 2
E1 Activating Enzyme 100nM 25nM 50nM
E2 Conjugating
Enzyme, UbcH5b
2.5µM 0.25µM 0.5µM
GST-RING domain 4µM 1µM 2.5µM
Ten µL of each reaction product was electrophoresed in 4-12% gradient polyacrylamide
gels followed by western blotting for biotinylated ubiquitin. All reactions yielded
multiple protein bands, corresponding to ubiquitinated proteins, with most of these of a
similar appearance and intensity in reactions containing GST-RMND5A, GST-
RMND5A (C356A/H358A), GST-RMND5B and GST-RMND5B (C358A/H360A)
RING domain proteins. However, a band at ~38kDa, corresponding to
monoubiquitinated GST-RMND5 RING domains which was present at both enzyme
concentrations tested (Table 4.2), appeared to be reduced in in vitro ubiquitination
reactions containing the GST-RMND5A (C356A/H358A) RING and GST-RMND5B
(C358A/H360A) RING domains compared to those reactions containing wild-type
GST-RMND5A and GST-RMND5B RING domains (Section 3.14.1, Figure 4.27).
Additionally, high molecular weight protein bands at >100kDa corresponding to
polyubiquitinated proteins were of a greater intensity in reactions containing the wild-
type GST-RMND5B RING domain compared to the GST-RMND5B (C358A/H360A)
RING domain.
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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Experiments were repeated using Condition 1 (Table 4.2), along with negative control
reactions lacking ATP or the GST-RMND5 RING domain, and 10µL of each product
was electrophoresed in 4-12% gradient gels (Section 3.14.1, 3.15). Western blotting for
biotinylated ubiquitin and GST were used to detect ubiquitinated proteins and GST-
fusion proteins, respectively (Section 3.15, Figure 4.28). Reactions lacking ATP yielded
a single band corresponding to free ubiquitin, whilst reactions lacking the GST-RMND5
RING yielded bands corresponding to ubiquitinated E2 enzyme only (Figure 4.28).
Again, multiple biotinylated ubiquitin bands of similar intensity were present in
reactions containing GST-RMND5A RING, GST-RMND5B RING, GST-RMND5A
(C356A/H358A) RING and GST-RMND5B (C358A/H360A) RING domains.
However, in reactions containing the GST-RMND5A (C356A/H358A) RING and GST-
RMND5B (C358A/H360A) RING domain proteins, the protein band at ~38kDa
corresponding to monoubiquitinated GST-RING domain proteins was reduced
compared to reactions containing wild-type GST-RMND5A RING and GST-RMND5B
RING domain proteins (Figure 4.28). GST western blotting identified expression of the
GST-RING domains, while high molecular weight bands corresponding to
polyubiquitinated proteins (>100kDa) were not visible in this set of in vitro
ubiquitination reaction products at the short x-ray film exposure times used to visualise
differences in the appearance and intensity of monoubiquitinated GST-RMND5 RING
domain protein bands (Figure 4.28). These results are consistent with the in vivo
ubiquitination assay results where mutation of the RMND5B RING domain resulted in
a larger reduction in ubiquitination activity compared to analogous mutations of the
RMND5A RING domain (Section 4.2.7.6) and are consistent with the RMND5A
(C356A/H358A) and RMND5B (C358A/H360A) mutations disrupting RMND5 RING
domain activity.
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
168
Condition 1 Condition 2
Monoubiquitinated RMND5A RING domain
MonoubiquitinatedRMND5B RING domain
WT WT
C356A/H358A
A
B
WT WT
C358A/H360A
C358A/H360A
C356A/H358A
GST-RMND5A RING domain
GST-RMND5B RING domain
Figure 4.27: Optimisation of in vitro ubiquitination assays for GST-RMND5A
(C356A/H358A) RING and GST-RMND5B (C358A/H360A) RING. The
concentrations of E1, E2 and GST-RING domains used in in vitro ubiquitination
assays were optimised, with condition 1 containing 25nM E1, 0.25µM E2 and
1µM GST-RING, whilst condition 2 contained 50nM E1, 0.5µM E2 and 2.5µM
GST-RING. The in vitro ubiquitination assays were carried using either (A) GST-
RMND5A RING or GST-RMND5A (C356A/H358A) RING or (B) GST-
RMND5B RING or GST-RMND5B (C358A/H360A) RING. Western blotting for
biotinylated ubiquitin identified protein bands corresponding to ubiquitinated
proteins in reactions containing both wild-type and mutant GST-RMND5 RING
domains, including a band corresponding to monoubiquitinated GST-RMND5
RING domains in each reaction. (WT= wild-type).
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
169
Figure 4.28: In vitro ubiquitination activity of GST-RMND5A (C356A/H358A) and GST-RMND5B (C358A/H360A). In vitro ubiquitination assays using GST-RMND5A RING, GST-RMND5B RING, GST-RMND5A (C356A/H358A) RING or GST-RMND5B (C358A/H360A) RING were performed and 10µL of each product was electrophoresed in 4-12% gradient gels. GST western blotting identified the presence of GST-RING domain proteins in all reactions except the negative control lacking E3 enzyme. Western blotting for biotinylated ubiquitin yielded multiple bands including a ~38kDa band corresponding in size to monoubiquitinated GST-RMND5 RING domains that was reduced in reactions containing GST-RMND5A (C356A/H358A) RING and GST-RMND5B (C358A/H360A) RING compared to reactions containing GST-RMND5A or GST-RMND5B, respectively. The experiment was performed twice and representative results are shown. (WT= wild-type).
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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4.3 Discussion
The homologues RMND5A and RMND5B are highly conserved between diverse
species ranging from mammals to Arabidopsis. In this thesis, analysis of RMND5
protein domain composition and amino acid sequence identified that each contains 4
protein-protein interaction domains, a LisH, CTLH, CRA and RING domain and that
the amino acid variation between RMND5A and RMND5B is not confined to a
particular protein domain but rather is scattered throughout the protein sequences,
including areas of the proteins that do not contain identifiable protein domains.
Bioinformatics analyses indicated that the general protein domain architecture of
RMND5 proteins was similar to that of other LisH domain containing proteins, which
also commonly possess a CTLH and CRA domain, and that the protein, EMP has the
same protein domain architecture as human RMND5A and RMND5B (Schultz et al.,
1998). The six human proteins that possess a LisH, CTLH and CRA domain either form
part of the human CTLH complex or are orthologues of the known CTLH complex
members, suggesting that the presence of this particular protein domain architecture
allows the proteins to function in complementary cellular pathways or roles (Santt et al.,
2008).
The existence of orthologues of the CTLH complex members such as RMND5B and
RanBP10 (orthologue of RanBPM) provides evidence that these proteins may be able to
replace or join their paralogue in the CTLH complex, perhaps altering the function or
substrates of the complex. Alternatively, the function of the orthologues may have
diverged, as has been documented for other paralogous genes (Sahdev et al., 2008;
Singh and Hannenhalli, 2008). The LisH and CTLH domains are co-expressed in
RMND5A and RMND5B in addition to many as yet uncharacterised proteins,
suggesting that the domain pair functions together in its interaction with cellular
proteins or in alternative activities. However, although the LisH, CTLH and CRA
domains are proposed protein-protein interaction domains, their functions are not well
characterised, with the majority of information inferred from the activities of proteins
that contain these domains. For example, involvement of LisH domain containing
proteins in microtubule based protein transport was hypothesised from the function of
LIS1, the protein in which the LisH domain was originally identified (Emes and
Ponting, 2001).
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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The C-termini of RMND5A and RMND5B that contain the RING domain are the most
highly conserved regions of the two proteins, indicating that the RING domain performs
similar functions in both proteins. This hypothesis is consistent with the findings of this
study that the RING domains of both RMND5 proteins possess E3 ubiquitin ligase
activity. Bioinformatics analyses of the RING domains of RMND5A and RMND5B
identified that although all eight residues predicted to be required for zinc coordination
are not the canonical cysteine and histidine residues, they are identical to those present
in yeast RMD5, a functional E3 ubiquitin ligase (Santt et al., 2008). Furthermore,
comparison of the RING domains of RMND5A and RMND5B to RING domain
consensus sequences identified that additional residues in the RING domains of
RMND5 proteins were conserved, with these residues present in other well
characterised E3 ubiquitin ligases such as MDM2 and plant E3 ubiquitin ligases (Stone
et al., 2005). Arabidopsis E3 ubiquitin ligases are well studied, with nine RING domain
variants characterised that differ in their zinc coordinating and intervening residues,
providing support for the existence of additional RING domain variants still to be
identified in human proteins (Stone et al., 2005).
The 34 amino acid residues located immediately amino-terminal to the RING domain
share 84% amino acid conservation between RMND5A and RMND5B. Since this
region is adjacent to the RING domain it may be important for the E3 ubiquitin ligase
activity of RMND5 proteins, as identified in CBL where the linker region, which is
located N-terminal to the RING domain, is important for interaction with the E2
enzyme UbcH7 (Zheng et al., 2000). As such, this region of RMND5A and RMND5B
may perform similar functions, potentially contributing to E2 enzyme binding, and the
high degree of homology indicates that both RMND5 proteins may be able to interact
with similar E2 enzymes via this region. Alternatively, the domain may be required for
the regulation of RING domain activity or structure. The amino-terminal of RMND5
proteins is not as well conserved as the carboxy-terminal, with the LisH and CTLH
region sharing between 60-68% amino acid identity and the carboxy-terminal located
CRA domain sharing 74% amino acid homology. Gene duplication, through which
RMND5A and RMND5B may have arisen, has been suggested to allow one copy of the
gene to maintain the normal gene function, whilst the other gene is able to undergo
divergence and acquire new functions, thereby allowing the evolution of new
morphology (Mazet and Shimeld, 2002). Thus, RMND5 proteins may have arisen from
the same gene and over time they have acquired different cellular substrates or binding
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
172
partners to allow divergent roles. This, together with the lower amino acid homology of
the LisH and CTLH domains, suggests that these domains may have diverged in their
protein recognition or interaction roles, indicating that RMND5 proteins may utilise
these domains to interact with different proteins. The interacting proteins may be
substrates of RMND5 E3 ubiquitin ligase activity, however, the protein domain
architecture of RMND5 proteins with the RING domain located carboxy-terminally and
the 3 additional protein-protein interaction domains located towards the amino-terminus
suggests that the amino-terminal domains may be able to mediate additional cellular
roles. This has been suggested for other RING domain containing proteins with a
similar multi-domain architecture, including BARD1. The RING domain-containing
protein BARD1, which functions with and enhances the activity of the functional E3
ubiquitin ligase and tumour suppressor BRCA1, contains an amino-terminal RING
domain and two additional protein-protein interaction domains towards the C-terminus
of the protein, it required for cell viability and has tumour suppressor properties of its
own (Irminger-Finger and Jefford, 2006). In addition to its activity with BRCA1,
BARD1 interacts with the mRNA polyadenylation cleavage factor CSTF1 and represses
its cellular polyadenylation activity, a mechanism by which BARD1 is proposed to
regulate cell proliferation (Kleiman and Manley, 1999; Irminger-Finger and Jefford,
2006).
The amino-termini of RMND5 proteins, which do not contain identifiable protein
domains or localisation signals exhibit a high degree of amino acid identity (76%),
which suggests that this stretch of 113 amino acids is important in RMND5 protein
function. According to the SBASE protein domain prediction database, RMND5
proteins also contain putative protein domains in their amino-terminal region which are
loosely conserved. RMND5A contains a ribulose phosphate 3-epimerase-like domain
(amino acids 11-40) and a GAT-like domain (amino acids 42-94), whilst RMND5B
contains a myosin tail like domain (amino acids 26-91) (Vlahovicek et al., 2005).
Although these domains are only weakly conserved in RMND5A and RMND5B, their
functions are interesting. Ribulose phosphate 3-epimerase is activated by Zn2+ binding,
similar to the RING domain, and functions in the pentose phosphate pathway (Akana et
al., 2006). The domain is also present in many other enzymes that use phosphorylated
proteins as substrates (Akana et al., 2006). The presence of this putative domain is
intriguing as many E3 ubiquitin ligases contain phosphorecognition motifs to allow
recognition and binding to their substrates for ubiquitination. For example, CBL
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
173
recognises phosphorylated receptor tyrosine kinases via its SH2 domain, which it then
ubiquitinates (Joazeiro et al., 1999). Therefore it would be interesting to determine
whether the ribulose phosphate 3-epimerase-like domain, although perhaps non-
functional with regards to epimerase activity, retains phosphate binding ability. The
GGA and Tom1 (GAT) domain is a ubiquitin binding domain that is present in GGA
clathrin coat adaptors, which function in the trans-Golgi network by sorting
monoubiquitinated cargo (Scott et al., 2004). While GAT domains are also found in
proteins involved in the sorting of ubiquitinated proteins in multi-vesicular bodies, it is
presently unknown whether this putative domain facilitates binding of ubiquitinated
proteins with RMND5A (Scott et al., 2004; Hurley et al., 2006). As will be discussed in
Chapter 6, a member of the CTLH complex, ARMC8α interacts with the endosomal
sorting protein Hrs which binds ubiquitinated proteins, thus the CLTH complex with
RMND5A as a member may ubiquitinate proteins and/or transport ubiquitinated
proteins to the endosomal system (Tomaru et al., 2010).
Finally, RMND5B contains a putative myosin tail like domain, the function of which
varies between myosin family members, either interacting with other myosin tails or
binding cargo proteins functioning in the intracellular trafficking of organelles (Sellers,
2000). Myosin motors traffic organelles along actin “tracks” whilst dyneins and
kinesins are involved in intracellular transport using microtubules, which consist of α-
tubulin and β-tubulin (Nelson et al., 2005). The presence of this putative domain in
RMND5B is interesting as the LisH domain is also implicated in organelle trafficking
by binding cytoplasmic dynein heavy chain and regulating microtubule function (Emes
and Ponting, 2001). Additionally, members of the yeast Vid30 complex, the human
orthologues of which contain LisH and CTLH domains, are involved in the vacuole-
based transport of FBPase and also interact with actin patches, thereby merging the
vacuole with the endocytic system (Brown et al., 2010; Alibhoy et al., 2012). The
presence of the LisH, CTLH, putative ribulose phosphate 3-epimerase-like, GAT-like
and myosin tail-like domains, which may recognise and transport ubiquitinated proteins
to the endosome/lysosome for degradation, implicate RMND5 proteins and members of
both the yeast Vid30 and human CTLH complexes in the intracellular transport of
cargo. As RMND5 proteins are E3 ubiquitin ligases, they may play dual roles, either
ubiquitinating proteins for degradation or recognising ubiquitinated proteins and
sorting/transporting them within the cell. In this study, proteasome inhibitors were used
to determine the accumulation of ubiquitinated proteins upon overexpression of
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
174
RMND5 proteins in vivo, however given the aforementioned information, future
experiments may more extensively investigate the use of lysosome inhibitors to
determine whether RMND5 proteins are involved in the lysosomal degradation of
proteins.
The presence in both RMND5A and RMND5B of the LisH and RING domains, which
function as dimerisation motifs, indicates that RMND5 proteins may use these domains
to form homodimers or heterodimers with each other, as demonstrated in this thesis
(Chapter 6), or with other similar proteins such as EMP (Kim et al., 2004). RING
domains are commonly used as dimerisation motifs, often resulting in their enhanced
activity, especially where RING domain heterdimersation is involved (Brzovic et al.,
2001; Linke et al., 2008). For example, dimerisation of BRCA1/BARD1 enhances the
activity of BRCA1 as does the association of MDM2 with MDMX, while RING domain
homodimerisation is also essential for the functioning of some E3 ubiquitin ligases
including the E3 enzyme RNF4 (Brzovic et al., 2001; Liew et al., 2010; Pant et al.,
2011). Thus it is feasible that RMND5 proteins interact with other RING domain
containing proteins to enhance their own E3 ubiquitin ligase activity or that of an
interacting RING domain containing protein. This hypothesis is consistent with findings
in this thesis that although mutant RMND5A (C356A/H358A) and RMND5B
(C358A/H360A) exhibited reduced RING domain automonoubiquitination in vitro, in
in vivo ubiquitination assays no reduction in the activity of the mutant RMND5A was
detected.
RING domain E3 ubiquitin ligases may function as single subunit or multi-subunit
(complex) proteins. Whether RMND5 proteins are able to function in either or both
modalities is yet to be determined. As mentioned previously, RMND5A has been shown
form to part of the large multi-protein CTLH complex and as such could impart its
activity to the complex (Kobayashi et al., 2007). However, both RMND5 proteins
contain LisH, CTLH and CRA domains in addition to the RING domain, and therefore
the additional protein-protein interaction domains could act in a substrate recognition
capacity, allowing RMND5A and/or RMND5B to function as E3 ubiquitin ligases
independently of the CTLH complex. The tissue distribution or endogenous levels of
RMND5A and RMND5B proteins are unknown, although both genes are expressed in
the majority of tissue types (NCBI Unigene EST Profiles
ftp://ftp.ncbi.nih.gov/repository/UniGene/Homo_sapiens/Hs75277, Hs27222). Further
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
175
investigation of the tissue distribution of these proteins will help to elucidate the cellular
roles of RMND5A and RMND5B and to identify whether they function together or
have divergent targets in different tissues. The LisH domain has been documented to aid
in the formation of other protein complexes, including E3 ubiquitin ligase complexes
such as the Cullin4A-RING DCAF1 complex (Cerna and Wilson, 2005; Choi et al.,
2008; Ahn et al., 2011). As other LisH, CTLH and CRA domain containing proteins
form part of the CTLH complex, the LisH domain may similarly be responsible for
complex formation (the CTLH complex will be discussed in detail in Chapter 6).
RMND5A and RMND5B were found to be localised in both the nucleus and cytoplasm,
however in approximately 60% of transfected cells, RMND5B exhibited a punctate
cytoplasmic distribution. A similar intracellular distribution was also displayed by
RMND5A in a lower proportion of GFP-RMND5A overexpressing cells. Due to the
lack of suitable commercially available antibodies against either RMND5A or
RMND5B, the intracellular distribution of endogenous RMND5A or RMND5B proteins
is unknown at this stage and as such it is not clear whether their punctate appearance is
due to a normal cellular function or results from their overexpression following
transfection. The punctate appearance of RMND5A and RMND5B has now been
identified in multiple cell lines, including both breast and prostate cancer cell lines (not
shown) and for RMND5B, following overexpression with multiple N and C terminal
protein tags, suggesting that the localisation is specific to RMND5 proteins (Dawson,
2006). The cellular localisation of RMND5A has been reported previously as diffusely
nuclear and cytoplasmic in HEK293 cells, which is in agreement with the findings in
this study, including those cells that displayed a punctuate cytoplasmic distribution of
RMND5A (Kobayashi et al., 2007).
The well characterised E3 ubiquitin ligase, Siah1 is also reported to exhibit a punctate
cytoplasmic distribution, due to its association with mitochondria, however cells
expressing Siah1 RING deletion mutants no longer displayed this staining, implicating
the RING domain structure or function in its punctate distribution (Hu and Fearon,
1999). As such, RMND5 proteins could be localising to these punctate speckles as part
of a normal cellular activity, which may be due to their RING domain and/or E3
ubiquitin ligase activity or unrelated cellular role, and which may be investigated in
future studies using RING domain deletion mutants of RMND5A and RMND5B. To
determine the specific localisation of RMND5 proteins, organelle stains may also be
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
176
used, for example early endosomal markers (EEA1, Rab4, Rab5 antibodies), late
endosome markers (Rab7, Rab9 antibodies) lysosomal markers (LAMP1, LAMP2
antibodies, Lyso Tracker® (Life Technologies)) or mitochondrial markers (Mito
Tracker® (Life Technologies)). Localisation of RMND5 proteins in the lysosome for
example may suggest either that they play a particular role associated with the
lysosome, or that they are packaged and degraded in the lysosome when overexpressed,
therefore additional functional studies will be required to characterise the relationship
between RMND5 protein intracellular localisation, activity and processing. RMND5
proteins are degraded at least in part by the proteasome as they show accumulation upon
proteasome inhibition (Chapter 6), and although mutation of the RING domains did not
markedly alter RMND5 protein localisation, their localisation may be determined by
their interaction with other proteins through either the RING domain or via one of their
other protein-protein interaction domains, the LisH, CTLH or CRA domains. Therefore,
single or multiple domain deletion mutants of RMND5A or RMND5B may enable the
determination of protein domain(s) that direct their intracellular localisation, including
their punctate distribution.
To characterise the E3 ubiquitin ligase activity of RMND5 proteins in this study, full
length RMND5A was cloned into the pGEX-2TK expression vector to allow the
expression of full length GST-tagged RMND5 proteins for use in in vitro ubiquitination
assays. However, the expression and purification of both GST-RMND5A and GST-
RMND5B fusion proteins was hindered by the packaging of the proteins into inclusion
bodies in E. coli BL21 bacterial cells. The misfolding of expressed proteins in bacteria
into insoluble aggregates, which are biologically inactive, has been widely documented
(Francis and Page, 2010). Proteins may be misfolded due to their requirement for longer
folding times or the need for chaperones to facilitate correct folding and, due to the
inability of bacteria to accomplish numerous eukaryotic post-translational modifications
such as phosphorylation, larger proteins and specific protein domains may be difficult to
obtain as soluble proteins (Villaverde and Carrio, 2003; Esposito and Chatterjee, 2006;
Francis and Page, 2010). The production of insoluble proteins may also be due to weak
promoter sequences, inefficient initiation of translation and the presence of rare codons
(codon bias) which may be rectified by the modification of promoter and translation
initiation sequences and the use of codon optimised bacterial cells, respectively (Sahdev
et al., 2008; Malhotra, 2009). In these experiments, western blotting of whole cell
lysates of the transformed bacteria indicated that GST-RMND5 fusion proteins were
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
177
produced in large quantities in the bacteria, suggesting that promoter sequences and
translation initiation were not causing the production of insoluble aggregates.
The E. coli BL21 strain utilised for the present studies was codon optimised and the
amount of full-length (insoluble) protein produced by the bacteria indicated that the low
yields of purified protein were not likely to be due to codon bias. Furthermore, a second
codon optimised strain, Rosetta BL21 cells was also tested, producing similar results.
The use of BL21 cells for routine production of fusion proteins has been suggested due
to their codon optimisation and the absence of expression of specific intracellular
proteases, thereby promoting stabilisation of the fusion proteins (Sahdev et al., 2008).
The GST protein tag, which is 211 amino acids in length, is also considered desirable as
it acts as a chaperone to aid in protein folding and can increase the solubility of the
fusion protein (Malhotra, 2009). In addition, the GST-fusion proteins can be
immobilised on glutathione agarose beads which have a high affinity for GST ensuring
low non-specific binding and therefore reducing contamination of the final purified
product with other proteins (Malhotra, 2009; Harper and Speicher, 2011). As an
alternative to the GST tag, smaller tags including a His-tag (6-10 histidine residues) or
Strep-II tag (8 amino acid residues WSHPQFEK) or an amino-terminal fusion with a
highly translated native protein such as maltose binding protein (396 amino acids) or
thioredoxin (109 amino acids) could have been tested to maximise solubility of the
fusion protein (Yasukawa et al., 1995; Malhotra, 2009).
Modification of expression conditions may also be used to increase solubilisation of
proteins, including lowering of the induction temperature, increasing aeration and the
co-expression of chaperones, however many proteins will remain insoluble (Esposito
and Chatterjee, 2006; Lorick et al., 2006). At lower temperatures, the expression of
chaperones is induced, the activity of proteases is reduced, and protein interactions
aiding in inclusion body formation and toxic phenotypes of fusion proteins are
suppressed (Sahdev et al., 2008). In this study, expression conditions were modified by
increasing aeration, reducing induction temperature and optimising GST-fusion protein
induction time, however altering these conditions did not increase solubilisation of
GST-RMND5A. The medium in which the bacterial cells were grown was also
optimised to obtain soluble protein by the addition of ZnCl2 to aid correct folding of the
RING domain. This modification of growth medium conditions has been suggested to
enhance protein solubilisation for the production of RING domain containing and other
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
178
proteins (Lorick et al., 2006; Sahdev et al., 2008). A further option was to purify the
inclusion bodies containing the misfolded proteins, disaggregate the proteins by
denaturation using buffers containing urea and then following dialysis, refold the
proteins. However this is a long process which is not considered optimal as the protein
yield is often low and the production of correctly folded proteins must be stringently
optimised and is not guaranteed, therefore potentially yielding non-functional proteins
or proteins with variably reduced activity (Middelberg, 2002; Rabhi-Essafi et al., 2007;
Sahdev et al., 2008). As such, this option for the production of GST-RMND5A was not
pursued.
Cell free systems may also be used for the production of proteins for biochemical assays
and can be advantageous because the in vitro system is directed to the production of a
single protein in comparison to in vivo systems that require the synthesis of normal
cellular proteins as well as the protein of interest, which may be cytotoxic to the cells
(Iskakova et al., 2006). Such methods involve the production of fusion proteins from
mRNA or DNA templates using coupled or linked in vitro transcription and translation.
These systems combine the use of a prokaryotic phage RNA polymerase and promoter
(e.g. T7, T3) with eukaryotic or prokaryotic extracts from human cells, wheat germ or
rabbit reticulocytes (for example) to provide the transcription and translation machinery
and are supplemented with energy regenerating solutions, amino acids and accessory
proteins (Stueber et al., 1984; Mikami et al., 2008). Newer methods for the production
of larger proteins such as the 200kDa Dicer, which possess biological activity have been
developed (Mikami et al., 2008). The use of human cell line extracts and advances in
cell free protein production systems are proposed to facilitate proper protein folding and
post-translational modification of the fusion proteins by the addition of reagents and
chaperones. Although these methods are an improvement on prokaryotic cell free
systems, which have been found to produce proteins with low activity, the production of
proteins that require post-translational modifications remains a challenge as it is
difficult to obtain homogeneously modified protein products in cell free systems
(Katzen et al., 2005; Iskakova et al., 2006). While these methods often involve the use
of radioactively labelled amino acids they can be modified to produce unlabelled tagged
proteins, with in vitro transcribed/translated proteins reported to be successfully used in
in vitro ubiquitination assays (Lorick et al., 2006; Jin et al., 2009).
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
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An alternative to the production of proteins in bacterial cells would be their production
in mammalian, yeast or insect cells such as Trichoplusia or Spodoptera. For these
methods, insect cells are infected with the baculovirus expression vector system, and
this method has been used extensively for the production of complex proteins, including
membrane bound proteins, as well as in the pharmaceutical industry for the manufacture
of animal and human vaccines such as Cervarix (Kost et al., 2005; Cox, 2012).
Establishment of the method is time-consuming as the insect cells must be cultured and
infected with the baculovirus at particular stages of cell growth to achieve maximal
infection and protein production, the type of insect cells must be selected and the ability
of the cells to produce the protein of choice must be verified (Reuveny et al., 1993). The
use of insect cells is advantageous as they are often able to produce large quantities of
heterologous protein, and the cells can be cultured at room temperature, do not require
CO2 and in most cases can be grown in serum free media (Kost et al., 2005). Although
insect cells are eukaryotic, they are not able to perform all types of mammalian post-
translational modifications such as glycosylation, however insect cells engineered to
produce glycosylated proteins by the incorporation of mammalian genes encoding N-
glycan activity have now made it possible to produce glycosylated proteins in insect
cells (Hollister et al., 2002; Kost et al., 2005). Baculovirus expression systems
containing mammalian cell-active expression cassettes (BacMam), can also be utilised
to infect mammalian cells for the expression of recombinant proteins, and human
osteosarcoma and hepatic cells have been reported to produce high levels of gene
expression, however not all cell lines are able to be transduced or show effective
expression of the gene/protein of interest (Gao et al., 2002; Song et al., 2003; Kost et
al., 2005). There are therefore a number of alternative means for the production of large
quantities of recombinant proteins, which are misfolded such as RMND5 proteins or
that are quickly degraded in bacterial cells. However, due to time constraints an
alternative means to examine the activity of RMND5 proteins was to clone smaller,
soluble peptides containing the active domain of interest, the RING domain, into the
pGEX-2TK expression vector.
GST-RING domain fusion proteins were therefore used in in vitro ubiquitination assays
for this thesis in order to test the E3 ubiquitin ligase activity of RMND5 proteins. In
vitro ubiquitination assays are routinely employed to determine the E3 ubiquitin ligase
activity of putative ubiquitin ligases, and although where possible full length proteins
are used, shorter peptides encompassing the active domain, RING/HECT/U-box or
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
180
PHD domain, may also be used (Furukawa et al., 2005). The use of the GST moiety can
be advantageous when testing autoubiquitination activity as the GST tag dimerises,
which can aid in the detection of E3 activity as many RING domains also dimerise
(Lorick et al., 2006). However, in vitro auto-ubiquitination assays are prone to artefacts
due to non-specific protein aggregation formed during incubation and there are a
number of methods to minimise these, which were used where possible in this study. A
positive control in the form of CBL was utilised, and reactions lacking ATP and GST-
RING domains (negative controls) were included as part of the evaluation of the panel
of 11 E2 enzymes. Although the positive results expected from the in vitro assay
included a single high molecular weight band representing polyubiquitinated proteins
and lower molecular weight bands corresponding to mono- or multi-ubiquitinated
proteins, multiple additional bands corresponding to thioester linked proteins were also
visualised. Thus controls were used to investigate the identity of the additional bands
evident in ubiquitin western blots of the reaction products, specifically whether their
presence was related to the addition of GST-RMND5 RING domains and therefore
represented their ubiquitination products. Moreover, a full set of control reactions was
performed with each reaction omitting a reaction ingredient, following selection of the
E2 enzyme UbcH5b for further study. In this way the in vitro ubiquitination assays
consistently identified specific ubiquitination patterns (including bands corresponding
to auto-monoubiquitination) when the GST-RMND5 RING domains were included in
the reactions. These bands were not present when GST-CBL was used as a positive
control or when reaction ingredients were omitted, suggesting that the results were
specific to the presence of the RMND5 RING domains.
The in vitro ubiquitination assays performed using the RMND5 RING domains that
were carried out for this thesis provided a starting point for the analysis of RMND5
protein E3 ubiquitin ligase activity in vitro, and formed the preliminary information
required for further assessment of RMND5 protein E3 ubiquitin ligase activity.
However, a number of other assays may be performed in the future to further assess
RMND5 E3 ubiquitin ligase activity in vitro. Longer isoforms of GST-RMND5A or
GST-RMND5B that include additional domains required for substrate recognition, that
facilitate protein folding or that increase the number of lysine residues available for
ubiquitination (assisting in the visualisation of (larger) ubiquitinated proteins) may be
used in future studies (Zhang et al., 2009). To improve visualisation of ubiquitinated
proteins, in vitro ubiquitination assays may be performed with the GST fusion proteins
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
181
attached to GST beads, allowing the removal (by washing) from the beads of non-
specific reaction products and E1 or E2 enzymes with thioester linked ubiquitin
(Furukawa et al., 2005; Lorick et al., 2006). Alternatively, RMND5 proteins may be
overexpressed by transfection of mammalian cells, immunoprecipitated and the purified
proteins used in in vitro assays, which also overcomes the problem of insoluble full
length protein production in bacteria (Furukawa et al., 2002; Burger et al., 2005; Lorick
et al., 2006). An advantage to this method is that the protein has been produced in
mammalian cells and as such would have undergone the correct post-translational
modifications, and in addition, if the E3 ubiquitin ligase requires binding partners or
cofactors, these may be coimmunoprecipitated, strengthening the in vitro assays (Lorick
et al., 2006).
Most E3 ubiquitin ligases are able to interact with more than one E2 conjugating
enzyme, with at least one of the UbcH5 family of E2 enzymes interacting with most E3
ligases. However, the identity of E2 enzymes that interact with an E3 cannot be
predicted and need to be determined in functional studies such as the in vitro
ubiquitination assays used in this thesis, which utilised a panel of 11 E2 enzymes. For a
variety of reasons, in vitro ubiquitination assays may not identify all possible E2
enzymes that interact with RMND5 proteins to mediate ubiquitin transfer. For example,
the RING domain alone was used in these assays and as such areas outside of the
domain required for E2 interaction would not be present, including regions immediately
adjacent to the RING domain that are known to be important for E2-E3 interactions
(Zheng et al., 2000). In addition, the interaction between the E2 and E3 may not be
sufficient to result in ubiquitin transfer in vitro, for example CBL can interact with
UbcH5b and UbcH7, but in vitro only the interaction with UbcH5b results in ubiquitin
transfer, although CBL uses UbcH7 in vivo (Huang et al., 2009).
In this study, in vitro ubiquitination assays were performed using a single set of
conditions with only 11 E2 enzymes evaluated and as such, RMND5 proteins may be
able to interact in vivo with other E2 enzymes that were or were not present in the panel.
In humans, ~40 E2 enzymes have been characterised, many of which are ubiquitously
expressed and exhibit both cytoplasmic and nuclear localisation, similar to both
RMND5A and RMND5B (van Wijk and Timmers, 2010). Of the E2 enzymes in the
panel that did not interact with RMND5 proteins in in vitro ubiquitination assays, it is
possible with optimal experimental conditions, specific cellular environments or the
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
182
presence of cofactors or complex components including the RING domains of other
proteins that they are be able to interact with RMND5 proteins to mediate ubiquitin
transfer. Furthermore, as some E2 enzymes are chain initiating enzymes, the use of only
the RMND5 RING domains and single E2 enzymes may have failed to detect
interactions between RMND5 proteins and important chain elongating E2 enzymes that
require mono- or poly-ubiquitinated substrates for their activity (Windheim et al., 2008;
Ye and Rape, 2009). For example, TRAF6 utilises UbcH5 to initiate chain formation by
monoubiquitating the substrate, and this is followed by its interaction with the chain
elongating E2 enzyme UBE2N-UBE2V1 to polyubiquitinate the substrate NF-κB. As
the E2 enzymes UBE2N-UBEV1 and UBE2S cannot perform ubiquitin chain initiation,
their substrate activity is dependent on substrate chain initiation by other E2 enzymes
(Christensen et al., 2007; Petroski et al., 2007; Windheim et al., 2008).
Finally, the in vitro assays used in this project assessed the auto-ubiquitination activity
of RMND5 RING domains and therefore did not allow evaluation of potential
interactions of full-length RMND5 proteins with different E2 enzymes depending on the
substrate and cellular environment. To identify E2 enzymes that interact with RMND5
proteins in vivo, future studies may use immunoprecipitation assays from whole cell
lysates to determine E2-E3 interaction pairs, which may be confirmed by GST pull-
down assays and colocalisation microscopy (Lorick et al., 2006). Additionally, the type
of ubiquitin linkage attached to the substrate following the E3 interaction with specific
E2 enzymes can be assessed in either in vitro or in vivo assays with the use of linkage
specific antibodies or ubiquitin mutants (Lorick et al., 2006).
The use of GST-RMND5A and GST-RMND5B RING domains in in vitro assays
showed that RMND5 proteins were able to interact with a number of E2 enzymes to
mediate ubiquitin transfer and that both RMND5A and RMND5B were able to interact
with UbcH5b to mediate their auto-monoubiquitination. The finding that RMND5A and
RMND5B interacted with members of the UbcH5 family of E3 ubiquitin ligases was
not unusual as almost all E3 ubiquitin ligases are able to interact with at least one of the
family members, including the E3 ubiquitin ligase AO7, which only interacts with
members of this E2 enzyme family (Hakli et al., 2004; Lorick et al., 2006). The UbcH5
E2 conjugating enzyme family contains only the ubiquitin conjugating domain and as
such, members of this family are classified as class I E2 enzymes. UbcH5 members are
able to interact with a multitude of E3 ubiquitin ligases to mediate the transfer of
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
183
ubiquitin to many different substrates, and in most cases the enzymes are not specific
for the target lysine residues on the substrate that are ubiquitinated (Kirkpatrick et al.,
2006; Ye and Rape, 2009). UbcH5 enzymes are associated with monoubiquitination and
are therefore known as chain initiating E2 enzymes, responsible for the initial
monoubiquitination of the substrate but subsequently replaced on the E3 interacting site
by a chain elongating E2 enzyme, which is specific for the type of ubiquitin chain
linkages that are formed (Ye and Rape, 2009). However, members of the UbcH5 family
are also able to form ubiquitin chains through lysine 11, 48 or 63 in vitro and are not
specific for the ubiquitin chain linkages that they form (Windheim et al., 2008; Boname
et al., 2010; Dynek et al., 2010). It is therefore proposed that UbcH5 enzymes are able
to perform polyubiquitination under some conditions but usually perform
monoubiquitination.
In a global yeast two hybrid screen for E2-E3 interaction pairs, RMND5B was
identified to interact with UbcH5 family members but not with UbcH2, consistent with
the results of the present study (van Wijk et al., 2009). Given the function of UbcH5
enzymes, RMND5A and RMND5B may utilise these enzymes for chain initiation and
use other E2 enzymes to extend these chains in elongation reactions. In contrast to
RMND5B, under the assay conditions used for this study, RMND5A was able to
interact with UbcH2 to mediate ubiquitin transfer, suggesting that RMND5 proteins
target different substrates or direct alternative outcomes of their ubiquitinated substrates
due to their interaction with different E2 enzymes. Interestingly the yeast orthologue of
UbcH2, Ubc8 (Gid 3) is reported to be the E2 enzyme associated with the Vid30
complex, and as RMND5A is a proposed member of the CTLH complex, human
orthologue of the Vid30 complex, it is feasible that the CTLH complex similarly utilises
UbcH2 (Kaiser et al., 1994; Santt et al., 2008). UbcH2, a class 3 E2 conjugating
enzyme, is associated with the formation of lysine 11 and 48 linked ubiquitin chains
which target substrate proteins for degradation by the proteasome, indicating that if
RMND5A is able to utilise this E2 enzyme in vivo, its ubiquitinated protein substrates
may be similarly targeted for proteasomal degradation (Santt et al., 2008).
In order to confirm E3 ubiquitin ligase activity of RMND5 proteins, site directed
mutagenesis of the RING domains was performed. As described previously (Section
1.7.2), the RING domain contains eight conserved cysteine or histidine residues that are
required to chelate two zinc ions which hold the RING domain in a cross brace structure
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
184
for the correct functioning of the domain (Deshaies and Joazeiro, 2009). In order to
show that the RING domain is responsible for the E3 ubiquitin ligase activity of a
protein, the RING domain is mutated to reduce or abolish E2 enzyme binding, with
these mutants used in in vitro and in vivo ubiquitination assays. Conventionally, one or
two of the eight zinc coordinating residues are mutated to disrupt domain function,
however not all mutations will disrupt the RING structure, as was shown in this study.
For these experiments, a conserved amino acid (C379S), mutation of which in yeast
RMD5 rendered it unable to ubiquitinate its substrate fructose-1,6-bisphosphatase, was
mutated in the RMND5 proteins, however this mutation did not grossly affect RMND5
protein E3 ubiquitin ligase activity. Despite the lack of major effects on RING domain
function, the mutation may affect the ability of the RING domains to ubiquitinate
specific substrates as was reported for yeast RMD5 (Santt et al., 2008). In agreement
with this, the RMND5A (C356S) mutant appeared to preferentially mediate auto-
monoubiquitination compared to wild-type RMND5A, consistent with a difference in a
specific activity of the mutant. Disruption of a single amino acid would leave 7 other
zinc coordinating residues and potentially other conserved residues in the RING domain
that maintain protein folding. Results of the experiments indicated that the single
mutation permitted E2 enzyme binding but may have distorted the positioning of the
ubiquitin molecules and their attachment to each other upon elongation of the
monoubiquitin chain, thereby rendering the mutant RMND5A (C356S) RING domain
with a preference for auto-monoubiquitination.
As the C356S mutation was not sufficient to disrupt E2 enzyme binding of the
RMND5A RING domain, and a similar result was obtained for RMND5B (C358S), two
amino acid residues were mutated in both RMND5A and RMND5B. For these studies,
the 2 amino acids, C356A/H358A (RMND5A) and C358A/H360A (RMND5B) were
chosen as each binds a separate zinc residue in both RING domains. Therefore
theoretically, mutation of both residues in the RING domains could reduce or negate the
ability to bind two zinc residues, disrupting the structure and E3 ubiquitin ligase activity
of the domain, as has been observed following mutation of the equivalent amino acids
in other RING domain containing proteins in the literature (Zhang et al., 2009). The
residues mutated in RMND5A and RMND5B were also canonical cysteine and histidine
RING domain residues, although not all 8 coordinating residues present in the RING
domains of RMND5 proteins are these conserved amino acids (Section 4.2.1).
Alternatively, if the site of E2 interaction was known, the coordinating amino acids of
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
185
the RING domain important for E2 interaction could be mutated for investigation of E2-
E3 interaction as this strategy would be more likely to inhibit E2-E3 interaction and
disrupt substrate ubiquitination. In this case, the amino acid residues required for
UbcH5B interaction were unknown, however, bioinformatics analysis and comparison
of the common E2 interaction residues in RING domains in different E3 ubiquitin
ligases has subsequently identified that RMND5 proteins both contain conserved E2
interaction residues within their RING domains (not shown) which could be mutated in
further studies to abolish RING domain activity (Deshaies and Joazeiro, 2009). For
example, mutation of a conserved tryptophan residue in the RING domains of CBL
(W408A) and TOPORS (W131A) renders these E3 ubiquitin ligases inactive, and this
residue is hypothesised to be critical for the physical interaction of the E3 with its
cognate E2 (Joazeiro et al., 1999; Rajendra et al., 2004).
In in vitro ubiquitination assays, the RMND5 RING domains were able to auto-
monoubiquitinate, although the physiological relevance of this observation, if any,
remains to be determined. Due to their enzymatic activity, E3 ubiquitin ligases are able
to ubiquitinate their own lysine residues either as a by-product of their activity or as a
means of auto-regulation. For example, MDM2 ubiquitinates itself and additionally
ubiquitinates p53, targeting both proteins for degradation (Fang et al., 2000). Whether
RMND5 proteins are able to auto-ubiquitinate their RING domains in vivo, the type of
ubiquitin chains formed (as the E2 enzyme UbcH5b used in in vitro ubiquitination
assays prefers monoubiquitination) and the outcome of ubiquitination will need to be
addressed in future studies.
Mutation of the RING domain can result in the altered cellular localisation of the E3
ubiquitin ligase, however mutant RMND5A (C356A/H358A) and RMND5B
(C358A/H360A) exhibited a similar nuclear and cytoplasmic localisation compared to
the wild-type protein under the culture conditions used in this study. In contrast,
mutation of the first RING domain in Parkin was reported to result in its mislocalisation
and packaging into aggresomes in which misfolded proteins are localised, but did not
cause the abnormal localisation of wild-type Parkin (Cookson et al., 2003). Similarly,
deletion of the RING domain of Siah1 also resulted in its altered cellular localisation
from a punctuate cytoplasmic distribution to a diffuse cytoplasmic localisation (of the
mutant but not endogenous wild-type Siah1) (Hu and Fearon, 1999). These findings
suggest that the mutant RMND5 proteins are still able to maintain interactions with
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
186
endogenous RMND5 proteins or other cellular binding partners that determine their
cellular distribution, which may or may not be involved in their E3 ubiquitin ligase
activity (e.g. CTLH complex members).
In in vitro ubiquitination assays performed to assess RMND5A (C356A/H368A) mutant
activity, a small reduction in activity was observed, however no differences in the
activity of the wild-type and mutant RMND5A proteins were detected in the in vivo
ubiquitination assays used in this thesis project. Mutation of the RING domains of E3
ubiquitin ligases can result in the stabilisation of their substrates as well as their own
stabilisation as they are no longer able to regulate their own activity by
autoubiquitination or that of their substrate (Hu and Fearon, 1999; Fang et al., 2000).
Therefore, if RMND5 proteins form homo- or hetero-dimers in vivo, mutant RMND5
proteins may interact with wild-type functional RMND5A or RMND5B or other
proteins and increase their stability, resulting in no loss of E3 ubiquitin ligase activity in
vivo, as was observed for the RMND5A (C356S) and (C356A/C358A) mutants.
Although the RMND5A (C356A/H358A) mutant did not exhibit a general reduction in
E3 ubiquitin ligase activity in vivo, its ability to ubiquitinate specific substrates may be
disrupted, as was described previously for yeast RMD5 (Santt et al., 2008).
Additionally, whilst the RMND5A (C356A/H358A) mutation may have disrupted the
RING domain and its interaction with UbcH5b in vitro, the mutation may not have
affected the mutant RMND5A binding to other E2 enzymes in vivo, with several E2
enzymes such as Rad18 and gp78 reported to interact with E2 enzymes outside of the
RING domain (Bailly et al., 1997; Das et al., 2009; Li et al., 2009). Another
explanation for the apparently unaltered association of the RMND5A (C356A/H358A)
mutant with ubiquitinated proteins in vivo is that disruption of the RMND5A RING
domain resulted in the misfolding of RMND5A and its recognition and ubiquitination
by proteins functioning in quality control. Therefore, RMND5A (C356A/H358A) may
seem to be maintaining its E3 ubiquitin ligase activity in vivo whilst the (unidentified)
ubiquitinated proteins detected in the assay may represent increased ubiquitination of
the misfolded RMND5A (C356A/H358A). Alternative methods to abolish the E3
ubiquitin ligase activity of RING domain containing proteins are available, for example,
RING domain deletion mutants are commonly used in the literature for in vivo or in
vitro studies, or for in vitro ubiquitination assays, zinc chelators such as EDTA or
TPEN may be used to inhibit the RING domain activity (Lorick et al., 2006; Gao et al.,
2009).
Chapter 4 Characterisation of RMND5 E3 Ubiquitin Ligase Activity
187
The activity of RMND5B (C358A/H360A) was markedly reduced in in vitro and in vivo
assays, suggesting that mutation of the two amino acids residues resulted in substantial
disruption of RING domain function. Alternatively, in in vivo ubiquitin assays the
structure of RMND5B as a whole may have been altered by mutation of the RING
domain, thereby disrupting other RMND5B protein-protein interactions in addition to
that of the RMND5B RING domain interaction with E2 enzymes. E3 ubiquitin ligase
RING domain mutants have been reported to function in a dominant negative manner,
for example, a RING domain deleted Siah1 mutant without E3 ubiquitin ligase activity
was shown to accumulate in cells due to its inability to auto-ubiquitinate and target
itself for degradation (Hu and Fearon, 1999). Furthermore, the mutant Siah1 protein was
still able to associate with other Siah and Sina proteins and a Siah substrate DCC.
Mutant Siah1 was proposed to prevent substrate ubiquitination and degradation by the
proteasome due to its sequestration of the substrate away from wild-type Siah1, thereby
providing evidence that the mutant Siah1 functions in a dominant negative manner (Hu
and Fearon, 1999). However, mutation of the RING domain may result in the enhanced
degradation of the misfolded protein and in this study it was noted that it was difficult to
immunoprecipitate equal amounts of mutant RMND5 proteins compared to wild-type
proteins which may be due to an increased rate of degradation of the misfolded mutants
(or alternatively a reduction in the production of these mutants).
The results from this chapter have shown that RMND5A and RMND5B are
multidomain proteins with a similar protein domain architecture including a RING
domain, typically present in E3 ubiquitin ligases. These studies have provided evidence
that both RMND5 proteins function as E3 ubiquitin ligases in vitro and in vivo in
LNCaP cells. As RMND5B was originally identified to interact with NKX3.1, the
ability of RMND5A and RMND5B to bind and ubiquitinate NKX3.1 was investigated.
Chapter 5 RMND5 Proteins Ubiquitinate NKX3.1
Chapter 5: RMND5 Proteins Ubiquitinate
NKX3.1
Chapter 5 RMND5 Proteins Ubiquitinate NKX3.1
188
5.1 Introduction
Expression of the prostatic tumour suppressor NKX3.1 is reduced in many high grade
prostate cancers and is undetectable in up to 80% of metastatic prostate tumours
(Section 1.2). Loss of heterozygosity (LOH) at the 8p21.2 chromosomal locus that
includes the NKX3.1 gene is common, however inactivating mutations in the coding
region of the remaining allele are not yet reported (Voeller et al., 1997; Xu et al., 2000;
Ornstein et al., 2001). NKX3.1 gene promoter hypermethylation is also not widely
detected in prostate tumours and studies have reported increased NKX3.1 mRNA levels
and discordance between NKX3.1 mRNA and protein levels in prostate tumours,
suggesting that the NKX3.1 gene is transcribed (Voeller et al., 1997; Ornstein et al.,
2001; Asatiani et al., 2005; Lind et al., 2005; Bethel et al., 2006; Bethel and Bieberich,
2007). As such, the mechanism(s) by which NKX3.1 protein levels are reduced or
undetectable in prostate cancer cells are not well understood but provide evidence for
altered regulation of NKX3.1 at the translational or post-translational level (Bethel et
al., 2006). In addition, it has been documented that NKX3.1 is aberrantly localised in
the cytoplasm in a proportion of prostate tumours, indicating its inability to perform
transcriptional regulatory roles and suggesting that deregulation of proteins involved in
the post-translational regulation of NKX3.1 may contribute to prostate carcinogenesis
(Kim et al., 2002b).
The transcriptional regulation of NKX3.1 by the androgen receptor (AR), retinoic acid
receptor (RAR), ETS1 and ERG/ESE has been characterised, however post-
transcriptional and post-translational regulation of NKX3.1 in the normal prostate and
in prostate tumour cells are less well understood (Section 1.2.3) (He et al., 1997;
Prescott et al., 1998; Kunderfranco et al., 2010; Thomas et al., 2010; Preece et al.,
2011). Phosphorylation and ubiquitination are closely related post-translational
modifications, with many E3 ubiquitin ligases and substrates requiring phosphorylation
prior to activation or ubiquitination, respectively (Yamamoto et al., 2005; Lin et al.,
2006; Suizu et al., 2009). The relationship between post-translational modification and
protein processing is evident in the regulation of NKX3.1, which has been documented
to undergo phosphorylation on a number of serine and threonine residues that affect
protein ubiquitination and stability. NKX3.1 levels are regulated by the protein kinase
casein kinase 2 (CK2), with the CK2α’ catalytic subunit of CK2 specifically
phosphorylating NKX3.1 on threonine residues 89 and 93 (Thr89, Thr93) resulting in
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NKX3.1 protein stabilisation (Figure 5.1) (Li et al., 2006). Reduction of CK2 activity
by siRNA or specific inhibitors decreases NKX3.1 protein levels, which are restored
following proteasome inhibition, thereby demonstrating that CK2-mediated
phosphorylation prevents NKX3.1 degradation by the proteasome (Li et al., 2006). The
mechanisms by which Thr89 and Thr93 phosphorylation in the NKX3.1 acidic domain
affect NKX3.1 stability have been investigated by nuclear magnetic resonance imaging,
identifying that the homeodomain associates with the acidic domain and upon DNA
binding, this association is relieved (Ju et al., 2006; Ju et al., 2009). Phosphorylation of
the acidic domain enhances its interaction with the homeodomain and increased stability
of the interaction is predicted to prevent ubiquitination of NKX3.1, which is
hypothesised to occur in the NKX3.1 homeodomain (Ju et al., 2009). These events
inhibit ubiquitin-mediated degradation of NKX3.1 and therefore NKX3.1 levels are
stabilised.
Conversely, the inflammatory cytokines, tumour necrosis factor α (TNFα) and
interleukin 1β accelerate the degradation of NKX3.1 by phosphorylating NKX3.1 on
serine 196 (Ser196) in the carboxy-terminal, promoting ubiquitination (Figure 5.1)
(Markowski et al., 2008). NKX3.1 is not believed to be ubiquitinated at its carboxy
terminus near Ser196, although NKX3.1 has been proposed to be ubiquitinated on
lysine residues within its homeodomain, which is located close to the carboxy terminus
(Markowski et al., 2008; Ju et al., 2009). The lysine residues of NKX3.1 that are
ubiquitinated have not been investigated, and in addition, the kinase and ubiquitin ligase
responsible for cytokine-mediated degradation of NKX3.1 have not been identified
(Markowski et al., 2008). The finding that inflammatory cytokines are involved in
NKX3.1 degradation is interesting as Nkx3.1 expression is markedly reduced in
bacterial prostatitis and inflammation has been implicated in prostate carcinogenesis,
although the drivers and mediators of these events have not been elucidated (De Marzo
et al., 2007; Khalili et al., 2010). Phosphorylation of NKX3.1 can also occur on the
closely located serine residues Ser185 and Ser195 (Figure 5.1) (Markowski et al.,
2008). Phosphorylation of NKX3.1 at Ser185 promotes its ubiquitination and
degradation under steady state conditions, with Ser185Ala mutants exhibiting increased
stability, while Ser195 phosphorylation enhances both cytokine mediated Ser196 and
steady state Ser185 phosphorylation, leading to NKX3.1 degradation (Markowski et
al., 2008). NKX3.1 phosphorylation on serine 48 (Ser48) was originally investigated
due its proximity to arginine 52 (R52), which is substituted with cysteine (R52C) due to
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the C154T polymorphism in ~11% of healthy men (Gelmann et al., 2002). In prostate
cancer patients, the presence of this polymorphism (R52C) is associated with a ~1.8
fold increased risk of stage C or D disease (or Gleason Score of 7 or greater) (Gelmann
et al., 2002). R52 is important for directing Ser48 phosphorylation by protein kinase C
(PKC) and mutation of either of these residues (R52C or S48A) results in reduced
NKX3.1 phosphorylation. Consistent with previous studies reporting that NKX3.1
phosphorylation facilitates NKX3.1 self-association thereby reducing NKX3.1 DNA
binding (through Ser89 and Ser93), phosphorylation at this site affects NKX3.1 DNA
binding (Gelmann et al., 2002). Both R52C and S48A mutants exhibit enhanced binding
compared to the wild-type NKX3.1 protein on an NKX3.1 consensus DNA sequence
(Gelmann et al., 2002), therefore suggesting that NKX3.1 phosphorylation on multiple
residues may aid in its self-association. Although the R52C polymorphism alters
NKX3.1 phosphorylation and DNA binding it does not affect NKX3.1 coactivation of
SRF transcriptional activity, however the transcriptional activity of other NKX3.1
cofactors may be affected. Supporting this, a more recent study has found that men who
were either heterozygous or homozygous for the C form of the R52C polymorphism
were at 1.6x risk for enlarged prostate size with a high proportion also developing
benign prostatic hyperplasia (Rodriguez Ortner et al., 2006).
Figure 5.1: Post-translational modification of NKX3.1. NKX3.1 is reported to be phosphorylated on serine 48 (Ser48) (Gelmann et al., 2002), threonine residues 89 and 93 (Thr89, Thr 93) (Li et al., 2006), and serine residues 185, 195 and 196 (Ser185, Ser195, Ser196) (Markowski et al., 2008) and to undergo ubiquitination on lysine residues within the homeodomain (Ju et al., 2009).
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Although its ubiquitination has been incompletely characterised, NKX3.1 is
ubiquitinated by the E3 ubiquitin ligase TOPORS both in vitro and in vivo, resulting in
its proteasome dependent degradation in vivo (Guan et al., 2008). Supporting these
findings, siRNA mediated knockdown of TOPORS resulted in NKX3.1 protein
accumulation and its prolonged half-life (Guan et al., 2008). Using NKX3.1 deletion
proteins, it was determined that the homeodomain was required for the interaction
between NKX3.1 and TOPORS, and although the amino-terminus of NKX3.1 was
sufficient for binding to TOPORS, the interaction was weaker than that of proteins
containing either the homeodomain and the amino-terminus, or the homeodomain and
the carboxy-terminus (Guan et al., 2008). Consistent with previous reports that the
homeodomain was a possible site of NKX3.1 ubiquitination, the study also
demonstrated that the homeodomain alone was ubiquitinated by TOPORS, although to a
lesser degree than other proteins containing the homeodomain and additional protein
sequences. These results supported a hypothesis that the homeodomain is likely to be
the site of ubiquitination, with other residues outside of the homeodomain increasing the
efficiency of NKX3.1 interaction with TOPORS (Guan et al., 2008). NKX3.1 contains
fourteen lysine residues which are potential ubiquitination sites with nine of these
located in the homeodomain, although the specific lysine residues in the homeodomain
that are ubiquitinated by TOPORS or by other E3 ubiquitin ligases, and the
ubiquitination of other lysine residues in the NKX3.1 protein remain uncharacterised.
Interestingly, both TOPORS and NKX3.1 interact with the DNA Helicase
Topoisomerase I (TOPO I). TOPORS was originally identified as a TOPO I binding
partner and acts as a SUMO-1 E3 ligase that regulates TOPO I and chromatin binding
protein activity (Hammer et al., 2007; Pungaliya et al., 2007). TOPORS was
hypothesised to interact with TOPO I, recruiting it to RNA polymerase II transcriptional
complexes, whilst NKX3.1 was found to enhance TOPO I DNA helicase activity
(Haluska et al., 1999; Bowen et al., 2007). Additionally, upon exposure to DNA
damaging agents, NKX3.1 and TOPO I comigrate within the nucleus (Bowen et al.,
2007). These studies implicate both NKX3.1 and TOPORS in the regulation of TOPO I
activity, and therefore suggest their ability to regulate DNA replication, gene expression
and DNA repair.
RMND5B was originally identified in our laboratory as an NKX3.1 binding partner in a
yeast two-hybrid screen. In order to determine the possible outcome(s) of the interaction
between RMND5B and NKX3.1, the function of RMND5B and its homologue
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RMND5A was investigated in this thesis. Both RMND5 proteins were determined to
function as E3 ubiquitin ligases (Chapter 4), leading to the hypothesis that NKX3.1 may
be a substrate of RMND5 mediated ubiquitination. Therefore, the ubiquitination of
NKX3.1 by RMND5A and RMND5B and the outcome of NKX3.1 ubiquitination was
investigated in prostate cancer cells.
Chapter 5 RMND5 Proteins Ubiquitinate NKX3.1
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5.2 Results
5.2.1 RMND5 Proteins Interact with NKX3.1 in LNCaP Prostate Cancer Cells
5.2.1.1 RMND5A Interacts with NKX3.1
RMND5B was initially identified in our laboratory due to its interaction with the
prostatic tumour suppressor NKX3.1 (Dawson, 2006), however the interaction between
RMND5A and NKX3.1 had not been determined. To investigate NKX3.1 interaction
with RMND5A, LNCaP cells growing in 10cm petri dishes were cotransfected with
plasmids encoding GFP-RMND5A and NKX3.1-V5 (Section 3.1.4). At 48 hours post-
transfection, the cells were lysed, an aliquot (50µL) of the cell lysate was taken, and the
remaining lysate was subjected to either GFP or V5 immunoprecipitation (Section
3.13). The immunoprecipitation reaction products were electrophoresed in 12%
polyacrylamide gels and western blotting was performed for GFP or V5 (Section 3.15).
For GFP-RMND5A immunoprecipitations, GFP-RMND5A was present at the expected
size of ~70kDa in the immunoprecipitate, but not in the input lysate indicating that the
expression levels of GFP-RMND5A were below the sensitivity of detection of the
antibody in this dilute fraction. GFP-RMND5A was not detected in the mock
immunoprecipitation control, indicating successful immunoprecipitation (Figure 5.2A).
Western blotting of the lysates for NKX3.1-V5 identified a ~35kDa protein band
corresponding to NKX3.1-V5 in both the input lysate and immunoprecipitate, with no
bands in the mock immunoprecipitated control, indicating that RMND5A and NKX3.1
interact in LNCaP cells. When NKX3.1-V5 was immunoprecipitated, western blotting
for NKX3.1-V5 identified a band of ~35kDa corresponding to NKX3.1-V5 in the input
lysate and immunoprecipitate, whilst western blotting for GFP-RMND5A resulted in a
band of ~70kDa in the input lysate and immunoprecipitated fractions (Figure 5.2A). No
bands were detected in the untransfected or mock immunoprecipitated controls and
together these results indicated that RMND5A and NKX3.1 interact in LNCaP cells.
5.2.1.2 RMND5B Interacts with NKX3.1
To confirm the interaction between RMND5B and NKX3.1, LNCaP cells growing in
10cm petri dishes were cotransfected with plasmids encoding GFP-RMND5B and
NKX3.1-V5, lysed at 48 hours post-transfection, an aliquot of the cell lysate taken, and
the remaining lysate immunoprecipitated using GFP antibodies (Sections 3.1.4, 3.13).
Immunoprecipitated proteins were electrophoresed in 12% polyacrylamide gels and
western blotting was performed for GFP-RMND5B and NKX3.1-V5 (Section 3.15). A
Chapter 5 RMND5 Proteins Ubiquitinate NKX3.1
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Figure 5.2: NKX3.1 interacts with RMND5A and RMND5B in prostate cancer cells. (A) LNCaP cells were cotransfected with plasmids encoding GFP-RMND5A and NKX3.1-V5. At 48 hours post-transfection, (A) (i) GFP-RMND5A was immunoprecipitated from the cells using GFP antibodies or (ii) NKX3.1-V5 was immunoprecipitated using V5 antibodies. Western blotting for GFP and V5 was performed, identifying co-immunoprecipitation of GFP-RMND5A and NKX3.1-V5. (B) LNCaP cells were cotransfected with plasmids encoding GFP-RMND5B and NKX3.1-V5 and 48 hours post-transfection, GFP-RMND5B was immunoprecipitated using anti-GFP antibodies. Western blotting for GFP-RMND5B and NKX3.1-V5 identified both proteins in the immunoprecipitated samples. Each experiment was performed twice and representative results are shown.
Chapter 5 RMND5 Proteins Ubiquitinate NKX3.1
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band corresponding to GFP-RMND5B was present in the immunoprecipitate,
identifying successful GFP-RMND5B immunoprecipitation (Figure 5.2B). GFP-
RMND5B bands were not detected in the input lysate, indicating that GFP-RMND5B
levels were below the sensitivity of the antibody in this (dilute) fraction, and similarly
no bands were detected in the untransfected and mock immunoprecipitated control
reactions (Figure 5.2B). Western blotting identified a ~35kDa band corresponding in
size to NKX3.1-V5 in the immunoprecipitated and input lysate samples (Figure 5.2B).
These results confirmed the interaction between RMND5B and NKX3.1 and with
results described in Section 5.2.1.1 indicated that both RMND5A and RMND5B are
able to interact with NKX3.1 in LNCaP cells.
5.2.2 RMND5 Proteins Colocalise with NKX3.1 in LNCaP Cells
The localisation and colocalisation of NKX3.1 and RMND5 proteins in LNCaP cells
were investigated using fluorescence microscopy. For these studies, LNCaP cells
growing on coverslips were cotransfected with plasmids encoding NKX3.1-V5 and
either GFP-RMND5A or GFP-RMND5B, then cultured for 48 hours post-transfection
prior to V5 immunostaining of the cells (Section 3.1.3, 3.1.4, 3.16). When coexpressed
with NKX3.1-V5, both GFP-RMND5A and GFP-RMND5B exhibited a predominantly
nuclear localisation, with some diffuse cytoplasmic staining (Figure 5.3). In contrast, in
the absence of NKX3.1-V5 overexpression, GFP-RMND5A and GFP-RMND5B
exhibited a diffuse nuclear and cytoplasmic distribution (Section 4.2.6.4, 4.2.7.5), whilst
in cells expressing NKX3.1-V5 alone, NKX3.1 displayed a predominantly nuclear
localisation (Figure 5.3). In cells coexpressing NKX3.1-V5 and GFP-RMND5A or
GFP-RMND5B, NKX3.1-V5 exhibited a mainly cytoplasmic localisation which also
contrasted previous reports and findings in our laboratory, including those observed in
this study (in the absence of RMND5 protein overexpression) where NKX3.1 and
NKX3.1-V5 were predominantly nuclear (Asatiani et al., 2005, Dawson, 2006). In these
experiments, colocalisation of NKX3.1 with GFP-RMND5A or GFP-RMND5B was
evident predominantly in the cytoplasm of LNCaP cells (Figure 5.3). To investigate
whether the reduced nuclear localisation of NKX3.1-V5 in RMND5A/RMND5B
overexpressing cells resulted from its increased ubiquitin-mediated degradation by the
proteasome, these experiments were repeated using LNCaP cells treated with 10µM
MG132, a proteasome inhibitor (Section 5.2.3) for the final 3 hours of culture prior to
preparation of the cells for viewing by microscopy at 48 hours post-transfection
(Section 3.1.3, 3.1.4, 3.15). Under these conditions, NKX3.1-V5 exhibited a similar
Chapter 5 RMND5 Proteins Ubiquitinate NKX3.1
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cytoplasmic localisation in cells co-expressing GFP-RMND5A/GFP-RMND5B, which
was predominantly nuclear (not shown). Under these culture conditions, GFP-
RMND5A/GFP-RMND5B and NKX3.1-V5 colocalisation occurred mainly in the
cytoplasm of LNCaP cells (not shown). The lack of MG132 effects on NKX3.1
localisation may be due in part to the short MG132 treatment period, however, longer
MG132 treatment of LNCaP cells growing on coverslips resulted in cytotoxic effects
due to proteasome inhibition (Section 5.2.3). Fluorescence microscopy to determine the
localisation and colocalisation of GFP-RMND5A (C356A/H358A) or GFP-RMND5B
(C358A/H360A) and NKX3.1-V5 determined that similar to wild-type GFP-RMND5
proteins, GFP-RMND5A (C356A/H358A) and GFP-RMND5B (C358A/H360A)
exhibited diffuse nuclear and cytoplasmic distribution, with GFP-RMND5B
(C358A/H360A) also exhibiting a punctate cytoplasmic appearance when expressed
alone (Section 3.1.3, 3.1.4, 3.15, 4.2.7.5). Coexpression of GFP-RMND5A
(C356A/H358A) or GFP-RMND5B (C358A/H360A) with NKX3.1-V5 identified that
NKX3.1-V5 exhibited a mainly cytoplasmic distribution whilst GFP-RMND5A
(C356A/H358A) and GFP-RMND5B (C358A/H360A) displayed a predominantly
nuclear cellular localisation with some cytoplasmic staining (not shown). GFP-
RMND5A (C356A/H358A) and GFP-RMND5B (C358A/H360A) colocalised with
NKX3.1-V5 predominantly in the cytoplasm of LNCaP cells (not shown), in a similar
manner to the colocalisation of wild-type GFP-RMND5A or GFP-RMND5B with
NKX3.1-V5. Furthermore, in cells coexpressing GFP-RMND5A (C356A/H358A) or
GFP-RMND5B (C358A/H360A) and NKX3.1-V5, 3 hours of treatment with 10µM
MG132 did not markedly alter the intracellular distribution or colocalisation of the
exogenously expressed proteins (not shown). These experiments may be further
optimised in future studies to allow for more extensive investigation of the effects of
RMND5 protein overexpression on the intracellular localisation of NKX3.1, including
the analysis of the fluorescence intensity of NKX3.1-V5 following proteasome
inhibition which may indicate its accumulation in the cell.
5.2.3 Regulation of NKX3.1 Expression in Prostate Cancer Cells
In order to determine the effects of RMND5 proteins on NKX3.1 expression, NKX3.1
protein half-life was initially investigated by determination of NKX3.1 levels following
treatment of cultures with 10µg/mL cycloheximide for 0 – 240 minutes (Section 3.1.5).
Chapter 5 RMND5 Proteins Ubiquitinate NKX3.1
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Figure 5.3: RMND5 proteins colocalise with NKX3.1 in LNCaP cells. To investigate the localisation and colocalisation of RMND5 proteins and NKX3.1, LNCaP cells growing on coverslips were transfected with plasmids encoding (A) NKX3.1-V5, (B) GFP-RMND5A and NKX3.1-V5, (C) GFP-RMND5B and NKX3.1-V5 or (D) untransfected. At 48 hours post-transfection, cells were fixed, permeabilised and stained for NKX3.1-V5 using an anti-V5 primary antibody and secondary anti-goat AlexaFluor® 546 antibody. Coverslips were viewed by fluorescence microscopy, identifying that GFP-RMND5A and GFP-RMND5B displayed a predominantly nuclear localisation with some diffuse cytoplasmic staining and that NKX3.1-V5 exhibited a predominantly nuclear localisation when expressed alone whilst when coexpressed with either GFP-RMND5A or GFP-RMND5B in the cell, NKX3.1-V5 displayed a predominantly cytoplasmic cellular localisation. Colocalisation of NKX3.1-V5 with GFP-RMND5A or GFP-RMND5B was predominantly detected in the cytoplasm of LNCaP cells (Magnification x1000). The experiment was performed three times and representative results are shown.
Overlay
NKX3.1-V5 GFP-RMND5A Hœchst 33258 Overlay
NKX3.1-V5 GFP-RMND5B Hœchst 33258 Overlay
B
C
NKX3.1-V5 488 channel Hœchst 33258 Overlay A
Hœchst 33258 No 1° Ab D Overlay
Chapter 5 RMND5 Proteins Ubiquitinate NKX3.1
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Under these culture conditions (RPMI/PS/10% FCS), the half-life of NKX3.1 was
estimated to be ~15.8 minutes, which is consistent with a previous report where the
NKX3.1 protein half-life was determined to be ~25 minutes (Section 3.1.5, 3.15, Figure
5.4) (Thomas et al., 2006).
Regulation of NKX3.1 levels by the proteasome has been reported previously (Guan et
al., 2006, Li et al., 2006), and to establish experimental conditions for these studies,
degradation of NKX3.1 by the proteasome and lysosome were examined. To
demonstrate NKX3.1 degradation by the proteasome, NKX3.1 levels were examined
following treatment of LNCaP cells with the proteasome inhibitors lactacystin and
MG132. For these experiments, 10µM lactacystin was added to cultures growing in
RPMI/PS/10%FCS for 8 hours prior to lysis, followed by NKX3.1 and β-actin western
blotting (Section 3.1.5, 3.15). Under these culture conditions, β-actin levels remained
relatively constant during the initial 6 hours of lactacystin treatment while NKX3.1
protein levels were not markedly altered until 6 hours of exposure to lactacystin (Figure
5.5). By 8 hours, both β-actin and NKX3.1 levels were decreasing and, due to the light
microscopic appearance of the cultures (not shown), it was likely that these results were
in part due to the cytotoxicity of the lactacystin.
A second proteasome inhibitor, MG132 was also utilised to investigate NKX3.1 protein
degradation by the proteasome and for these experiments LNCaP cells growing in 6
well plates in RPMI/PS/10%FCS were treated with 10µM MG132 for either 8 or 24
hours prior to western blotting for NKX3.1 and β-actin (Section 3.1.5, 3.15). In initial
experiments, NKX3.1 protein levels were similar in treated and untreated cultures (data
not shown), therefore since NKX3.1 expression is androgen regulated (He et al., 1997;
Prescott et al., 1998), NKX3.1 protein levels were monitored during androgen treatment
of LNCaP cells, which were grown in RPMI/PS/5%CSS for 24 hours to deplete the
cultures of androgens, then treated with 10-8M 5α-dihydrotestosterone (DHT) in
conjunction with 10µM MG132 for 8 or 24 hours (Section 3.1.5, 3.15). NKX3.1 protein
levels were low in cultures depleted of androgens (RPMI/PS/5%CSS) and in the
presence of androgens, NKX3.1 levels were markedly increased (not shown). As such,
accumulation of NKX3.1 protein levels due to proteasome inhibition were concealed by
the strong induction of NKX3.1 expression by DHT (not shown). Therefore, NKX3.1
protein levels were examined during androgen depletion of LNCaP cells cultured in
RPMI/PS/5%CSS and 10µM MG132 for 8 or 24 hours (Section 3.1.5, 3.15). Under
Chapter 5 RMND5 Proteins Ubiquitinate NKX3.1
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β-actin
NKX3.1
15 30 60 120 240
Cycloheximide
00.20.40.60.8
11.2
0 15 30 60 120 240
Norm
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dN
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3.1
Pro
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Lev
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Cycloheximide Treatment (minutes)
(minutes)
A
B
0
Figure 5.4: Determination of NKX3.1 half-life. The half-life of NKX3.1 in LNCaP cells was estimated by treatment of cultures with 10µg/mL cycloheximide for 0-240 minutes to inhibit new protein production. (A) Cells were lysed at the indicated time points and the lysates electrophoresed in 12% polyacrylamide gels for NKX3.1 and β-actin western blotting. (B) Quantitation of normalised NKX3.1 protein levels indicated that the half-life of NKX3.1 was ~15.8 minutes under these culture conditions. The experiment was performed twice and representative results are shown.
Chapter 5 RMND5 Proteins Ubiquitinate NKX3.1
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(hours)NKX3.1
β-actin
~30kDa
~44kDa
2 4 6 8
0
0.5
1
1.5
Vehicle 2 4 6 8
Nor
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NK
X3.
1 P
rote
in
Leve
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10µM Lactacystin (hours)
Lactacystin
β-actin
NKX3.1
8 24 8 24
Control MG132
0
1
2
3
4
5
Vehicle 10µM MG132
Nor
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8 24
~30kDa
~44kDa
(hours)
hours
A
B
0
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Control
Figure 5.5: Degradation of NKX3.1 by the proteasome. LNCaP cells were treated with 10µM of the proteasome inhibitors, lactacystin or MG132. Following treatment, cells were harvested, lysates were electrophoresed in 12% polyacrylamide gels and western blotting was performed for endogenous NKX3.1 and β-actin. (A) When cells were cultured in RPMI/PS/10% FCS, NKX3.1 protein levels were not altered during 2-4 hours of lactacystin treatment, but were reduced to ~40% of controls by 8 hours. (B) When LNCaP cells were cultured in RPMI/PS/5%CSS for 8 hours, NKX3.1 protein levels were initially low due to androgen deprivation, but were increased following 8 hours of culture in RPMI/PS/5%CSS with MG132 treatment. By 24 hours of culture with MG132, NKX3.1 levels were decreased, potentially due to nonspecific toxic effects of MG132. Experiments were performed twice and representative results are shown.
Chapter 5 RMND5 Proteins Ubiquitinate NKX3.1
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these culture conditions, NKX3.1 remained at low levels in vehicle treated cells (as
expected due to androgen depletion) however, following 8 hours of MG132 treatment,
NKX3.1 protein levels were markedly increased (Figure 5.5). This result is therefore
consistent with findings in the literature that NKX3.1 undergoes proteasomal
degradation. By 24 hours of MG132 treatment, both NKX3.1 and β-actin levels were
reduced and, similar to cultures treated with lactacystin for longer time periods, there
was evidence of cytotoxicity owing to long term proteasome inhibition. For these
reasons, LNCaP cells were treated with lactacystin and MG132 for only short periods of
time.
To assess whether NKX3.1 may also undergo degradation by the lysosome, NKX3.1
protein levels were monitored following treatment of cells with two lysosome
inhibitors, NH4Cl or choloroquine. For these experiments, LNCaP cells growing in 6
well plates were treated with 10mM or 20mM NH4Cl for either 6 or 24 hours prior to
western blotting for NKX3.1 and β-actin (Section 3.1.5, 3.15). Under these culture
conditions, NKX3.1 levels were reduced to similar levels at 6 and 24 hours of treatment
with 10mM or 20mM NH4Cl, results that were in part due to nonspecific cytotoxic
effects of NH4Cl on LNCaP cells (Figure 5.6). Preliminary testing of chloroquine
indicated an optimum concentration of 25µM, with 50µM or 100µM chloroquine
treatments resulting in marked cytotoxicity and large reductions in NKX3.1 protein
levels (Section 3.1.5, 3.15). For these experiments, NKX3.1 protein levels in LNCaP
cells treated with 25µM chloroquine for up to 48 hours were found to increase ~1.5 fold
following 6 hours of chloroquine treatment (Figure 5.6). The increased NKX3.1 levels
were maintained until 48 hours of treatment, providing evidence that NKX3.1
degradation may also be regulated in part by the lysosome (Figure 5.6). NKX3.1 protein
levels were similarly monitored in cultures that had been androgen depleted in
RPMI/PS/5%CSS for 24 hours prior to the addition of 10-8M DHT and 10mM or 20mM
NH4Cl, or 25μM chloroquine for 6 or 24 hours (Section 3.1.5, 3.15). However similar to
proteasome inhibition, changes in NKX3.1 protein levels due to lysosome inhibition
were not evident due to the rapid accumulation of NKX3.1 protein after DHT treatment
(not shown). NKX3.1 levels were then monitored during androgen withdrawal by
culture of LNCaP cells in RPMI/PS/5%CSS with lysosome inhibition using 10mM or
20mM NH4Cl, or 25μM chloroquine for 6 or 24 hours (Section 3.1.5, 3.15). In these
experiments, low levels of NKX3.1 were evident in vehicle treated cells (as expected
Chapter 5 RMND5 Proteins Ubiquitinate NKX3.1
202
(Hours)
Control10mM NH4Cl
20mM NH4Cl
6 6 624 24 24
NKX3.1
β-actin
~30kDa
~44kDa
β-actin
4 6 8 24 482
NKX3.1~30kDa
~44kDa
Chloroquine
00.40.81.21.6
2
Nor
mal
ised
NK
X3.
1 P
rote
in L
evel
s
25µM Chloroquine (hours)
0
0.5
1
1.5
Control 10mM NH4Cl20mM NH4Cl
Nor
mal
ised
N
KX
3.1
Pro
tein
Le
vels
6 hours 24 hours
(hours)
A
B
10mM NH4Cl
20mM NH4Cl
0
Figure 5.6: Lysosomal processing of NKX3.1. To determine whether NKX3.1 was degraded by the lysosome, endogenous NKX3.1 levels were monitored following treatment of LNCaP cells with (A) 10mM or 20mM NH4Cl for 6 or 24 hours, or (B) 25µM chloroquine for 2-48 hours. Following treatment, cells were harvested and lysates electrophoresed in 12% polyacrylamide gels then analysed by western blotting for NKX3.1 and the housekeeping protein β-actin. NKX3.1 levels were reduced at 6 and 24 hours of NH4Cl treatment, whilst cells treated with chloroquine showed a ~1.5-fold increase in NKX3.1 protein levels at 6 hours which was maintained until 48 hours of treatment. Experiments were performed twice and representative results are shown.
Chapter 5 RMND5 Proteins Ubiquitinate NKX3.1
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due to androgen depletion) and upon lysosome inhibition NKX3.1 protein levels
remained at low levels compared to controls at both time points, providing no additional
evidence for lysosomal degradation of NKX3.1 (not shown). The contribution of the
lysosome in the regulation of NKX3.1 levels was not investigated further for this thesis
and subsequent experiments examined the role of RMND5 proteins in proteasome-
mediated NKX3.1 degradation. However, lysosomal degradation of NKX3.1 may be
investigated in future studies to determine whether it plays a direct or indirect role in the
control of NKX3.1 expression.
5.2.4 RMND5 Protein Effects on NKX3.1 Protein Expression
5.2.4.1 RMND5A and RMND5B Reduce NKX3.1 Protein Levels
To determine whether NKX3.1 was a substrate of RMND5 protein ubiquitination,
NKX3.1 protein levels in LNCaP cells were monitored following overexpression of
either RMND5A or RMND5B. For these studies, LNCaP cells growing in 6 well plates
were transfected with increasing concentrations (0-4µg) of plasmids encoding GFP-
RMND5A or GFP-RMND5B, cultures were lysed at 48 hours post-transfection and
analysed by western blotting for GFP and endogenous NKX3.1 (Section 3.1.4, 3.15).
GFP western blotting confirmed increasing expression of GFP-RMND5A or GFP-
RMND5B in cultures transfected with increasing amounts of the expression plasmids
(Figure 5.7). While western blotting for NKX3.1 identified a band at the expected size
of ~30kDa in lysates from GFP-RMND5A and GFP-RMND5B overexpressing cells,
NKX3.1 levels were reduced as the concentration of GFP-RMND5A or GFP-RMND5B
increased. These results indicated that overexpression of either RMND5A or RMND5B
downregulated NKX3.1 levels (Figure 5.7).
To investigate whether the reduction in NXK3.1 levels following RMND5
overexpression was due to its proteasomal degradation, LNCaP cells transfected with
plasmids encoding GFP-RMND5A or GFP-RMND5B were treated with 10µM MG132
for the final 6 hours prior to harvesting of cells at 48 hours post-transfection and
western blotting for GFP and endogenous NKX3.1 (Section 3.1.4, 3.1.5, 3.15). NKX3.1
levels were reduced following either GFP-RMND5A or GFP-RMND5B
overexpression, with these effects partially reversed when GFP-RMND5A or GFP-
RMND5B overexpressing cells were treated with MG132 (Figure 5.8). GFP-RMND5A
and GFP-RMND5B were also increased in MG132 treated cultures, indicating that they
Chapter 5 RMND5 Proteins Ubiquitinate NKX3.1
204
Figure 5.7: Overexpression of RMND5A or RMND5B reduces NKX3.1 levels. LNCaP prostate cancer cells were transfected with increasing concentrations of plasmids encoding either (A) GFP-RMND5A or (B) GFP-RMND5B and at 48 hours post-transfection, cells were harvested for western blotting. GFP western blotting confirmed increasing expression of GFP-RMND5 proteins, whilst NKX3.1 protein levels were reduced as GFP-RMND5A and GFP-RMND5B expression increased.
Chapter 5 RMND5 Proteins Ubiquitinate NKX3.1
205
- + - + - +Control
pEGFP-RMND5A
pEGFP-RMND5B
10µM MG132
NKX3.1
GFP
β-actin
0
0.5
1
1.5
2
2.5
Vehicle RMND5A RMND5B
Norm
alise
dNK
X3.1
Pr
otei
n Le
vels
Ethanol MG132
~30kDa
~44kDa
~74kDa
NKX3.1
GFP
β-actin
- + - + - +Control
pEGFP-RMND5A
pEGFP-RMND5B
10µM MG132~30kDa
~44kDa
~74kDa
0
0.4
0.8
1.2
1.6
Vehicle RMND5A RMND5B
Norm
alise
dNK
X3.1
Pr
otei
n Le
vels Ethanol MG132
A
B
Control
Control
Vehicle
Vehicle
Figure 5.8: Proteasome inhibition restores NKX3.1 protein levels following RMND5 overexpression. LNCaP cells were transfected with plasmids encoding GFP-RMND5A or GFP-RMND5B and at 42 hours post-transfection the medium was replaced with (A) RPMI/PS/10%FCS or (B) RPMI/PS/5%CSS and the culture treated with 10µM MG132 as indicated. Cells were harvested at 48 hours post-transfection and analysed by western blotting for NKX3.1, GFP-RMND5A or GFP-RMND5B. Under both conditions, NKX3.1 protein levels were reduced following RMND5 overexpression and NKX3.1 protein levels were increased following MG132 treatment, as were the protein levels of GFP-RMND5A and GFP-RMND5B. Experiments were performed twice and representative results are shown.
Chapter 5 RMND5 Proteins Ubiquitinate NKX3.1
206
too are regulated by proteasomal degradation (Figure 5.8). As NKX3.1 expression is
regulated by androgens, the above experiment was repeated and the cells were grown in
medium depleted of androgens (RPMI/PS/5%CSS) for the final 6 hours of culture,
during which time they were treated with MG132 (Section 3.1.4, 3.1.5, 3.15). Western
blotting of the lysates for NKX3.1 determined that upon androgen depletion, NKX3.1
protein levels were low, consistent with previous experiments (Section 5.2.3, Figure
5.8). Under these conditions, the reductions in NKX3.1 protein levels following GFP-
RMND5A or GFP-RMND5B overexpression were diminished or not evident, however
the accumulation of NKX3.1 in MG132-treated GFP-RMND5A and GFP-RMND5B
overexpressing or control cultures was enhanced (Figure 5.8). Together, these results
provided evidence that RMND5 proteins were involved in the targeting of NKX3.1 for
degradation by the proteasome, and that RMND5 proteins are themselves degraded by
the proteasome.
5.2.4.2 RMND5A (C356S and C356A/H358A) and RMND5B (C358S and
C358A/H360A) Reduce NKX3.1 Protein Levels
To determine the effects of mutant RMND5A (C356S and C356A/H358A) and
RMND5B (C358S and C358A/H360A) on NKX3.1 expression, NKX3.1 protein levels
were monitored following transfection of LNCaP cells with 4µg pEGFP-RMND5A,
pEGFP-RMND5A (C356S), pEGFP-RMND5A (C356A/H358A), pEGFP-RMND5B,
pEGFP-RMND5B (C358S) or pEGFP-RMND5B (C358A/H360A). Cells were lysed at
48 hours post-transfection and GFP-RMND5 protein and endogenous NKX3.1 levels
were determined by western blotting (Section 3.1.5, 3.15). In these experiments, GFP
western blotting resulted in the identification of ~70kDa bands in lysates from all
cultures transfected with GFP-RMND5 proteins, confirming expression of both wild-
type and mutant GFP-RMND5A and GFP-RMND5B (Figure 5.9). NKX3.1 was
expressed in all cultures, with NKX3.1 protein levels reduced following wild-type
RMND5 protein overexpression, consistent with previous findings (Figure 5.9).
Surprisingly, the reduction in NKX3.1 protein levels was greater in lysates expressing
mutant GFP-RMND5A (C356S or C356A/H358A) or GFP-RMND5B (C358S or
C358A/H360A). Although the western blotting technique is only semi-quantitative and
these results were not evaluated statistically, NKX3.1 levels in cultures overexpressing
mutant RMND5 proteins were consistently lower than NKX3.1 levels in cultures
overexpressing wild-type RMND5 proteins (and control cultures). Therefore, while
Chapter 5 RMND5 Proteins Ubiquitinate NKX3.1
207
NKX3.1
GFP
β-actin
pEGFP-RMND5A
pEGFP-RMND5B
WT WT
~30kDa
~70kDa
~44kDa
0
0.2
0.4
0.6
0.8
1
1.2
Untransfected RMND5A RMND5B
Nor
mal
ised
NKX
3.1
Prot
ein
Leve
ls WT
(C356S)/(C358S)
(C356A/H358A)/(C358A/H360A)
Figure 5.9: Overexpression of wild-type and mutant RMND5 proteins reduces NKX3.1 levels. LNCaP cells were transfected with plasmids encoding GFP-RMND5A, GFP-RMND5B, GFP-RMND5A (C356S), GFP-RMND5B (C358S), GFP-RMND5A (C356A/H358A) or GFP-RMND5B (C358A/H360A) and at 48 hours post-transfection cells were harvested and analysed by western blotting for NKX3.1, GFP and β-actin. Endogenous NKX3.1 protein levels were reduced by >30% following overexpression of wild-type or mutant GFP-RMND5 proteins. Experiment was performed twice and representative results are shown.
Chapter 5 RMND5 Proteins Ubiquitinate NKX3.1
208
overexpression of RMND5 proteins reduced NKX3.1 protein levels, mutant RMND5
proteins resulted in greater decreases in NKX3.1 protein levels.
5.2.5 NKX3.1 is Ubiquitinated in LNCaP Cells
5.2.5.1 RMND5 Proteins Ubiquitinate NKX3.1
To determine whether RMND5A and RMND5B ubiquitinate NKX3.1, targeting it for
degradation by the proteasome, in vivo ubiquitination assays were carried out. For these
experiments, LNCaP cells were cotransfected with plasmids encoding NKX3.1-V5,
HA-ubiquitin and either GFP-RMND5A or GFP-RMND5B, and 48 hours post-
transfection, cells were lysed, input lysate samples were collected and NKX3.1-V5 was
immunoprecipitated with anti-V5 antibodies (Section 3.1.4, 3.13). Immunoprecipitation
products were electrophoresed in 4-12% gradient polyacrylamide gels and analysed by
HA (ubiquitin), V5 (NKX3.1) and GFP (RMND5A/RMND5B) western blotting
(Section 3.15). Western blotting of the input lysate samples for NKX3.1-V5 produced
bands at the expected size of ~35kDa in all lysates, and GFP western blotting identified
GFP-RMND5A and GFP-RMND5B in cultures transfected with the respective plasmids
(with the GFP-RMND5B protein band exhibiting low intensity due to dilution in the
total cell lysate) (Figure 5.10 (i)). In V5-immunoprecipitated samples, the presence of
~35kDa bands following V5 western blotting indicated successful immunoprecipitation
of V5-tagged NKX3.1. In the absence of GFP-RMND5A or GFP-RMND5B
overexpression, HA-ubiquitin western blotting detected multiple bands ranging in size
from ~35kDa – 175kDa, consistent with the presence of ubiquitinated NKX3.1 in the
V5 immunoprecipitation samples, however few bands corresponding to ubiquitinated
NKX3.1-V5 were detected when GFP-RMND5A or GFP-RMND5A was overexpressed
(Figure 5.10 (i)). To verify that this was not due to failure of transfections with the HA-
Ubiquitin expression plasmid, HA western blotting was performed on the input lysates
from these experiments, demonstrating that all transfections were successful (Section
3.15, Figure 5.10 (i)). These results suggested that although NKX3.1 is ubiquitinated in
vivo, overexpression of RMND5 proteins resulted in their rapid degradation, markedly
reducing detection of ubiquitinated NKX3.1 in the cultures.
To investigate this hypothesis, the experiments were repeated and cells were treated
with 10µM MG132 for the final 3 hours prior to harvest to allow the accumulation of
ubiquitinated proteins (Section 3.1.4, 3.1.5, 3.13). In these experiments, V5-
Chapter 5 RMND5 Proteins Ubiquitinate NKX3.1
209
Figure 5.10: Ubiquitination of NKX3.1 in vivo by RMND5A and RMND5B. (i) LNCaP cells were cotransfected with plasmids encoding NKX3.1-V5, HA-Ubiquitin and GFP-RMND5A or GFP-RMND5B, and at 48 hours post-transfection NKX3.1-V5 was immunoprecipitated using anti-V5 antibodies. Western blotting for HA-ubiquitin demonstrated that NKX3.1 was ubiquitinated in vivo and that there was a reduction in NKX3.1-associated ubiquitination following RMND5A or RMND5B overexpression that was potentially due to its rapid degradation in the proteasome. (ii) The experiment was repeated with cells treated with the proteasome inhibitor 10µM MG132 for the final 3 hours of culture prior to immunoprecipitation of NKX3.1-V5 with anti-V5 antibodies at 48 hours post-transfection. Western blotting for HA-ubiquitin indicated an increase in NKX3.1-associated ubiquitination following RMND5A and RMND5B overexpression. Experiments were performed twice and representative results are shown.
Input lysate (2%)
+ + + - + + + +
- - - -
- - +
+
- - - -
IP
HA (Ubiquitin, ~35 – 175kDa)
V5 (NKX3.1, ~35kDa)
NKX3.1-V5
GFP-RMND5A
GFP-RMND5B
HA-Ubiquitin
10µM MG132 + + + +
+ + + - + + + +
- - - -
- - +
+
HA (Ubiquitin, 20 – 200kDa)
V5 (NKX3.1, ~35kDa)
GFP (RMND5A/RMND5B, ~70kDa)
(i) (ii)
Chapter 5 RMND5 Proteins Ubiquitinate NKX3.1
210
immunoprecipitated and total cellular input samples were electrophoresed in 4-12%
gradient polyacrylamide gels and western blotting was performed for HA (ubiquitin),
V5 (NKX3.1) and GFP (RMND5A/RMND5B) (Section 3.13, 3.15). GFP western
blotting of the total cellular inputs identified GFP-RMND5A and GFP-RMND5B
expression in lysates of cells transfected with the appropriate plasmids and V5 western
blotting of all total cellular inputs and V5 immunoprecipitation samples indicated
successful transfection and immunoprecipitation of the ~35kDa NKX3.1-V5 protein,
respectively (Figure 5.10 (ii)). Following proteasome inhibition by MG132,
accumulation of high molecular weight HA-ubiquitin corresponding to ubiquitinated
and polyubiquitinated proteins was evident in the total cellular input fractions and HA-
ubiquitinated NKX3.1 (or NKX3.1-associated proteins) were evident in V5
immunoprecipitated samples (Figure 5.10 (ii)). In contrast to experiments performed in
the absence of MG132 treatment (Figure 5.10 (i)), the levels of HA-ubiquitinated
NKX3.1 (or NKX3.1-associated proteins) in GFP-RMND5A and GFP-RMND5B
overexpressing cells were markedly increased in comparison to cells that did not
overexpress RMND5 proteins (Figure 5.10 (ii)). These results indicated that both
RMND5A and RMND5B promoted the ubiquitination of NKX3.1, leading to its
proteasome-mediated degradation.
5.2.5.2 RMND5A (C356A/C358A) and RMND5B (C358A/H360A)
Ubiquitinate NKX3.1
Previous experiments had identified that overexpression of mutant RMND5A
(C356A/H358A) or RMND5B (C358A/H360A) was associated with reduced NKX3.1
protein levels (Section 5.2.4.2). To examine whether RMND5A (C356A/H358A) and
RMND5B (C358A/H360A) were able to ubiquitinate NKX3.1, NKX3.1 ubiquitination
was assessed following transfection of cells with plasmids encoding NKX3.1-V5, HA-
Ubiquitin and GFP-RMND5A, GFP-RMND5B, GFP-RMND5A (C356A/H358A) or
GFP-RMND5B (C358A/H360A) (Section 3.1.4). Cells were treated with 10µM MG132
for 3 hours prior to harvesting, total cellular input samples were taken and NKX3.1-V5
was immunoprecipitated with anti-V5 antibodies (Section 3.1.4, 3.1.5, 3.13).
Immunoprecipitates and total cellular input samples were electrophoresed in 4-12%
gradient polyacrylamide gels and analysed by western blotting for V5 (NKX3.1), HA
(Ubiquitin) and GFP (RMND5 proteins) (Section 3.15). Western blotting of the total
cellular input samples demonstrated that HA-ubiquitin, NKX3.1-V5 and (wild-type and
Chapter 5 RMND5 Proteins Ubiquitinate NKX3.1
211
mutant) GFP-RMND5A proteins were expressed at the expected molecular weights in
the lysates from transfected cells, however (wild- type and mutant) GFP-RMND5B was
barely visible in the (dilute) input lysates samples (Figure 5.11). Western blotting of
lysates following V5 immunoprecipitation showed that NKX3.1-V5 was successfully
immunoprecipitated and HA-ubiquitin western blotting of the immunoprecipitated
samples identified smears of ubiquitinated proteins ranging in size from ~35kDa -
~175kDa (Figure 5.11). Ubiquitinated NKX3.1 (or NKX3.1 associated proteins) were
evident in all lysates, however NKX3.1 ubiquitination was enhanced in lysates of
cultures that overexpressed GFP-RMND5A, GFP-RMND5B or mutant GFP-RMND5B
(C358A/H360A), whilst a reduction in NKX3.1 ubiquitination following
overexpression of GFP-RMND5A (C356A/H358A) was observed (Figure 5.11). These
experiments demonstrated that mutation of key amino acid residues in RMND5A
(C356A/H368A) reduced its ability to ubiquitinate NKX3.1-V5, however similar
mutations in RMND5B (C358A/H360A) did not markedly alter its ability to
ubiquitinate of NKX3.1-V5.
Chapter 5 RMND5 Proteins Ubiquitinate NKX3.1
212
Figure 5.11: In vivo ubiquitination of NKX3.1 following overexpression of wild-type and mutant RMND5 proteins. LNCaP cells were cotransfected with plasmids encoding NKX3.1-V5, HA-Ubiquitin and GFP-RMND5A, GFP-RMND5B, GFP-RMND5A (C356A/H358A) or GFP-RMND5B (C358A/H360A). At 48 hours post-transfection, NKX3.1-V5 was immunoprecipitated using anti-V5 antibodies. Western blotting for HA-ubiquitin showed that NKX3.1 was ubiquitinated in vivo and that there was an increase in NKX3.1-associated ubiquitination following RMND5A and RMND5B overexpression. HA western blotting also demonstrated that NKX3.1 ubiquitination was reduced following overexpression of GFP-RMND5A (C356A/H358A) compared to GFP-RMND5A. In contrast, levels of NKX3.1 ubiquitination following GFP-RMND5B (C358A/H360A) or GFP-RMND5B overexpression were similar. The experiment was performed twice and representative results are shown.
GFP (RMND5A/RMND5B, ~70kDa)
NKX3.1-V5
GFP-RMND5A
GFP-RMND5B
HA-Ubiquitin
MG132
+ + + - + + + +
-
-
- -
- -
+
+
GFP-RMND5A (C356A/H358A)
GFP-RMND5B (C358A/H360A)
+
+
- - - - - - -
- - - - - - -
+ +
+ +
+ + + + + +
HA (Ubiquitin, 35kDa – 175kDa)
V5 (NKX3.1, ~35kDa)
HA (Ubiquitin, ~20 – 200kDa)
V5 (NKX3.1, ~35kDa)
IP
Input Lysate (2%)
Chapter 5 RMND5 Proteins Ubiquitinate NKX3.1
213
5.3 Discussion Expression of the prostatic tumour suppressor, NKX3.1 is reduced or undetectable in up
to 80% of prostate tumours, and although loss of heterozygosity at the NKX3.1 locus is
common, mutation or epigenetic inactivation of the remaining allele, which could
account for the loss of NKX3.1 expression is believed to be a rare occurrence (Voeller
et al., 1997; Bowen et al., 2000; Asatiani et al., 2005). Discordance between NKX3.1
mRNA and protein levels in prostate tumours and prostate cancer cell lines has led to
the hypothesis that aberrant post-translational modification of NKX3.1 may contribute
to the low protein levels detected in prostate tumour cells, however mechanisms
involved in this process have not been described (Xu et al., 2000; Bethel et al., 2006).
RMND5B was originally identified in our laboratory to interact with NKX3.1 in a yeast
two hybrid screen and this binding was initially confirmed using GST-pulldown and
coimmunoprecipitation assays (Dawson, 2006). In this thesis, RMND5B and its
homologue RMND5A were identified to function as E3 ubiquitin ligases (Chapter 4)
and therefore characterisation of RMND5 protein function was extended to investigate
whether NKX3.1 was a ubiquitination target of either or both RMND5 proteins.
Initially, interaction between RMND5B and NKX3.1 was confirmed and the interaction
between RMND5A and NKX3.1 was investigated using coimmunoprecipitation assays,
establishing that both RMND5 proteins interacted with NKX3.1 in LNCaP cells. In this
thesis, only immunoprecipitation assays were used to identify NKX3.1 interaction with
the RMND5 proteins although protein-protein interactions are usually confirmed using
a variety of methods. These most commonly include yeast two-hybrid assays, which
enable the identification of novel interactions as well as validation of protein-protein or
protein domain interactions, GST-pulldown, Far western blotting,
coimmunoprecipitation and bioluminscence resonance energy transfer (BRET) or
fluorescence resonance energy transfer (FRET) assays (Fuks et al., 2003; Ciruela et al.,
2010). GST-pulldown assays are useful in situations where expression of high levels of
a protein of interest is limiting, for example where the protein product inhibits
mammalian cell growth or reduces cell viability. However, as proteins are produced in
bacteria, posttranslational modifications such as phosphorylation, which may be
required for specific protein-protein interactions may not occur, limiting the usefulness
of the method (Sahdev et al., 2008). Although yeast-two hybrid assays are performed in
eukaryotic cells, cell type specific factors including cofactors required for protein-
Chapter 5 RMND5 Proteins Ubiquitinate NKX3.1
214
protein interactions may not be present, therefore some interactions may not be detected
(Luo et al., 1997). Other limitations can include the generation of false positive binding
as interacting proteins in yeast may not be localised in the same cellular compartments
in mammalian cells (Luo et al., 1997). Far western blotting is similar to western blotting
but instead of using antibodies for the detection of a particular protein (bait), another
protein (prey) is utilised, thereby detecting specific protein-protein interactions (Wu et
al., 2007b). FRET and BRET rely on the transfer of energy from a
fluorescent/luminescent donor to a fluorescence acceptor to detect protein-protein
interactions where proteins are within 1-10nm of each other (Wu and Brand, 1994; Xu
et al., 1999; Ciruela et al., 2010). An advantage of BRET/FRET is that the techniques
can be utilised to determine protein-protein interactions in real time in living cells,
including interactions that are transient or unstable and which therefore would be
difficult to detect using coimmunoprecipitation or GST pulldown assays (Pfleger and
Eidne, 2006; Kocan et al., 2010). While the present studies have provided good
evidence for RMND5 protein interaction with NKX3.1 in prostate cancer cells, future
studies that characterise the protein-protein interaction domains of either protein and the
physiological conditions that promote or inhibit these interactions may use one or more
additional methods to investigate or confirm results.
Following identification of the interaction between RMND5A/RMND5B and NKX3.1,
fluorescence microscopy was used to determine their localisation/colocalisation and
therefore the potential location of their interaction in prostate cancer cells. The results
obtained were interesting as the localisation of both NKX3.1 and the RMND5 proteins
was altered upon co-expression of either RMND5 protein with NKX3.1. As discussed
previously, RMND5A and RMND5B generally display a diffuse nuclear and
cytoplasmic distribution (Section 4.2.6.4, 4.2.7.5) with a proportion of cells exhibiting a
punctate cytoplasmic localisation. NKX3.1 is an androgen regulated transcription factor
and as such it localises predominantly to the nucleus in the presence of androgens
(Bowen et al., 2007; Guan et al., 2008). Overexpression of NKX3.1 with either
RMND5A or RMND5B resulted in the cytoplasmic localisation of NKX3.1 and the
predominantly nuclear localisation of RMND5 proteins, although some colocalisation in
the cytoplasm was observed. NKX3.1 protein levels are reduced in prostate tumours,
however NKX3.1 cytoplasmic mislocalisation has also been hypothesised as a possible
contributor to loss of NKX3.1 function, a finding that is consistent with the results of
this study where overexpression of RMND5 proteins resulted in the cytoplasmic
Chapter 5 RMND5 Proteins Ubiquitinate NKX3.1
215
mislocalisation of NKX3.1 (Kim et al., 2002b). The mechanism by which RMND5
proteins are localised to the nucleus and NKX3.1 is localised to the cytoplasm under
these culture conditions is unknown. It is feasible that nuclear RMND5A and RMND5B
promote the ubiquitination of NKX3.1 and its relocalisation to the cytoplasm for
degradation or an as yet unidentified cytoplasmic role. Alternatively, ubiquitinated
NKX3.1 may be degraded in the nucleus. As the functions of RMND5 proteins are
incompletely characterised, it is also possible that their effects on the intracellular
localisation of NKX3.1 do not involve their E3 ubiquitin ligase activity.
Mechanisms regulating the intracellular distribution of RMND5 proteins in cells
transiently overexpressing RMND5A or RMND5B with or without NKX3.1
overexpression are also unknown at this time, however it is conceivable that this
involves post-translational modification. RMND5 protein auto-ubiquitination which
was observed in this study, may result in their altered intracellular localisation. In
addition, the increased RMND5 protein levels detected following proteasome inhibition
indicate that RMND5 protein levels are regulated by proteasomal degradation, which
may be related to their auto-ubiquitination or to ubiquitination by other E3 ubiquitin
ligases. E3 ubiquitin ligase auto-regulation has been widely documented with MDM2
and TRAC-1 two examples of ubiquitin ligases that regulate their own protein levels by
auto-ubiquitination, thereby resulting in their own proteasome dependent degradation
(Fang et al., 2000; Giannini et al., 2008). The findings of this study that RMND5
proteins are able to mono- and poly-auto-ubiquitinate their RING domains suggests that
RMND5A and RMND5B may regulate their own activity, where for example
monoubiquitination may result in altered cellular function or localisation and
polyubiquitination could lead to proteasome-dependent degradation. The type of
ubiquitin linkages, which direct the fate of RMND5 proteins may be ascertained in
future studies with the use of linkage specific antibodies or ubiquitin mutants (Wu-Baer
et al., 2003; Newton et al., 2008).
RMND5 protein effects on NKX3.1 may be related to their level of expression
following transient transfection of LNCaP cells. Such findings have been observed for
MDM2, which ubiquitinates p53 and depending upon the levels of MDM2, the type of
ubiquitination and the outcome of p53 ubiquitination differ (Boyd et al., 2000, Geyer et
al., 2000, Li et al., 2003). When MDM2 protein levels are high, p53 is ubiquitinated
and targeted for nuclear degradation, however, lower levels of MDM2 result in p53
Chapter 5 RMND5 Proteins Ubiquitinate NKX3.1
216
monoubiquitination and its nuclear export and cytoplasmic localisation where it is
unable to function as a transcription factor (Boyd et al., 2000; Geyer et al., 2000; Li et
al., 2003). Since the levels and cellular localisation of endogenous RMND5 proteins are
unknown, it is not possible at present to determine the contribution of RMND5A and/or
RMND5B to steady-state or androgen-induced NKX3.1 levels and localisation.
However, these may be investigated in future studies following development of
antibodies to the endogenous proteins (Section 7.2). Similarly, the dose-dependent
effects of RMND5 proteins on NKX3.1 levels and localisation may also be examined
using inducible methods of RMND5 protein overexpression. Interestingly, muskelin and
RanBPM, members of the CTLH complex (along with RMND5A) are hypothesised to
function in nucleocytoplasmic shuttling, and it is therefore feasible that changes in the
intracellular localisation of NKX3.1 following RMND5 overexpression are mediated in
part by the CTLH complex (Section 6.1.1, 6.1.4.3) (Kobayashi et al., 2007;
Valiyaveettil et al., 2008). Following RMND5 overexpression, a dose-dependent
reduction in NKX3.1 protein levels was observed, although changes in NKX3.1 mRNA
were not investigated but could be determined in future studies to assess whether
RMND5A and RMND5B regulate NKX3.1 solely at the protein level. As RMND5B
has been demonstrated to exert transcriptional repressor effects on an NKX3.1
responsive element, one of which is present in the NKX3.1 promoter, the nuclear
localisation of RMND5 proteins may reflect alternative cellular functions of RMND5A
and RMND5B (Dawson, 2006).
In order to examine RMND5 protein regulation of NKX3.1 levels, proteasomal and
lysosomal degradation of NKX3.1 were initially investigated. Culture of cells with the
proteasome inhibitor, lactacystin initially increased NKX3.1 protein levels, supporting
its processing by the proteasome, with progressive reduction in protein levels over eight
hours of treatment likely due to in part to nonspecific toxic effects of lactacystin or the
expected damaging effects of longterm disruption of the normal regulation of protein
processing. The concentration of 10µM lactacystin to inhibit the proteasome in LNCaP
cells has been commonly used in the literature, however duration of exposure to
lactacystin is generally limited due to the accumulation of nonspecific effects (Huang et
al., 2005b; Shirley et al., 2005; Chen et al., 2011a). Cells treated with a second
proteasome inhibitor MG132 did not show an accumulation of NKX3.1 under normal
culture conditions (RPMI medium supplemented with FCS) nor did cells cultured in
medium depleted of steroid hormones and supplemented with physiological levels of
Chapter 5 RMND5 Proteins Ubiquitinate NKX3.1
217
the androgen metabolite DHT (RPMI supplemented with charcoal stripped FCS and
DHT), highlighting the importance of performing studies under experimental conditions
where proteasomal processing of the target protein is likely to be enhanced. Since
NKX3.1 is an androgen regulated gene, NKX3.1 protein levels were monitored
immediately following androgen depletion (RPMI medium supplemented with charcoal
stripped FCS) when NKX3.1 levels are known to decline rapidly (He et al., 1997;
Prescott et al., 1998). Again, the concentration of 10µM MG132 used was obtained
from previous studies (Huang et al., 2005b; Chen et al., 2011a) and under these culture
conditions, the rapid reduction of NKX3.1 protein levels was reversed following
MG132 treatment. These experiments therefore demonstrated that NKX3.1 was
degraded by the proteasome and that androgen deprivation of LNCaP cells promoted
proteasome-mediated degradation of NKX3.1. Proteasome inhibitors such as MG132
and ALLN, which are peptide aldehydes, predominantly inhibit the chymotrypsin-like
activity of the proteasome and generally do not affect cell viability or growth for 10-20
hours of treatment (Rock et al., 1994). However, peptide aldehydes can also inhibit
some lysosomal cysteine proteases and calpains, thereby affecting lysosomal activity
(Lee and Goldberg, 1998). Lactacystin is an irreversible proteasome inhibitor that is a
more complete inhibitor of proteasome activity than the peptide aldehydes due to its
ability to inhibit both the chymotrypsin and trypsin-like activities of the proteasome,
however it also inhibits cathepsin A, another lysosomal protease (Craiu et al., 1997).
Validation of the mechanism of protein degradation using more than one proteasome
inhibitor is important and in this study both lactacystin and MG132 were tested, with
results both comparable between the two treatments (Section 4.2.5, 4.2.6.5, 4.2.7.6) and
consistent with previous studies that identified regulation of NKX3.1 levels by
proteasomal degradation (Li et al., 2006; Guan et al., 2008).
Lysosomal degradation of NKX3.1 was examined in cells treated with the lysosome
inhibitors ammonium chloride and chloroquine, although both proteasome inhibitors
tested are also able to inhibit lysosomal activity to some extent. While studies using
ammonium chloride found no evidence of lysosomal processing of NKX3.1, in
experiments using chloroquine, the increased NKX3.1 protein levels suggested that
under specific culture conditions, NKX3.1 may be degraded by a lysosomal mechanism.
In contrast to results from the experiments testing proteasome inhibitors, there was no
evidence that lysosomal degradation mediated the rapid decrease in NKX3.1 levels
following removal of androgens from the culture medium. Both chloroquine and
Chapter 5 RMND5 Proteins Ubiquitinate NKX3.1
218
ammonium chloride inhibit lysosomal acidification and thus to confirm that NKX3.1 is
able to undergo degradation by the lysosome, a lysosomal inhibitor that functions by
inhibiting lysosomal proteases such as leupeptin or E-64 may be utilised (Lee and
Goldberg, 1998). Findings in this thesis of degradation of NKX3.1 by the proteasome
are consistent with the canonical pathway in which up to 90% of short-lived cellular
proteins undergo proteasomal degradation (Hicke, 1999). In contrast, those proteins that
undergo lysosomal degradation are generally membrane bound proteins (Lee and
Goldberg, 1998; Hicke, 1999), although a number of proteins have been reported, for
example α-synuclein and connexin43, whose degradation is mediated by both protein
degradation pathways (Tofaris et al., 2001; Qin et al., 2003; Webb et al., 2003). Both
the proteasomal and lysosomal pathways of protein degradation are regulated by
ubiquitination and cross-talk between the two pathways of protein degradation has been
reported (Qiao and Zhang, 2009; Ciechanover, 2012). Inhibition of the proteasome
frequently leads to an increase in lysosomal proteases and therefore function, however
lysosomal inhibition most commonly results in a reduction in proteasomal function but
an increase in heat shock proteins, suggesting that lysosome inhibition enhances
autophagy that is mediated by chaperones (Rideout et al., 2004; Pandey et al., 2007;
Qiao and Zhang, 2009). Therefore, similar to other cellular proteins, it is feasible that
depending upon the cellular environment and the presence of NKX3.1 regulators,
NKX3.1 may be degraded by either the proteasome or lysosome, although proteasomal
degradation is likely to predominate.
Overexpression of RMND5A or RMND5B resulted in reduced NKX3.1 protein levels
that were partially restored upon proteasome inhibition, indicating that RMND5
proteins regulated cellular NKX3.1 levels and that this was in part mediated by
promotion of NKX3.1 ubiquitination and proteasomal degradation. This mechanism
was further supported by in vivo ubiquitination assays which determined that upon
RMND5 overexpression, the levels of ubiquitinated NKX3.1 were markedly enhanced.
NKX3.1 ubiquitination and proteasomal degradation may be mediated by a direct
interaction of RMND5A or RMND5B with NKX3.1 or may occur via an intermediate
RMND5 protein regulated pathway. For example, Runx2 is reported to be ubiquitinated
by the E3 ubiquitin ligase Smurf1, however deletion of the PY domain of Runx2 with
which Smurf1 interacts resulted in only a partial reduction of Runx2 degradation (Shen
et al., 2006). In this study it was determined that Smad6 interacted with both Smurf1
and Runx2 and enhanced Smurf1 induced Runx2 proteasomal degradation, providing a
Chapter 5 RMND5 Proteins Ubiquitinate NKX3.1
219
mechanism by which Smurf1 induced Runx2 proteasomal degradation both directly and
indirectly (Shen et al., 2006). The protein-protein interaction domains of RMND5
proteins and NKX3.1 have not been mapped, however their elucidation would enable
mutation studies to be performed to differentiate between direct or indirect mechanisms.
Although endogenous RMND5A or RMND5B protein levels in LNCaP cells are
unknown due to the lack of suitable commercially available antibodies, further
confirmation of the involvement of RMND5 proteins in the normal regulation of
NKX3.1 may be achieved with the use of siRNA for knockdown of RMND5 protein
levels. Increased NKX3.1 levels following RMND5 knockdown, which has been
performed to confirm the effect of other E3 ubiquitin ligases on their substrates would
indicate an important role of RMND5 proteins in the regulation of NKX3.1 levels that
may have significant implications in normal physiology and in pathological conditions
including prostate cancer (Guan et al., 2008; Tatham et al., 2008). Whether the outcome
of RMND5 mediated ubiquitination of NKX3.1 solely results in the proteasomal
degradation of NKX3.1 remains to be determined. The type and therefore outcome of
NKX3.1 ubiquitination may be investigated by western blotting using the linkage
specific antibodies to characterise the specific ubiquitin linkages associated with
NKX3.1 during RMND5-mediated ubiquitination (Newton et al., 2008). Additionally,
in vitro ubiquitination assays could also be performed to verify NKX3.1 ubiquitination
by RMND5 proteins. This would require the immunoprecipitation of both
RMND5A/RMND5B and NKX3.1 from mammalian cells as neither full length GST
fusion protein is able to be purified in sufficient quantities from bacterial cells. An
advantage to this assay is that the type of NKX3.1-associated ubiquitin linkages
promoted by RMND5 proteins can be more specifically determined incorporating use of
ubiquitin mutants or linkage specific antibodies (Wu-Baer et al., 2003; Newton et al.,
2008).
Overexpression of mutant RMND5 proteins in LNCaP cells resulted in similar
reductions in NKX3.1 protein levels compared to that associated with overexpression of
wild-type RMND5 proteins. These unexpected results may reflect residual activity of
the mutant RMND5 proteins which was evident in in vitro ubiquitination assays using
RMND5A (C356A/H358A) and RMND5B (C358A/360A) RING domains (Section
4.2.7.7). Alternatively, the reduced E3 ubiquitin ligase activity of RMND5 proteins may
be compensated for in vivo by RMND5 binding partners that possess E3 ubiquitin ligase
activity or that augment the activity of RMND5 proteins in mammalian cells. For
Chapter 5 RMND5 Proteins Ubiquitinate NKX3.1
220
example RMND5A forms part of the CTLH complex which contains a second putative
RING domain protein, EMP that may contribute E3 ubiquitin ligase activity to the
complex, thereby maintaining all or part of its E3 ubiquitin ligase activity following
mutation of RMND5A (Santt et al., 2008). E3 ubiquitin ligases may function as homo-
or heterodimers (Hashizume et al., 2001; Linke et al., 2008; Johnson et al., 2012) and
therefore the reduced enzymatic activity of the mutant RMND5 proteins may be
compensated for by the E3 ubiquitin ligase activity of its interacting partner, including
wild-type endogenous RMND5 proteins. The reduction in NKX3.1 protein levels upon
RMND5 mutant protein overexpression may also indicate that RMND5 proteins are
able to reduce NKX3.1 protein levels, in part, by a mechanism unrelated to their E3
ubiquitin ligase activity and that RING domain inactivation may enhance this activity.
Finally, the in vivo function of RMND5 proteins in relation to NKX3.1 ubiquitination
may be dependent on specific environmental conditions such as androgen withdrawal or
activity of signalling pathways, and therefore changes in NKX3.1 levels following
overexpression of mutant RMND5 proteins were not evident using the experimental
parameters employed for these studies.
The similar effects of mutant RMND5A and RMND5B on NKX3.1 levels in LNCaP
cells were expected to be reflected by similar levels of NKX3.1 ubiquitination in cells
overexpressing each of RMND5A (C356A/H358A) and RMND5B (C358A/H360A).
However, under the experimental conditions used, overexpression of RMND5A
(C356A/H358A) was associated with reduced levels of ubiquitinated NKX3.1, while
overexpression of RMND5B (C358A/H360A) was associated with similar levels of
NKX3.1 ubiquitination compared to LNCaP cells overexpressing wild-type RMND5B.
These findings for RMND5A (C356A/C358A) are consistent with previous studies
where mutation of E3 ubiquitin ligases typically results in a reduction in substrate
ubiquitination, including the finding by Santt et al. (2008) that mutant RMD5 (C379S)
no longer ubiquitinates its substrate FBPase (Qiu et al., 2000). Thus the experiments
performed for this thesis provided evidence that NKX3.1 is a ubiquitination target of
RMND5A and to confirm that RMND5A is able to ubiquitinate NKX3.1, in vitro
ubiquitination assays could be carried out in future studies using full length RMND5A
and RMND5A (C356A/H358A). Ubiquitination of NKX3.1 by RMND5A and reduced
levels of NKX3.1 ubiquitination by RMND5A (C356A/H358A) would verify that the
specific RING domain mutations introduced reduced activity toward this substrate as
has been demonstrated in the literature for further E3 ligases (Ryu et al., 2011).
Chapter 5 RMND5 Proteins Ubiquitinate NKX3.1
221
Contrary to results of in vitro ubiquitination assays and to its association with
ubiquitinated proteins in vivo (Section 4.2.7.6, 4.2.7.7), overexpression of the RMND5B
(C358A/H360A) mutant did not alter NKX3.1 ubiquitination compared to wild-type
RMND5B in LNCaP cells. While the reasons for this cannot be ascertained without
further studies, it is feasible that this may be due to the maintenance of residual E3
ubiquitin ligase activity of the RMND5B (C358A/H360A) mutant which was evident in
in vitro ubiquitination assays and supported by the reduction in NKX3.1 protein levels
following RMND5B (C358A/H360A) overexpression. Mutant RMND5B may interact
with another E3 ubiquitin ligase that is able to ubiquitinate NKX3.1, allowing
accumulation of ubiquitinated NKX3.1 to similar levels that accompany overexpression
of wild-type RMND5B. In these studies, the levels of ubiquitinated NKX3.1 were
determined 48 hours after transfection of cells to overexpress RMND5B
(C358A/H360A), and it is also feasible that alterations in NKX3.1 ubiquitination are
more clearly evident at earlier time points. Although RMND5A and RMND5B are
highly homologous proteins that are likely to have overlapping substrates, a proportion
of their targets are expected to be unique. Similarly, the mutations introduced into each
of RMND5A and RMND5B may have differentially affected their affinity for NKX3.1
and the efficiency of their ability to ubiquitinate NKX3.1. Each of these aspects of
RMND5 protein activity may be evaluated in future studies using in vitro and in vivo
binding and ubiquitination assays.
The regulation of NKX3.1 at the protein level and the mechanisms by which NKX3.1
protein levels are reduced in prostate tumours are not well understood and it is likely
that a number of proteins including protein kinases and E3 ubiquitin ligases regulate the
post-translational modification and degradation of NKX3.1. The E3 ubiquitin ligase,
TOPORS plays a role in NKX3.1 ubiquitination and proteasomal degradation (Guan et
al., 2008), and results of this thesis indicate that both RMND5 proteins and TOPORS
may regulate NKX3.1 levels in the prostate, potentially under different environmental
conditions. Many cellular proteins including p53 are ubiquitinated by more than one E3
ubiquitin ligase resulting in their degradation by the proteasome, and deregulation of
individual E3 ubiquitin ligases may produce minimal or profound changes in the
regulation of cellular levels of the target protein due to contributions of the other E3
ubiquitin ligases (Scheffner et al., 1993; Kubbutat et al., 1997; Esser et al., 2005).
Although it is unknown whether the RMND5A and RMND5B gene loci are themselves
disrupted in prostate cancer, the RMND5B gene is located at chromosome 5q35, a
Chapter 5 RMND5 Proteins Ubiquitinate NKX3.1
222
prostate cancer heritability locus, and the chromosomal regions of both RMND5A and
RMND5B are disrupted in a number of cancer types, with RMND5A overexpressed in
ovarian cancer (Xu et al., 2005; Li et al., 2008; Christensen et al., 2010). In the absence
of well-characterised RMND5 antibodies, the effects of RMND5 loss of expression or
overexpression on NKX3.1 expression in human prostate tumours remain to be
determined. Previous studies have shown that the TOPORS chromosomal locus
undergoes LOH in 21.8%-50% of prostate tumours, however as TOPORS levels or
function have not been characterised in human prostate tumours, the relationship
between TOPORS LOH and reduced NKX3.1 levels are unknown (Perinchery et al.,
1999). It is also likely that the reduced NKX3.1 levels seen in prostate tumours results
from the dysregulation of other proteins that mediate NKX3.1 post-translational
modifications and stability, including CK2, which phosphorylates NKX3.1, increasing
its half-life (Li et al., 2006). The chromosomal loci encoding the CK2α and α’ catalytic
subunits are deleted in a proportion of prostate tumours, with further studies required to
determine correlations between alterations in CK2 expression or activity and NKX3.1
levels (Best et al., 2005; Jin et al., 2011).
Results from these studies have therefore identified that both RMND5A and RMND5B
interact with NKX3.1 and that NKX3.1 is a ubiquitination target of RMND5 proteins in
prostate cancer cells. Although aberrant RMND5 expression has not been investigated
in prostate tumours, the findings that RMND5A is overexpressed in ovarian tumours and
that the RMND5A and RMND5B loci are amplified in a number of cancers suggests that
RMND5 overexpression may contribute, in part, to the reduced NKX3.1 levels present
in prostate tumours. As NKX3.1 expression is largely restricted to the prostate in the
adult and according to the NCBI Gene Expression Omnibus Database, RMND5 proteins
are ubiquitously expressed, it is likely that RMND5 proteins have additional cellular
substrates and binding partners. These binding partners may be substrates of
RMND5A/RMND5B induced ubiquitination or may be involved in additional activities
of RMND5 proteins that are potentially mediated by the LisH, CTLH and CRA
domains found in both proteins. To further characterise RMND5 protein function,
additional RMND5 protein binding partners were therefore investigated, in both the
context of the previously reported CTLH complex and in the identification of novel
interactors.
Chapter 6 Characterisation of RMND5 Binding Partners
Chapter 6: Characterisation of RMND5
Protein Binding Partners
Chapter 6 Characterisation of RMND5 Protein Binding Partners
223
6.1 Introduction The CTLH complex is the proposed human orthologue of the yeast E3 ubiquitin ligase
Vid30 complex (Santt et al., 2008). Although the biological activity of the RMND5A-
containing CTLH complex has not yet been characterised, the function of RMND5A as
an E3 ubiquitin ligase and the protein domain architecture of other CTLH complex
components suggests that this complex may also function as an E3 ubiquitin ligase
complex.
6.1.1 Characterisation of the CTLH Complex Components
RanBPM was originally identified to form part of a large 670kDa complex, with the
GTPase Ran coimmunoprecipitating with RanBPM as part of this complex (Nishitani et
al., 2001). Yeast two-hybrid studies characterising the members of the RanBPM
associated complex identified the muskelin, Twa1 and matrix metalloprotease 8
(HSMpp8) gene products as RanBPM binding partners, however only muskelin and
Twa1 were confirmed as complex members by subsequent co-immunoprecipitation and
gel filtration analyses (Umeda et al., 2003). Although Twa1 was described as a
predominantly nuclear protein, co-expression of muskelin with either Twa1 or RanBPM
resulted in the diffuse cytoplasmic and nuclear redistribution of Twa1 and RanBPM,
indicating the potential in vivo interaction between these proteins (Umeda et al., 2003).
In an effort to identify additional complex components, Kobayashi et al. (2007)
performed linear sucrose gradient centrifugation and immunoprecipitation of
endogenous RanBPM from HEK293 cells and identified the co-immunoprecipitating
proteins muskelin, p48EMLP (EMP/MAEA), p44CTLH (RMND5A) and the armadillo
repeat containing proteins ARMC8α and ARMC8β by mass spectrometry (Kobayashi et
al., 2007). Due to the finding that five of the CTLH complex components contained
similar protein domain architectures, including LisH and CTLH domains, the complex
was named the CTLH complex (Figure 6.1) (Kobayashi et al., 2007).
Three of the CTLH complex components were novel proteins including ARMC8α and
ARMCβ, alternatively spliced products of the same gene, with ARMC8α encoding the
larger isoform which was identified to co-immunoprecipitate with the other complex
components and with p44CTLH/RMND5A (Kobayashi et al., 2007). Although Twa1
was identified in the original screen, it was not detected as coimmunoprecipitating with
RanBPM and the associated CTLH complex, but was shown to interact with each
Chapter 6 Characterisation of RMND5 Protein Binding Partners
224
complex component in coimmunoprecipitation assays (Kobayashi et al., 2007). Using
these assays, it was determined that all complex components interacted with RanBPM,
Twa1, muskelin and ARMC8α and that these members were also able to self-associate
(Section 4.1.3.1) (Kobayashi et al., 2007). It was further demonstrated in
coimmunoprecipitation assays that RMND5A and p44EMLP interacted, that the
RMND5A amino-terminal and carboxy-terminal regions were necessary for this
interaction and that the CTLH domain was required for the interaction between
RMND5A and ARMC8α (Kobayashi et al., 2007). Fluorescence microscopy indicated
that endogenous RMND5A, ARMC8α, Twa1 and RanBPM displayed diffuse nuclear
and cytoplasmic localisation in HEK293 cells, p44EMLP exhibited a nuclear
distribution whilst muskelin was mainly cytoplasmic (Kobayashi et al., 2007). Although
individually not all components were expressed in the same cellular compartment, when
co-expressed, the cellular distribution of CTLH complex members was altered and the
proteins colocalised either in the nucleus or the cytoplasm or both (Kobayashi et al.,
2007).
6.1.2 Protein Domain Architecture of the CTLH Complex Members
Each of the CTLH complex components contains a similar protein domain architecture,
notably a LisH, CTLH and CRA domain, and it has been proposed that the proteins
utilise one or more of these domains to associate with each other (Figure 6.1). For
example, it has been demonstrated that RMND5A uses its CTLH domain and part of the
CRA domain to interact with ARMC8α (Kobayashi et al., 2007). As described
previously (Section 4.1.3.1), the LisH domain is a dimerisation motif and therefore
could be used by CTLH complex members to associate with each other. Alternatively,
the domain may be utilised for complex multimerisation in a similar manner to that of
the LisH domain of DCAF4, which forms Cullin4A RING E3 ubiquitin ligase
supramolecular complexes via its LisH domain (Ahn et al., 2011). The functions of the
CTLH and CRA domains are not well characterised, however they are also proposed
protein-protein interaction domains (Section 4.1.3.2, 4.1.3.3). Several CTLH complex
members contain additional protein domains including RMND5A which contains a
RING domain, RanBPM, which contains a SPRY domain, muskelin which contains a
discoidin-like and a Kelch repeat domain and ARMC8α/β, which possess Armadillo
repeat domains (Figure 6.1).
Chapter 6 Characterisation of RMND5 Protein Binding Partners
225
.
The SplA and ryanodine receptor (SPRY) domain is a protein-protein interaction
domain originally identified in the Dictyostelium discoideum tyrosine kinase spore lysis
A (SplA) and the mammalian Ryanodine receptor (Ponting et al., 1997). The domain is
found in a number of proteins including the tripartite motif (TRIM) RING domain
containing E3 ubiquitin ligases and is responsible for substrate binding of TRIM21
(Stacey et al., 2012). Additionally, suppressor of cytokine signalling (SOCS) box
proteins, e.g. SPSB2, which contain SPRY and/or WD40 repeats, function as substrate
recognition components in Cullin-Rbx1 E3 ubiquitin ligase complexes, with the SPRY
domain responsible for substrate recognition and binding (Kuang et al., 2010; Linossi
and Nicholson, 2012). The SPRY domains of SPSB1 to 4, are also able to bind the
intracellular domain of the human growth factor receptor MET (Wang et al., 2005).
Interestingly, the CTLH complex member RanBPM and its homologue RanBP10 also
use their SPRY domains to interact with MET, implicating the SPRY domain in similar
functions in these proteins and suggesting that this domain of RanBPM/RanBPM10
may function as a substrate recognition element in the CLTH complex (Wang et al.,
2002a; Wang et al., 2004).
Figure 6.1: Protein domain architecture of the CTLH complex components. The CTLH complex components possess a similar domain structure that includes Lissencephaly 1 homology (LisH), C-terminal to LisH (CTLH) and CT-11 RanBPM (CRA) domains as well as unique domains such as SplA and Ryanodine receptor (SPRY), discoidin-like domains, Kelch repeats and Armadillo repeat domains (Kobayshi et al., 2007).
(RMND5A)
Chapter 6 Characterisation of RMND5 Protein Binding Partners
226
The discoidin domain, which plays a role in membrane anchoring, is found in a variety
of extracellular and membrane bound proteins, with many being involved in cell
adhesion and migration, in particular during development (Baumgartner et al., 1998;
Arakawa et al., 2007). Kelch repeats form a β-propeller structure which is also formed
by the WD40 repeat domain and due to their similar structure, both domains possess
similar functions (Hudson and Cooley, 2008). WD40 repeats are known to bind
phosphoresidues on the substrate, however, this recognition activity of Kelch repeats is
uncharacterised (Pickart, 2001). Kelch and WD40 repeats are often present in F-box
proteins which are the substrate recognition components of SCF (SKP1, Cullin1-Rbx1,
F-box protein) E3 ubiquitin ligase complexes (Sun et al., 2007). The amino-terminal F-
box motif binds the SKP1 component of the complex whilst the carboxy-terminal
contains other protein-protein interaction motifs including the Kelch repeat or WD40
domain which bind the substrates of these complexes. For example, the F-box and
Kelch repeat containing protein Just one F-box and Kelch domain containing protein
(JFK) forms part of an SCF complex binding p53 through its Kelch domain and
resulting in its ubiquitination and targeting it for proteasomal degradation (Sun et al.,
2007; Sun et al., 2009). The Kelch repeat containing protein KLHL20 forms part of the
KLHL20-Cul3-Rbx E3 ubiquitin ligase complex and acts as the substrate recognition
component, using its Kelch repeat to interact with death associated protein kinase
(DAPK), resulting in the polyubiquitination of DAPK and its proteasome-mediated
degradation (Lee et al., 2010). This emerging function of Kelch repeat and SPRY
domains as substrate recognition domains for proteins forming part of E3 ubiquitin
ligase complexes is intriguing as little is known about the cellular functions of these
domains. The presence of the SPRY and Kelch repeat domains in RanBPM and
muskelin, respectively suggests that they may play roles as substrate recognition
components in the CTLH complex. Indeed, the yeast orthologue of RanBPM, Vid30
recognises and binds FBPase, a substrate of the Vid30 complex (Santt et al., 2008).
Armadillo repeats are found in a range of proteins involved in diverse cellular functions
including signalling, cytoskeleton formation/regulation and protein folding (Tewari et
al., 2010). The Armadillo repeat domain is also present in proteins involved in protein
degradation, such as the Arm-HECT E3 ubiquitin ligases, the F-box proteins Aardvark
and Arabidillo, the U-box ubiquitin ligase UFD2, and ARMC8α, which as will be
discussed shortly is involved in the degradation of α-catenin (Section 6.1.4.2) (Suzuki et
al., 2008; Tewari et al., 2010). Thus the presence of the LisH, SPRY, Kelch repeat and
Chapter 6 Characterisation of RMND5 Protein Binding Partners
227
Armadillo repeat domains within CTLH complex members provides some evidence that
the CTLH complex functions as an E3 ubiquitin ligase complex, similar to the yeast
Vid30 complex.
.
6.1.3 The Yeast Vid30 Complex
The yeast orthologue of the human CTLH complex is a well characterised E3 ubiquitin
ligase complex that ubiquitinates and targets for degradation a subset of gluconeogenic
enzymes including fructose 1,6 bisphosphatase (Section 4.1.1). The function of the
yeast Vid30 complex and the specific roles of each of the complex components may
indicate the function of the members of the human CTLH complex (Table 6.1). For
example, RMND5A, and potentially p44EMLP, yeast orthologues of which impart the
Vid30 complex with E3 ubiquitin ligase activity, may contribute this enzymatic activity
to the CTLH complex. Similarly, RanBPM, the human orthologue of Vid30 which
functions both as a core component and in substrate recognition of the complex, may
perform similar roles in the CLTH complex. Recently, Menssen et al. (2012) predicted
the architecture of the yeast Vid30 complex, and substitution of the Vid30 complex
members with the human CTLH complex components suggests the possible topology of
the CTLH complex as the human and yeast orthologues share the same protein domain
architecture (Figure 6.2) (Santt et al., 2008; Menssen et al., 2012).
The proposed CTLH complex architecture is consistent with the findings of Kobayashi
et al. (2007) in that each of the complex components are able to interact with each other
and RMND5A is able to interact with both ARMC8α and EMLP, with these interactions
potentially able to occur simultaneously as different protein domains are required
(Figure 6.2) (Kobayashi et al., 2007). Suzuki et al. (2008) determined that ARMC8α
and β are not essential for the formation of the CTLH complex, suggesting that they
function as adaptors (Suzuki et al., 2008). However, the yeast orthologue of ARMC8α,
Gid5 is a proposed core component of the Vid30 complex which binds the proposed E3
ubiquitin ligase components, Gid2/RMD5 (RMND5A) and Gid9 (p44EMLP) and links
the complex with Gid4, the activator of the Vid30 complex (Section 4.1.1) (Menssen et
al., 2012).
Chapter 6 Characterisation of RMND5 Protein Binding Partners
228
Table 6.1 – The human CTLH complex components and their yeast orthologues
The human orthologue of Gid4, C17orf39 has not been identified as a member of the
CTLH complex, suggesting either that the CTLH complex has been investigated in an
inactive form and requires ARMC8α to bind C17orf39 for activation of the complex,
which may be the reason for the finding that ARMC8α was not necessary for CTLH
complex formation (Suzuki et al., 2008). Or, alternatively the CTLH complex does not
require activation in mammalian cells. However, as Gid4 is only transiently associated
with the Vid30 complex, C17orf39 may not be identified as a cofactor for the CTLH
complex unless investigated under the specific set of environmental conditions required
for activation of the CTLH complex, which have yet to be determined.
CTLH Complex Component
Protein Domains
Protein Function/Cell Type Identified or Investigated
Yeast Orthologue
Yeast Orthologue Function in Vid30 Complex
RMND5A LisH, CTLH, CRA, RING
Proposed E3 ubiquitin ligase/HEK cells
Gid2/RMD5 E3 ubiquitin ligase
RanBPM SPRY, LisH, CTLH, CRA
Scaffolding protein; immune and neural cell interactions/many cell types
Gid1/Vid30 Proposed core component, substrate recognition component
Muskelin LisH, CTLH, Kelch Repeat
Mediator of cell spreading and morphology; neural cell and cardiomyocyte interactions/many cell types
Gid7/Moh2 Unknown
EMP LisH, CTLH, CRA
Mediates maturation of erythroblasts/HEK cells
Gid9/Fyv10 E3 ubiquitin ligase
ARMC8α/β Armadillo repeats/β-catenin-like repeats
Associates with HRS and is involved in α-catenin degradation/HEK cells
Gid5/Vid28 Proposed core component
Twa1 LisH, CTLH, CRA
Unknown/HEK cells Gid8/Dcr1 Unknown
C17orf39 Protein kinase-like domain
Unknown; Not identified as a CTLH complex component
Gid4/Vid24 Activator of Vid30 complex
Chapter 6 Characterisation of RMND5 Protein Binding Partners
229
The human orthologues of two proteins associated with the yeast Vid30 complex, the
E2 conjugating enzyme Ubc8 and the deubiquitinating enzyme Gid6/Ubp14, whose
human orthologues are UbcH2 and isopeptidase T, have not yet been determined to
interact with members of the human CTLH complex. However, in this thesis, RMND5A
was found to associate with UbcH2 in in vitro ubiquitination assays, and additional
studies may confirm UbcH2 interaction with the CTLH complex (Section 4.2.4.3).
These findings provide support further investigation of human CTLH complex function
as an E3 ubiquitin ligase complex that promotes protein degradation by the proteasome
and/or lysosome, as is the case for the yeast Vid30 complex.
6.1.4 CTLH Complex Components
The functions of most of the CTLH complex components have not been well
characterised, however reports in the literature have provided some indication of their
biological activities and thus their potential roles in the CTLH complex.
C17 orf39
ARMC8α
RMND5A EMLP
Twa1
RanBPM Muskelin
Yeast Vid30 Complex Human CTLH Complex
A B
Figure 6.2: Predicted Vid30 and CTLH complex topology. (A) Menssen et al. 2012 predicted the architecture of the yeast Vid30 complex by performing coimmunoprecipitation studies using full length and deletion constructs of the individual Vid30 complex members (B) Replacement of the Vid30 complex components with their proposed human orthologues in the predicted Vid30 complex structure indicates the possible structure of the CTLH complex. (Adapted from Menssen et al., 2012).
Chapter 6 Characterisation of RMND5 Protein Binding Partners
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6.1.4.1 Muskelin
Muskelin is a cytoplasmic and membrane associated protein that was originally
demonstrated to mediate the intracellular responses of cells growing on thrombospondin
by integrating cell spreading, adhesion and cytoskeletal organisation by as yet
uncharacterised mechanisms (Adams et al., 1998). Apart from its LisH and CTLH
domains, muskelin contains a carboxy-terminal Kelch repeat domain which forms a β-
propeller structure that functions as an actin binding motif in many proteins, and
although muskelin does not directly bind actin or tubulin, it is associated with the actin
cytoskeleton and modifies cytoskeletal organisation (Adams et al., 1998).
The muskelin amino-terminal contains a discoidin-like domain which, along with the
carboxy-terminal Kelch repeat domains, mediates self-association in a head to tail
orientation (Prag et al., 2004). PKC phosphorylates muskelin on two residues, Ser324
and Thr515, and mutation of these residues interferes with the ability of muskelin to
self-associate (Prag et al., 2007). The functional outcomes of this self-association are
incompletely characterised although other Kelch repeat containing proteins, including
Kelch, Actinfilin and Mayven undergo oligomerisation (Robinson and Cooley, 1997;
Soltysik-Espanola et al., 1999; Chen et al., 2002b). Multimerisation is required for the
mediation of actin filament crosslinking by Drosophila Kelch, the first identified Kelch
containing protein (Robinson and Cooley, 1997), whilst muskelin self-association has
recently been proposed to play a role in directing its cellular localisation (Valiyaveettil
et al., 2008). Although muskelin is predominantly localised in the cytoplasm, it is also
detectable in the nucleus, with studies in C2C12 skeletal myoblasts, COS-7 epithelial
cells and SW1222 carcinoma cells indicating that the LisH domain directs nuclear
localisation while the carboxy-terminal 35 amino acids localise muskelin to the
cytoplasm, even in the presence of the LisH domain (Valiyaveettil et al., 2008).
Muskelin self-association leads to autoinhibition, which is hypothesised to be relieved
by phosphorylation of Thr723, which releases the self-association and promotes nuclear
translocation (Valiyaveettil et al., 2008). The specific cellular environment required for
phosphorylation or dephosphorylation have not yet been determined.
Few muskelin binding partners have been reported, however those identified to date
support the involvement of muskelin in the endocytosis and intracellular transport of
cargo proteins as well as in cytoskeletal organisation. Muskelin interacts with the
Gamma-aminobutyric Acid A Receptor (GABAAR) α1 and aids in the transport of this
Chapter 6 Characterisation of RMND5 Protein Binding Partners
231
receptor to the lysosome for degradation. Muskelin and GABAAR α1 form a complex
with myosin VI and dynein, thereby facilitating the F-actin and microtubule based
internalisation and intracellular transport of GABAAR α1 to the lysosome for
degradation (Heisler et al., 2011). Delayed internalisation and intracellular transport of
GABAA R α1 are evident in muskelin knockout mice, which also exhibit diluted coat
colour, supporting a role for muskelin in intracellular transport in melanocytes (Heisler
et al., 2011). Muskelin associates with p39, a cyclin dependent kinase 5 (CDK5)
activator involved in the adhesion and migration of epithelial cells of the lens and
cornea (Ledee et al., 2005). p39 relocates muskelin to the cell periphery and is
hypothesised to link muskelin to the actin cytoskeleton by associating with α-actinin,
thereby facilitating its function in cytoskeletal organisation (Dhavan et al., 2002; Ledee
et al., 2005). Muskelin has also been identified as a prostaglandin EP3α receptor
binding partner that inhibits EP3α internalisation and promotes the association of this G
protein coupled receptor with Gi, enhancing Gi activity (Hasegawa et al., 2000).
6.1.4.2 ARMC8α
Although both ARMC8α and ARMC8β were identified as members of the CTLH
complex, ARMC8α is the longer isoform and is the isoform whose function has been
further investigated (Kobayashi et al., 2007; Suzuki et al., 2008; Tomaru et al., 2010).
ARMC8α is among several proteins including importin-α and β-catenin that contain
armadillo repeats, which are involved in intracellular transport and cell adhesion
(Franke et al., 1989; McCrea et al., 1991; Kobayashi et al., 2007). α-catenin colocalises
and interacts with ARMC8α and β-catenin at the cell membrane using its N-terminal
(amino acids 82-148). Overexpression of ARMC8α results in the rapid degradation of
α-catenin by the proteasome, whereas ARMC8α knockdown results in a prolonged α-
catenin half-life and reduced degradation (Suzuki et al., 2008). Together with β-catenin,
α-catenin forms part of the cadherin-catenin complex and is important in linking these
complexes to the actin cytoskeleton and directing actin assembly (Maiden and Hardin,
2011). ARMC8α also interacts with the endosomal protein, hepatocyte growth factor-
regulated tyrosine kinase substrate (HRS), which is involved in membrane protein
trafficking by recognising monoubiquitinated receptors such as the epidermal growth
factor receptor (EGFR), directing the ubiquitinated receptors for sorting through the
endosome and lysosomal degradation (Raiborg et al., 2003; Tomaru et al., 2010). The
interaction of HRS with ubiquitinated proteins through its ubiquitin interacting motif is
enhanced by ARMC8α and identification of the ubiquitinated proteins and the
Chapter 6 Characterisation of RMND5 Protein Binding Partners
232
mechanism by which ARMC8α is able to aid in their association with HRS will assist in
the understanding of ARMC8α function (Tomaru et al., 2010).
6.1.4.3 RanBPM
RanBPM has been reported to interact with at least 43 proteins involved in diverse
processes, identifying it to function as a protein stabiliser, transcriptional regulator, cell
cycle and neuronal function regulator and as a scaffolding/adaptor protein (Murrin and
Talbot, 2007; Suresh et al., 2012). RanBPM interacts with the cytoplasmic domain of
many membrane bound receptors, including MET, p75NTR, Axl/Sky, TrkA, TrkB and
with proteins associated with the intracellular domains of receptors including TRAF6
(Bai et al., 2003; Wang et al., 2004; Hafizi et al., 2005; Yuan et al., 2006; Yin et al.,
2010). Additionally, RanBPM interacts with other membrane bound proteins, including
the membrane transporter Dectin-1, the ecto-nuclease CD39 and the calcium CaV3.1
channel (Wu et al., 2006; Xie et al., 2006; Kim et al., 2009; Wang et al., 2012). The
interaction of RanBPM with specific receptors or associated proteins has been shown to
either enhance or repress signalling from that receptor. For example, RanBPM binding
to TRAF6 inhibits TRAF6 ubiquitination and its downstream signalling, leading to a
reduction in TRAF6 related NF-κB signalling (Wang et al., 2012). Alternatively,
RanBPM interaction with TrkB, a receptor tyrosine kinase for brain-derived
neurotrophic factor (BDNF), enhances BDNF-induced activation of the MAPK and Akt
signalling pathways and promotes neuronal survival (Yin et al., 2010).
RanBPM is also hypothesised to play a role in receptor endocytosis (Bai et al., 2003), it
reduces agonist-induced endocytosis of the mu opioid receptor without affecting
signalling from the receptor (Talbot et al., 2009), and by interacting with Plexin A,
RanBPM has been proposed to connect Plexin 3A receptors to retrograde transport
(Togashi et al., 2006). As well as binding to membrane bound receptors and proteins,
RanBPM interacts with steroid hormone receptors such as the AR, thyroid hormone
receptor and glucocorticoid receptor in which role it functions as a ligand dependent
coactivator. For example, RanBPM increases by 300% the transcriptional activity of
thyroid receptor alpha 1 on the thyrotropin releasing hormone and thyroid stimulating
hormone promoters (Rao et al., 2002; Poirier et al., 2006). RanBPM interacts with
numerous other transcription factors such as TAF4, which is involved in neural
development and the p53 associated nuclear transcription factor, p73α (Brunkhorst et
al., 2005; Kramer et al., 2005).
Chapter 6 Characterisation of RMND5 Protein Binding Partners
233
RanBPM may also play a role in the regulation of cytoskeletal organisation due to its
interaction with numerous proteins that mediate cell morphology, spreading and
migration, with RanBPM interaction with these proteins most frequently enhancing
these activities. For example, RanBPM has been characterised to interact with muskelin,
and knockdown of either of these proteins results in “protrusive cell morphologies with
enlarged perimeters” and altered F-actin distribution (Valiyaveettil et al., 2008). By
interacting with the lethal giant larvae (Mgl-1) tumour suppressor, RanBPM enhances
Mgl-1 associated cell migration and colony formation, and by interacting with MET, the
receptor tyrosine kinase for hepatocyte growth factor (HGF), RanBPM augments HGF-
MET signalling and enhances cell migration (Wang et al., 2004; Suresh et al., 2010). In
neural cells, RanBPM reduces cell proliferation, migration and neurite outgrowth by
interacting with and modulating the activity of CD39, the neural cell adhesion molecule
L1 and β1-integrin (Denti et al., 2004; Cheng et al., 2005; Wu et al., 2006). Together
with muskelin, RanBPM has been implicated in nucleocytoplasmic shuttling, as
supported by the original isolation of RanBPM as a Ran binding protein and by the roles
of RanBPM in escorting acetylcholinesterase and porphobilinogen deaminase (PBGD)
from the cytoplasm into the nucleus (Greenbaum et al., 2003; Gong et al., 2009).
The diverse activities of RanBPM have led to the hypothesis that it functions as an
adaptor or scaffolding protein, which is consistent with its proposed role in the CTLH
complex. The nucleocytoplasmic and cytoskeletal functions of RanBPM following its
interaction with muskelin suggest complementary or coordinated functions of these two
proteins, potentially including their roles as members of the CTLH complex.
6.1.4.4 Erythroblast Macrophage Protein (EMP)
Erythroblast macrophage protein (EMP/MAEA) was originally identified as a ~30kDa
protein mediating the attachment of erythroblasts to macrophages in erythroblast islands
(Hanspal and Hanspal, 1994). A number of lines of evidence have demonstrated that
EMP is required for the direct association of macrophages and erythroblasts in these
islands. Firstly, EMP possesses a short amino-terminal extracellular domain, and HeLa
cells expressing an amino-terminal deletion mutant are unable to associate with
erythroblasts (Hanspal et al., 1998). Additionally, Emp null mice display
haematopoietic defects, including the inability of macrophages to form erythroblastic
islands (Soni et al., 2006; Soni et al., 2008). In immature erythroblasts, EMP is
Chapter 6 Characterisation of RMND5 Protein Binding Partners
234
associated with the nuclear matrix whilst in mature erythroblasts, EMP is localised at
the cell membrane. Immature erythroblasts are unable to associate with macrophages,
suggesting that EMP must be attached to the cell membrane to directly mediate cell
attachment and that the protein may have additional functions in the nucleus (Soni et
al., 2007). The cell membrane association of EMP in HeLa cells indicates that EMP can
exhibit an extracellular amino-terminus in cell types other than macrophages, with this
domain potentially functioning in cell-cell or cell-matrix adhesion. Since the carboxy-
terminal intracellular domain of EMP contains multiple tyrosine residues, it has been
hypothesised that these residues may become phosphorylated, allowing association of
phospho-EMP with signalling kinases and therefore with intracellular signalling
pathways (Hanspal et al., 1998). Although the nuclear role of EMP has yet to be
investigated, in order to assess the cellular roles of EMP in non-haematopoietic cells,
Bala et al. (2006) determined the localisation of EMP in HEK cells. In these cells,
recombinant EMP was localised to either the nuclear matrix or cell membrane and
depending upon the stage of the cell cycle, EMP was localised to distinct nuclear
regions. During interphase, EMP colocalised with nuclear actin and SC35, a
spliceosome assembly factor whilst during mitosis, EMP localised to mitotic spindles
(Bala et al., 2006).
Another mechanism by which EMP is involved in erythroblast-macrophage maturation
is via its association with and regulation of the distribution of F-actin in erythroblasts
and macrophages, contributing to the enucleation of erythroblasts and the formation of
macrophage filopodia (Soni et al., 2006). These results are supported by the finding that
EMP null embyros exhibit little cytoplasmic F-actin, defects in erythropoiesis including
incomplete terminal differentiation of erythroblasts which display nuclei, and
macrophages that do not display cytoplasmic protrusions (Soni et al., 2006; Soni et al.,
2008). EMP additionally possesses anti-apoptotic functions and EMP deletion in
erythroblasts results in apoptosis (Hanspal et al., 1998). This activity is consistent with
that of its yeast orthologue, Fyv10/Gid9 which also plays an anti-apoptotic role
(Hanspal et al., 1998; Khoury et al., 2008). However, the mechanism by which EMP
inhibits apoptosis is yet to be established.
Members of the CTLH complex and their associated protein domains are therefore
implicated in broadly similar cellular pathways including cytoskeletal organisation,
protein trafficking and protein degradation. The widespread expression of many of the
Chapter 6 Characterisation of RMND5 Protein Binding Partners
235
CTLH complex members suggests that the complex may form in diverse cell types,
although the existence of the CTLH complex in cells other than HEK293 cells has not
been investigated. Additionally, the inclusion of orthologues of the complex members
RanBPM (RanBP10) and RMND5A (RMND5B) in the CTLH complex has not yet
been described. In addition to their proposed activities as part of the CTLH complex,
the multidomain CTLH complex members and their orthologues may also function in
additional roles. In this thesis, further characterisation of RMND5 protein function was
initiated by investigation of the expression of CTLH complex members in prostate
cancer cells and identification of RMND5A and RMND5B binding proteins.
Chapter 6 Characterisation of RMND5 Protein Binding Partners
236
6.2 Results
6.2.1 Transcripts encoding the CTLH Complex Components are Expressed
in Prostate Cancer Cells
In order to determine whether it was possible for the human CLTH complex to form in
prostate cancer cells, RT-PCR was used to identify whether the complex components
were expressed in LNCaP cells. Between 300bp-600bp of the coding region of each of
the CTLH complex components was PCR amplified using LNCaP cDNA template and
specific primer pairs for each CTLH complex member (Sections, 3.2, 3.3, Appendix II).
For RMND5A, RMND5B, RanBPM and muskelin, PCRs were carried out using 1.5mM
MgCl2 and an annealing temperature of 55°C for 35 cycles and the products
electrophoresed in a 2% agarose gel, identifying bands at the expected size for each
cDNA, RMND5A (~574bp), RMND5B (~393bp), RanBPM (~641bp) and muskelin
(~446bp) (Sections 3.4, 3.6, Figure 6.3A). Twa1 and EMP PCR amplification was
optimised using a gradient of annealing temperatures between 53°C and 60°C with
1.5mM MgCl2, and the PCR products were electrophoresed in a 2% agarose gel from
which single bands corresponding in size to Twa1 (~347bp) and EMP (~504bp) were
identified when higher annealing temperatures were used (Sections 3.4, 3.6, Figure
6.3B). ARMC8α was PCR amplified using a range of 1-2mM MgCl2 and an annealing
temperature of 55°C for 35 cycles, with analysis of the PCR products by electrophoresis
in a 2% agarose gel verifying the presence of the ~566bp ARMC8α fragment at all
MgCl2 concentrations tested (Sections 3.4, 3.6, Figure 6.3C). PCRs optimising the
amplification of C17orf39 were carried out using a range of annealing temperatures
from 53°C-60°C and 1-2mM MgCl2 concentrations for 35 cycles (Sections 3.4, 3.6).
Electrophoresis of the products identified a band corresponding in size to C17orf39
(~517bp) when an annealing temperature of 55°C and 2mM MgCl2 were used (Sections
3.4, 3.6, Figure 6.3D, E).
To confirm expression of each CTLH complex member in LNCaP cells and to ascertain
their expression in the DU145 prostate cancer and MCF-7 breast cancer cell lines, the
optimised PCR conditions for each complex component were used to amplify each
member using LNCaP, DU145 and MCF-7 cDNA, which was obtained by extraction of
the RNA from each cell line and reverse transcription of the RNA using oligo-dT
primers (Sections 3.2, 3.3). The PCR products were electrophoresed in 2% agarose gels,
verifying the presence of each complex component at the expected molecular sizes in
Chapter 6 Characterisation of RMND5 Protein Binding Partners
237
*
*
C Lane 1: MW Marker Lane 2: 1mM MgCl2 Lane 3: 1.5 mM MgCl2 Lane 4: 2mM MgCl2 Lane 5: Negative Control
Lane 1: MW Marker Lane 2: 53˚C Lane 3: 54˚C Lane 4: 55˚C Lane 5: 56˚C Lane 6: 57˚C Lane 7: Negative control
B
Lane 8: MW Marker Lane 9: 56˚C Lane 10: 57˚C Lane 11: 58.2˚C Lane 12: 59.2˚C Lane 13: 60˚C Lane 14: Negative Control
(i)
(ii)
Lane 1: MW Marker Lane 2: RMND5A (574bp) Lane 3: RMND5B (393bp) Lane 4: RanBPM (641bp) Lane 5: Muskelin (446bp) Lane 6: Negative Control
A
Chapter 6 Characterisation of RMND5 Protein Binding Partners
238
Figure 6.3: Optimisation of RMND5A, RMND5B, RanBPM, muskelin, Twa1, EMP, ARMC8α and C17orf39 PCR conditions. (A) PCRs for RMND5A, RMND5B, RanBPM and muskelin using LNCaP cDNA, 1.5mM MgCl2 and an annealing temperature of 55°C resulted in amplification of products of the expected size. (B) Gradient PCRs using 1.5mM MgCl2 and LNCaP cDNA were performed to optimise the amplification of (i) Twa1 (347bp) and (ii) EMP (504bp). Lower annealing temperatures resulted in the amplification of multiple bands, while higher temperatures yielded a single band of the expected molecular size (*) for both Twa1 and EMP. (C) PCRs to amplify ARCM8α (566bp) were carried out using an annealing temperature of 55°C, LNCaP cDNA and 1mM, 1.5mM or 2mM MgCl2, with all three conditions yielding products of the expected molecular size. (D) Gradient PCRs with annealing temperatures ranging from 53°C to 60°C were performed to amplify C17orf39 (517bp). (E) A prominent band corresponding to C17orf39 at ~500bp was amplified at an annealing temperature of 56°C, which was utilised in further PCRs using 1mM, 1.5mM and 2mM MgCl2. Ten µL of each reaction was electrophoresed in 2% agarose gels to visualise the products.
Lane 1: MW Marker Lane 2: 53˚C Lane 3: 54.2˚C Lane 4: 55˚C Lane 5: 56˚C Lane 6: 57.3˚C Lane 7: 58˚C Lane 8: 59.5˚C Lane 9: 60˚C Lane 10: Negative Control
D
E Lane 1: MW Marker Lane 2: 56˚C (1mM MgCl2) Lane 3: 56˚C (1.5 mM MgCl2) Lane 4: 56˚C (2mM MgCl2) Lane 5: Negative Control
Chapter 6 Characterisation of RMND5 Protein Binding Partners
239
each cell line (Section 3.4, 3.6, Figure 6.4). The amplified PCR products were not
sequenced at this stage to confirm that the appropriate cDNAs had been amplified,
however this could be performed in future studies to continue investigation of CTLH
complex members.
6.2.2 Cloning of RanBPM
The yeast orthologue of RanBPM, Gid1/Vid30, is a proposed core component of the
yeast Vid30 complex, suggesting that human RanBPM may fulfil a similar role in the
CTLH complex (Pitre et al., 2006). This is supported by the finding that RanBPM
interacts with each complex member in coimmunoprecipitation assays in Cos-7 cells
(Kobayashi et al., 2007). To investigate its similar function in prostate cancer cells, the
interaction between RMND5A and RanBPM was verified in the LNCaP cell line, and to
examine the potential for RMND5B to join or replace RMND5A in the CTLH complex,
interaction between RanBPM and RMND5B was also investigated. To perform these
assays, the RanBPM coding region was cloned into the pmCherry-C1 expression vector
to allow the expression of Cherry-RanBPM. Two isoforms of RanBPM have been
identified, a full length 90kDa isoform, and a smaller 55kDa isoform which has a
truncated amino-terminus but does not lack the currently identified protein domains
(Nakamura et al., 1998; Nishitani et al., 2001; Kobayashi et al., 2007) (Section
6.1.4.1). The PCR primers used to amplify RanBPM (Section 6.2.1) would not
distinguish between the 90kDa and 55kDa isoforms.
6.2.2.1 Cloning of Full Length RanBPM (90kDa)
To clone the 90kDa RanBPM isoform, PCR primers were designed to amplify the entire
RanBPM coding region using the RanBPM1-S and RanBPM2190-AS primers from
LNCaP, DU145 and MCF-7 cDNA, however, no products were amplified even after
extensive optimisation with a range of annealing temperatures, MgCl2 concentrations,
high fidelity and high GC content buffers, DMSO, the use of different RNA extraction
methods, reverse transcriptase enzymes and Taq polymerases (Section 3.4, 3.6, not
shown, and Figure 6.5). The second approach was to amplify the RanBPM coding
region in two fragments, and using an endogenous XbaI site, the two fragments were to
be ligated into the pGEM®T-Easy cloning vector (Figure 6.5). However, again, no
bands were present upon agarose gel electrophoresis of the PCR products (Section 3.4,
3.6, not shown). Therefore, amplification of the smaller 55kDa isoform of RanBPM
Chapter 6 Characterisation of RMND5 Protein Binding Partners
240
Figure 6.4: The CTLH complex components are expressed in prostate and breast cancer cells. RNA was extracted from the (A) LNCaP, (B) DU145 (prostate cancer) and (C) MCF-7 (breast cancer) cell lines, reverse transcribed and the resulting cDNA utilised in PCRs for each of the CTLH complex members. Ten µL of each reaction was electrophoresed in 2% agarose gels, identifying that all CTLH complex members were expressed in each cell line.
LNCaP
DU145
MCF-7
A
B
C
Lane 1: MW marker Lane 2: RMND5A (573bp) Lane 3: RMND5B (393bp) Lane 4: RanBPM (618bp) Lane 5: Twa1 (347bp) Lane 6: EMP (504bp) Lane 7: ARMC8α (566bp) Lane 8: Muskelin (446bp) Lane 9: C17orf39 (517bp) Lane 10: Negative Control
Chapter 6 Characterisation of RMND5 Protein Binding Partners
241
XbaI 1 2190 1190
RanBPM1-S
RanBPM 2190-AS
1 2190
1
2190
1259
1029
RanBPM1-S
RanBPM2190-AS
RanBPM 1029-S primer
RanBPM1259-AS
A
B
C
1 2190
SalI
SalI
SalI
HindIII
HindIII
HindIII
687 2190 HindIII SalI
RanBPM2190-AS
RanBPM687-S
D
Chapter 6 Characterisation of RMND5 Protein Binding Partners
242
Figure 6.5: Cloning of the RanBPM coding region. (A) Full length RanBPM is encoded by a 2190bp coding region that (B) could be amplified as a single fragment using the RanBPM1-S and RanBPM2190-AS primers (Appendix II). As the full length isoform could not be amplified in this manner, (C) the second method utilised the presence of an endogenous XbaI restriction site at position 1190bp to aid in the cloning of RanBPM as two fragments using the RanBPM1-S and RanBPM1259-AS primers to amplify the amino-terminal 1259bp and the RanBPM1029-S and RanBPM2190-AS primers to amplify the carboxy-terminal 1162bp of the RanBPM coding region which could both be ligated into the pGEM®T Easy cloning vector using TA cloning. The carboxy-terminal fragment could then be excised from the pGEM®T Easy cloning vector by restriction enzyme digestion with XbaI and SalI and the pGEM®T Easy cloning vector containing the amino-terminal fragment digested with XbaI to and SalI (at the 3’ end of the multiple cloning site of pGEM®T Easy) to allow the ligation of the second fragment into the cloning vector containing the first 1259bp fragment and thereby the production of full length RanBPM (2190bp). (D) If this could not be achieved, the smaller 55kDa isofom of RanBPM (55kDa) which arises from an alternative start site in the RanBPM coding region (687bp) could be PCR amplified using the RanBPM687-S and RanBPM2190-AS primers. The protein sequence encoded by the first 687bp of RanBPM does not contain identifiable protein domains or localisation signals.
Chapter 6 Characterisation of RMND5 Protein Binding Partners
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was pursued as it was not clear whether the failure to amplify full-length RanBPM was
due to technical difficulties, whether the full-length isoform was not expressed or was
expressed at low levels in LNCaP cells, or whether the full-length RanBPM mRNA was
not reverse transcribed efficiently under the conditions used. Thus, as the 55kDa
isoform of RanBPM has been used in previous studies characterising RanBPM function,
and the isoform retains all identified RanBPM protein domains, this approach was
considered acceptable for these studies (Figure 6.5) (Nakamura et al., 1998).
6.2.2.2 Cloning of the RanBPM 55kDa Isoform into pmCherry-C1
The RanBPM (55kDa) coding region was PCR amplified using the RanBPM687-S and
RanBPM2190-AS primers, a range of annealing temperatures from 52°C-60°C and
LNCaP cDNA (Section 3.2, 3.3, 3.4, Appendix II). The PCR products were
electrophoresed in a 1% agarose gel, identifying a product at the expected size of
~1.5kb at the annealing temperatures of 60°C, 58.6°C and 54.8°C (Figure 6.6A). The
PCR was repeated in quadruplicate at the optimum annealing temperature of 60°C, the
products combined, “A” tails added, the product purified and 5µL electrophoresed in a
1% agarose gel to confirm amplification of the ~1500bp product (Section 3.4, 3.6, 3.7.1,
Figure 6.6B).
To obtain pGEMT-RanBPM (55kDa), 40ng (4µL) purified RanBPM (55kDa) was
ligated into 50ng (1µL) pGEM®T-Easy and the ligation products were transformed into
competent E. coli DH5α cells (Sections 3.8.4, 3.8.5.1, 3.8.6). Colonies were selected by
growth on LB Agar/Ampicillin plates with blue/white colony selection and 6 white
colonies were picked and cultured in LB Broth/Ampicillin overnight (Section 3.8.6,
3.9). Plasmids were isolated by small scale plasmid purification, RNase treated and
EcoRI digested to release the RanBPM (55kDa) insert, and the products electrophoresed
in a 1% agarose gel (Section 3.6, 3.8.2, 3.9, Figure 6.6C). Inserts of the expected size
were identified in all clones, pGEMT-RanBPM (55kDa) clones 1 to 6 were purified and
2µL of each was electrophoresed in a 1% agarose gel (not shown). Based on the gel,
2µL of each of clones 1 to 3 was used in sequencing reactions using the M13-S, M13-
AS, RanBPM1029-S and RanBPM1259-AS primers (Appendix II, Section 3.12).
BLASTTM analysis of the sequencing chromatograms identified that pGEMT-RanBPM
(55kDa) clone 2 was mutation free (not shown).
Chapter 6 Characterisation of RMND5 Protein Binding Partners
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Figure 6.6: Cloning of RanBPM (55kDa) into the pGEM®-T Easy cloning vector. (A) To optimise amplification of the RanBPM (55kDa) isoform (~1500bp), PCRs containing 1.5mM MgCl2 and LNCaP cDNA were performed using annealing temperatures from 52°C to 60°C. (B) Using the optimum annealing temperature (60°C), four 25µL PCRs were performed, the amplified products pooled and purified. A 5µL aliquot of the purified product was electrophoresed in a 1% agarose gel from which the DNA concentration was estimated to be ~10ng/µL. (C) Purified RanBPM (55kDa) was ligated into the pGEM®-T Easy cloning vector, the ligation reaction transformed into DH5α cells and plasmid DNA extracted from 6 colonies. Plasmids isolated from clones 1 to 6 were digested with EcoRI and electrophoresed in a 1% agarose gel. Inserts were identified in all clones.
~3000bp
C
Lane 1: MW marker Lane 2, 4, 6, 8, 10, 12: Undigested cut pGEMT-RanBPM (55kDa) clones 1-6 Lane 3, 5, 7, 9, 11, 13: EcoRI digested pGEMT-RanBPM (55kDa) clones 1-6 Lane 14: MW marker
~540bp ~970bp
Lane 1: MW marker Lane 2: 60˚C Lane 3: 58.6˚C Lane 4: 57.8˚C Lane 5: 56.5˚C Lane 6: 54.8˚C Lane 7: 53.4˚C Lane 8: 52.5˚C Lane 9: 52˚C Lane 10: Negative Control
Lane 1: MW marker Lane 2: Purified RanBPM
(55kDa) Lane 3: Negative Control
A B ~1500bp ~1500bp
1 2 3 4 5 6 7 8 9 10
Chapter 6 Characterisation of RMND5 Protein Binding Partners
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To subclone RanBPM (55kDa) into pmCherry-C1, pGEMT-RanBPM (55kDa) clone 2
was digested with HindIII and SalI to release the insert, the insert was gel purified and
5µL of the purified product was electrophoresed in a 1% agarose gel (Section 3.6, 3.7.2,
3.8.2, Figure 6.7A). pmCherry-C1 was prepared by digestion with HindIII and SalI,
SAP treatment and 2µL product was electrophoresed in a 1% agarose gel (Section 3.6,
3.7.2, 3.8.2, Figure 6.7B). The ligation reaction containing 30ng (6µL) RanBPM
(55kDa) insert and 30ng (2µL) pmCherry-C1, was transformed into competent E. coli
DH5α cells and the transformed bacteria were selected by growth on LB
Agar/Kanamycin (Sections 3.8.4, 3.8.6). Plasmids extracted from 7 colonies were NdeI
digested and the purified products were electrophoresed in a 1% agarose gel (Section
3.6, 3.8.2, 3.9, Figure 6.7C), with products of the expected size identified in pmCherry-
RanBPM (55kDa) clones 1, 2, 3, 4 and 6 (Figure 6.7C). Clones 1-3 were purified and
sequenced using the RanBPM1-S, RanBPM1029-S, RanBPM2190-AS and
RanBPM1259-AS primers from which pmCherry-RanBPM (55kDa) clone 2 was
verified as mutation free using BLASTTM analysis (Section 3.12, Appendix III).
Large scale preparation of pmCherry-RanBPM (55kDa) clone 2 was performed to
obtain purified plasmid for transfection, the concentration of which was determined
using spectrophotometry to be 1.5µg/µL (Section 3.6, 3.10, Figure 6.7D). To confirm
expression from Cherry-RanBPM (55kDa) clone 2, LNCaP cells were transfected with
4µg pmCherry-RanBPM (55kDa), the cells were harvested at 48 hours post-transfection
and 10µL lysate was electrophoresed in a 12% polyacrylamide gel for western blotting
(Section 3.1.4, 3.15). Cherry western blotting identified a band at the expected
molecular size of ~85kDa in lysates from transfected cells, indicating successful
expression of the pmCherry-RanBPM (55kDa) construct (Figure 6.7E).
6.2.3 Interaction between RanBPM and RMND5A/RMND5B
6.2.3.1 RMND5A Interaction with RanBPM (55kDa)
To investigate the interaction of RMND5A with RanBPM, LNCaP cells growing in
10cm petri dishes were cotransfected with plasmids encoding GFP-RMND5A and
Cherry-RanBPM (55kDa), at 48 hours post-transfection the cells were lysed, an aliquot
was taken (total cellular input) and the lysate was immunoprecipitated with GFP
antibodies (Section 3.1.4, 3.13). The GFP immunocomplexes were electrophoresed in
12% polyacrylamide gels and GFP and Cherry western blotting was performed
Chapter 6 Characterisation of RMND5 Protein Binding Partners
246
Cherryβ-actin
~85kDa
~45kDa
Figure 6.7: Preparation of the pmCherry-RanBPM (55kDa) expression plasmid. (A) To prepare pmCherry-RanBPM (55kDa), pGEMT-RanBPM (55kDa) clone 2 was digested with HindIII and SalI to release the ~1500bp insert, which was gel purified. 5µL of the purified insert was electrophoresed in a 1% agarose gel from which the insert concentration was estimated to be ~5ng/µL. (B) To prepare pmCherry-C1, the plasmid was digested with HindIII and SalI, purified, SAP treated, re-purified and 2µL of the purified product was electrophoresed in a 1% agarose gel from which the concentration was estimated to be ~15ng/µL. (C) pmCherry-RanBPM (55kDa) was digested with NdeI, and the digested products electrophoresed in a 1% agarose gel. Clones 1, 2, 3, 4 and 6 contained an insert of the expected size (~1.16kb). (D) Large scale purification of the pmCherry-RanBPM (55kDa) plasmid was performed, and the purified plasmid was digested with NdeI and electrophoresed in a 1% agarose gel. (E) Expression of RanBPM (55kDa) from the pmCherry-RanBPM (55kDa) plasmid was determined by transfection of LNCaP cells with 4µg pmCherry-RanBPM (55kDa) and Cherry western blotting of lysates from cells harvested 48 hours post-transfection. A Cherry fusion protein of the expected size of ~85kDa was identified in transfected cultures.
E
Lane 1: MW Marker Lane 2: Undigested pmCherry-RanBPM (55kDa) Lane 3: NdeI digested pmCherry-RanBPM
(55kDa)
D
~1.16kb
~5kb
Lane 1: MW Marker Lane 2: 5µL gel purified
RanBPM (55kDa)
~1.5kb
A B
Lane 1: MW Marker Lane 2: 2µL Undigested pmCherry-C1 Lane 3: 2µL HindIII/SalI, SAP digested
pmCherry C1
~4.7kb
C
Lane 1: MW marker Lane 2, 4, 6, 8, 10, 12, 14: Undigested pmCherry-RanBPM55 clones 1-7 Lane 3, 5, 7, 9, 11, 13, 15: NdeI digested pmCherry-RanBPM55 clones 1-7 Lane 16: MW marker
~1.16kb
~5kb
Chapter 6 Characterisation of RMND5 Protein Binding Partners
247
(Section 3.15). A band corresponding in size to GFP-RMND5A at ~70kDa was present
in both the total cellular input and immunoprecipitated sample indicating successful
immunoprecipitation of GFP-RMND5A (Figure 6.8A). No bands were present in the
untransfected and mock immunoprecipitation samples. Western blotting for Cherry-
RanBPM (55kDa) identified a band at ~85kDa corresponding in size to Cherry-
RanBPM (55kDa) in the total cellular input and immunoprecipitated samples, indicating
successful co-immunoprecipitation of Cherry-RanBPM (55kDa) with GFP-RMND5A.
No bands were present in the untransfected and mock immunoprecipitation samples
(Figure 6.8A). These results confirmed an interaction between RMND5A and RanBPM
(55kDa).
6.2.3.2 RMND5B Interaction with RanBPM (55kDa)
To determine whether RMND5B and RanBPM interact in LNCaP cells, cells growing
in 10cm petri dishes were cotransfected with pEGFP-RMND5B and pmCherry-
RanBPM (55kDa) and at 48 hours post-transfection the cells were lysed, and the lysates
immunoprecipitated with GFP antibodies (Section 3.1.4, 3.13). Samples were
electrophoresed in 12% polyacrylamide gels and GFP western blotting verified the
presence of a ~70kDa band corresponding in size to GFP-RMND5B in the
immunoprecipitate, indicating successful GFP immunoprecipitation (Section 6.8B). No
GFP immunoreactive bands were present in the total cellular input indicating that GFP-
RMND5B was present at low levels in this (dilute) fraction (Figure 6.8B). Similarly,
GFP-RMND5B was not detected in the untransfected and mock immunoprecipitated
samples, while Cherry western blotting identified an ~85kDa band corresponding in size
to Cherry-RanBPM (55kDa) in both the total cellular input and immunoprecipitated
samples, with no bands visible in the untransfected and mock immunoprecipitated
controls (Figure 6.8B). These results indicated that RMND5B was also able to associate
with RanBPM (55kDa).
6.2.3.3 RanBPM (55kDa) Interaction with RMND5 proteins
To perform reciprocal coimmunoprecipitation studies to identify coimmunoprecipitation
of RMND5 proteins with Cherry-RanBPM, Cherry-RanBPM immunoprecipitation
using Protein G beads was optimised as the antibody subtype (rat IgG2a) is not
efficiently bound by Protein A (Section 3.15, 3.16, not shown). LNCaP cells growing in
10cm dishes were cotransfected with plasmids encoding GFP-RMND5A and Cherry-
Chapter 6 Characterisation of RMND5 Protein Binding Partners
248
WB: Cherry (RanBPM)
Untransfected
WB: GFP (RMND5B)
IP: GFP (RMND5B)Cotransfected
WB: GFP (RMND5A)
WB: Cherry (RanBPM)
IP: GFP (RMND5A)CotransfectedUntransfectedA
B
~70kDa
~85kDa
~70kDa
~85kDa
Figure 6.8: RMND5A and RMND5B interact with RanBPM in LNCaP cells. LNCaP cells were cotransfected with plasmids encoding Cherry-RanBPM (55kDa) and either GFP-RMND5A or GFP-RMND5B, the cells were harvested 48 hours post-transfection, proteins were immunoprecipitated from cell lysates using anti-GFP antibodies and Protein A beads and immunoprecipitated/coimmunoprecipitated proteins were detected by GFP and Cherry western blotting. (A) In cells cotransfected with GFP-RMND5A and Cherry-RanBPM (55kDa), western blotting detected both proteins in the total cell lysate and in immunoprecipitated samples. (B) In cells cotransfected with GFP-RMND5B and Cherry-RanBPM (55kDa), both proteins were detected in the immunoprecipitated sample and Cherry-RanBPM (55kDa) was detected in the total cell lysate. Levels of GFP-RMND5B were usually too low to be detected by GFP western blotting in total cell lysates (total input). Immunoprecipitated proteins were not detected in lysates of untransfected cells or in mock immunoprecipitation reactions (Mock). Experiments were performed twice and representative blots are shown.
Chapter 6 Characterisation of RMND5 Protein Binding Partners
249
RanBPM (55kDa) and 48 hours post-transfection, the cells were lysed and an aliquot
(total cellular input) taken (Section 3.1.4, 3.13). The remaining lysate was subjected to
immunoprecipitation using either GFP antibodies (mouse IgG1) or Cherry antibodies
(rat IgG2a), the immunoprecipitates were electrophoresed in 12% polyacrylamide gels
prior to GFP and Cherry western blotting (Section 3.13, Figure 6.9A, B). Although
both antibody subtypes, mouse IgG1 and rat IgG2a, should be recognised and bound by
Protein G, western blotting for both GFP-RMND5A or Cherry-RanBPM (55kDa)
identified only a small amount of either GFP-RMND5A or Cherry-RanBPM (55kDa) in
the immunoprecipitated samples, even though both proteins were readily detected in the
total cellular input samples (Figure 6.9A, B). These findings indicated that
immunoprecipitation using Protein G beads was inadequate. To further optimise the
methods, the experiment was repeated using Protein G beads from Miltenyi Biotec,
cells were lysed at 48 hours post-transfection and after an aliquot was taken (total
cellular input) the remaining lysate was immunoprecipitated with a Cherry antibody (rat
IgG2a) and Protein G beads (Section 3.1.4, 3.13). The Cherry immunocomplexes were
electrophoresed in 12% polyacrylamide gels and then analysed by Cherry and GFP
western blotting (Section 3.15, Figure 6.9C). Cherry western blotting identified a band
corresponding to Cherry-RanBPM (55kDa) at ~85kDa in the immunoprecipitated
samples, indicating successful immunoprecipitation. No bands were present in the total
cellular input samples, indicating that Cherry-RanBPM (55kDa) levels were below the
level of detection of the antibody. Similarly no bands were present in the untransfected
and unbound control fractions. GFP western blotting identified bands corresponding to
GFP-RMND5A and GFP-RMND5B in the immunoprecipitated samples at ~70kDa
(Figure 6.9C). Again, no bands corresponding to either GFP-RMND5A or GFP-
RMND5B were present in the total cellular input samples, indicating that these proteins
were present at low levels in this (dilute) fraction. Similarly, no bands were detected in
the untransfected and unbound control samples. These results confirmed the interaction
between RanBPM and RMND5A/RMND5B in LNCaP cells. Therefore, the studies
identified and confirmed RanBPM interaction with both RMND5A and RMND5B in
LNCaP cells.
6.2.4 Colocalisation of RanBPM with RMND5A and RMND5B
To investigate the localisation and colocalisation of RanBPM with RMND5A and
RMND5B using fluorescence microscopy, LNCaP cells growing on coverslips were
Chapter 6 Characterisation of RMND5 Protein Binding Partners
250
IP: Cherry (RanBPM)WB: Cherry (RanBPM)
Immunoprecipitating antibody: Cherry (rat IgG2a)
IP: GFP (RMND5A)WB: GFP (RMND5A)
Immunoprecipitating antibody: GFP (mouse IgG1) A
B
~70kDa
~85kDa
GFP-RMND5A GFP-RMND5B
WB: GFP
WB: Cherry (RanBPM)
IP: Cherry (RanBPM)
~70kDa
~85kDa
C
Figure 6.9: Optimisation of immunoprecipitation reactions using Protein G Sepharose. To optimise immunoprecipitation reactions using Protein G sepharose, LNCaP cells were cotransfected with GFP-RMND5A and Cherry-RanBPM (55kDa), harvested at 48 hours following transfection and immunoprecipitation reactions performed on the cell lysates using either (A) anti-GFP (mouse IgG1) antibody or (B) anti-Cherry (rat IgG2a), with western blotting using the same antibody performed to evaluate the immunoprecipitation reaction. Both of the immunoprecipitating antibodies are reported to be recognised by Protein G. The immunoprecipitated protein of interest was detected in both the total cell lysate and to a lesser extent in the immunoprecipitated samples. (C) Miltenyi Biotec Protein G sepharose was used to immunoprecipitate Cherry-RanBPM with an anti-Cherry antibody (rat IgG2a) and Cherry and GFP western blotting identified Cherry-RanBPM (55kDa) and either GFP-RMND5A or GFP-RMND5B in the immunoprecipitated samples (IP). Experiments were performed twice and representative blots are shown.
Chapter 6 Characterisation of RMND5 Protein Binding Partners
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cotransfected with plasmids encoding Cherry-RanBPM (55kDa) and either GFP-
RMND5A or GFP-RMND5B and at 48 hours post-transfection the cells were prepared
for microscopy (Section 3.1.3, 3.1.4, 3.16). Under these culture conditions, Cherry-
RanBPM (55kDa) displayed a diffuse nuclear and cytoplasmic cellular localisation
when coexpressed with GFP-RMND5A, which also exhibited a diffuse localisation in
the cytoplasm and nucleus (Figure 6.10). These findings are consistent with a previous
report investigating the intracellular localisation of RanBPM and RMND5A in HEK293
cells (Kobayashi et al., 2007). When coexpressed with GFP-RMND5B, both Cherry-
RanBPM (55kDa) and GFP-RMND5B exhibited a diffuse cellular localisation in the
nucleus and cytoplasm, however consistent with a previous study, GFP-RMND5B also
displayed punctate cytoplasmic staining in a proportion of cells (Dawson, 2006) (Figure
6.10). In these cells, Cherry-RanBPM (55kDa) colocalised with GFP-RMND5B in a
proportion of these speckles (Figure 6.10). The results identified that RMND5A and
RMND5B colocalise with RanBPM (55kDa) in LNCaP cells.
6.2.5 Interaction Between RMND5A and RMND5B
To determine whether RMND5 proteins interacted with each other, the
coimmunoprecipitation and colocalisation of RMND5A and RMND5B was investigated
in LNCaP cells. For these studies, RMND5B was initially cloned into the pmCherry
expression vector to allow the expression of a Cherry-RMND5B fusion protein.
6.2.5.1 Cloning of RMND5B into pmCherry-C1
To generate pmCherry-RMND5B, the RMND5B coding region was PCR amplified
using pEGFP-RMND5B as a template, the RMND5BBamHI1-S and RMND5BBamHI-
AS primers, a 55°C annealing temperature and 1-2mM MgCl2 for 35 cycles (Section
3.4). The PCR products were electrophoresed in a 1% agarose gel, identifying a ~1.2kb
product when 1.5mM MgCl2 and 2mM MgCl2 were used (Section 3.6, Figure 6.11A).
The optimised PCR conditions with 2mM MgCl2 were used to amplify RMND5B in
quadruplicate, the products were combined, “A” tails added, and 5µL of the purified
products electrophoresed in a 1% agarose gel (Section 3.4, 3.7.1, Figure 6.11B). To
obtain pGEMT-RMND5B, 80ng (2µL) RMND5B was ligated with 50ng (1µL)
pGEM®T-Easy and the ligation products were transformed into competent E. coli DH5α
(Section 3.8.4, 3.8.6). Transformants were selected by growth on LB Agar/Ampicillin
using blue/white colony selection, plasmids were purified from six white colonies,
Chapter 6 Characterisation of RMND5 Protein Binding Partners
252
Figure 6.10: Cherry-RanBPM (55kDa) colocalises with GFP-RMND5A and GFP-RMND5B. LNCaP cells were cotransfected with plasmids encoding Cherry-RanBPM (55kDa) and either GFP-RMND5A or GFP-RMND5B and the cells fixed and prepared for microscopy 48 hours post-transfection. The three fusion proteins exhibited a diffuse nuclear and cytoplasmic localisation in LNCaP cells with accumulations of GFP-RMND5B also having a punctate appearance. Cherry-RanBPM (55kDa) colocalised with (A) GFP-RMND5A and (B) GFP-RMND5B in both the nucleus and cytoplasm of LNCaP cells (C) Untransfected cells were imaged under the same conditions as the transfected cells (Magnification x1000). The experiment was performed twice and representative results are shown.
Cherry-RanBPM GFP-RMND5B Hoechst33258 Overlay
Cherry-RanBPM GFP-RMND5A Hoechst 33258 Overlay A
B
-ve control (546nm) Hœchst 33258 Overlay C -ve control (488nm)
Chapter 6 The CTLH Complex
253
Figure 6.11: Cloning of RMND5B into pGEM®-T Easy. (A) RMND5B was amplified from the pEGFP-RMND5B expression vector using 1-2mM MgCl2 and 55°C annealing temperature for 35 cycles. PCR products (10µL) were electrophoresed in a 1% agarose gel. (B) RMND5B was amplified using the optimised PCR conditions, the PCR products purified and 5µL electrophoresed in a 1% agarose gel from which the concentration was estimated to be 40ng/µL. (C) Plasmids isolated from pGEMT-RMND5B clones 1 to 6 were digested with BamHI to release the ~1.2kb insert and the products electrophoresed in a 1% agarose gel. Inserts were identified in all clones.
Lane 1: MW Marker Lane 2: 1mM MgCl2 Lane 3: 1.5mM MgCl2 Lane 4: 2mM MgCl2 Lane 5: Negative Control
Lane 1: MW Marker Lane 2: RMND5B Lane 3: Negative Control
~1.2kb
~1.2kb
A
Lane 1: MW Marker Lane 2, 4, 6, 8, 10, 12: Undigested pGEMT-RMND5B clones 1-6 Lane 3, 5, 7, 9, 11, 13: BamHI digested pGEMT-RMND5B clones 1-6
~1.2kb
~3kb
C
Chapter 6 Characterisation of RMND5 Protein Binding Partners
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RNase treated then BamHI digested to release the ~1.2kb insert and the products
electrophoresed in a 1% agarose gel (Section 3.6, 3.8.2, 3.9, Figure 6.11C). The ~1.2kb
RMND5B insert was identified in all 6 clones, and to confirm the correct insert
sequences, pGEMT-RMND5B clones 1-3 were purified then sequenced using M13-S
and M13-AS primers (Appendix II, Section 3.12), with BLASTTM analysis of the
chromatograms identifying pGEMT-RMND5B clone 1 to be mutation free (not shown).
To subclone RMND5B into the pmCherry-C1 expression vector, the RMND5B insert
from pGEMT-RMND5B clone 1 was released by BamHI digestion, the insert was gel
purified and 5µL purified plasmid was electrophoresed in a 1% agarose gel (Section 3.6,
3.7.2, 3.8.2, Figure 6.12A). pmCherry-C1 was prepared by BamHI digestion and SAP
treatment, and 5µL purified plasmid was electrophoresed in a 1% agarose gel (Section
3.6, 3.8.2, 3.8.3, Figure 6.12B). Sixty ng (3µL) RMND5B insert was ligated into 50ng
(1µL) pmCherry-C1 and the ligation products were transformed into competent E. coli
DH5α then selected by growth on LB Agar/Kanamycin (Section 3.8.4, 3.8.6). Plasmids
purified from 16 colonies were digested with RNase then BamHI and an aliquot of each
plasmid was electrophoresed in a 1% agarose gel, from which ~1.2kb (RMND5B)
inserts were identified in pmCherry-RMND5B clones 2, 7 and 11 (Section 3.6, 3.8.2,
3.9, Figure 6.12C). These clones were purified, digested with KpnI to determine insert
orientation and the products were electrophoresed in a 2% agarose gel (Section 3.6,
3.7.1, 3.8.2, Figure 6.12D). The presence of a ~1kb band upon digestion of pmCherry-
RMND5B clone 7 indicated that the RMND5B insert was in the sense orientation
(Figure 6.12D). pmCherry-RMND5B clone 7 was sequenced using the
RMND5BBamHI1-S, RMND5BBamHI1182-AS and RMND5B790-S primers and
BLAST™ analysis of the chromatograms identified that this clone was mutation free
(Section 3.12, Appendix II, Appendix III).
To prepare pmCherry-RMND5B for transfection, a large scale plasmid preparation of
pmCherry-RanBPM was performed and an aliquot of the purified plasmid
electrophoresed in a 1% agarose gel (Section 3.6, 3.10, Figure 6.12E). The plasmid was
also BamHI digested to verify the presence of the ~1.2kb RMND5B insert and the
concentration of the plasmid was determined to be 1.6µg/µL (Section 3.6, 3.8.2, 3.10,
Figure 6.12E). To investigate expression of Cherry-RMND5B, LNCaP cells were
transfected with 4µg pmCherry-RanBPM and at 48 hours post-transfection the cells
Chapter 6 The CTLH Complex
255
14
~5.9kb ~5.2kb
~1kb
~280bp
Lane 1: MW Marker Lane 2, 4, 6: Undigested pmCherry-RMND5B Clones 2, 3, 11 Lane 3, 5, 7: KpnI digested pmCherry-RMND5B Clones 2, 3, 11
D
~1.2kb
Lane 1: MW Marker Lane 2: RMND5B (clone 1)
~5kb
Lane 1: MW Marker Lane 2: Undigested pmCherry C1 Lane 3: BamHI/SAP digested pmCherry C1
A
C
~5kb ~1.2kb
~5kb
~1.2kb
Lanes 1, 18: MW Marker Lanes 2, 4, 6, 8, 10, 12, 14, 16, 19, 21, 23, 25, 27, 29, 31: Undigested pmCherry-RMND5B clones 1-16 Lanes 3, 5, 7, 9, 11, 13, 15,17, 20, 22, 24, 26, 28, 30, 32: BamHI digested pmCherry-RMND5B clones 1-16
1 2 3 4 5 6 7 8 9 10 11 12 13 15 16 17
18 19 20 21 23 24 25 26 27 28 29 30 31 32 22
B
Chapter 6 The CTLH Complex
256
Cherry
β-actin
~75kDa
~45kDa
F
Figure 6.12: Cloning of RMND5B into pmCherry-C1. (A) pGEMT-RMND5B clone 1 was digested with BamHI to release the insert, the ~1.2kb insert was gel purified and 5µL was electrophoresed in a 1% agarose gel from which the concentration was estimated to be ~20ng/µL. (B) pmCherry-C1 was prepared by digesting the plasmid with BamHI and SAP, followed by purification and electrophoresis of 5µL prepared plasmid in a 1% agarose gel. The plasmid concentration was estimated to be ~50ng/µL. (C) pmCherry clones 1-16 were digested with BamHI which identified that clones 2, 7 and 11 contained the ~1.2kb RMND5B insert. (D) To determine the orientation of the RMND5B insert, pGEMT-RMND5B clones 2, 7 and 11 were digested with KpnI and electrophoresed in a 1% agarose gel. Digestion of clones 2 and 11 produced bands at approximately 5.9kb and 280bp (antisense orientation), while KpnI digestion of clone 7 resulted in a ~1kb band (sense orientation). (E) Large scale plasmid preparation of pmCherry-RMND5B was performed and BamHI digestion released the ~1.2kb insert. (F) Expression of Cherry-RMND5B was confirmed by transfection of LNCaP cells with pmCherry-RMND5B and anti-Cherry western blotting performed using lysates prepared at 48 hours post-transfection.
~4.7kb
~1.2kb
Lane 1: MW Marker Lane 2: Undigested pmCherry-RMND5B Lane 3: BamHI digested pmCherry-RMND5B
E
Chapter 6 Characterisation of RMND5 Protein Binding Partners
257
were harvested, an aliquot electrophoresed in a 12% polyacrylamide gel and Cherry
western blotting was performed (Section 3.1.4, 3.15). Western blotting identified a
~75kDa band corresponding to Cherry-RMND5B in lysates from transfected cultures
only, confirming expression of the Cherry-RMND5B fusion protein (Figure 6.12F).
6.2.5.2 RMND5A and RMND5B Interact in LNCaP Cells
To examine whether RMND5 proteins interact in LNCaP cells, cells growing in 10cm
petri dishes were cotransfected with plasmids encoding GFP-RMND5A and Cherry-
RMND5B (Section 3.1.4). At 48 hours post-transfection, the cells were lysed, an aliquot
taken (total cellular input) and the lysate immunoprecipitated with GFP antibodies
(Section 3.13). GFP immunocomplexes were electrophoresed in 12% polyacrylamide
gels and GFP and Cherry western blotting was performed (Section 3.15). GFP western
blotting identified a ~70kDa band corresponding in size to GFP-RMND5A in the total
cellular input and in immunoprecipitated samples, indicating successful GFP
immunoprecipitation (Figure 6.13A). No bands were present in lysates from
untransfected cells or in mock immunoprecipitated samples. Cherry western blotting
identified a band corresponding to Cherry-RMND5B in the total cellular input and the
immunoprecipitated samples, indicating that RMND5A interacts with RMND5B
(Figure 6.13A). Again, no bands were detected in lysates from untransfected cells and
in mock immunoprecipitated samples.
6.2.5.3 RMND5A and RMND5B Colocalise in LNCaP Cells
To assess whether RMND5 proteins colocalise, LNCaP cells growing on coverslips
were cotransfected with plasmids encoding GFP-RMND5A and Cherry-RMND5B and
at 48 hours post-transfection the cells were prepared for fluorescence microscopy
(Section 3.1.3, 3.1.4, 3.16). Both RMND5 proteins exhibited a diffuse nuclear and
cytoplasmic distribution when coexpressed, which was consistent with previous results
in cells overexpressing either RMND5A or RMND5B alone (Section 4.2.6.4, 6.13B). In
a proportion of cells coexpressing GFP-RMND5A and Cherry-RanBPM, both proteins
also exhibited a punctate cytoplasmic distribution. This was more pronounced compared
to cells overexpressing only one of the RMND5 proteins although it was not possible to
quantitate these results due to a lack of suitable equipment (Figure 6.13B). The
colocalisation of RMND5 proteins in the nucleus and cytoplasm and in punctate
cytoplasmic speckles was indicated by yellow staining when the GFP-RMND5A and
Chapter 6 Characterisation of RMND5 Protein Binding Partners
258
WB: Cherry (RMND5B)
WB: GFP (RMND5A)
IP: GFP (RMND5A)
~75kDa
~70kDa
Figure 6.13: RMND5A and RMND5B interact and colocalise in LNCaP cells. (A) To assess whether RMND5A and RMND5B interact, LNCaP cells were transiently transfected with plasmids encoding GFP-RMND5A and Cherry-RMND5B. At 48 hours post-transfection, the cells were lysed and GFP-RMND5A was immunoprecipitated. GFP and Cherry western blotting identified that GFP-RMND5A and Cherry-RMND5B were both present in the total cell lysate and immunoprecipitated samples. (B) RMND5 protein colocalisation was determined by cotransfection of LNCaP prostate cancer cells with plasmids encoding GFP-RMND5A and Cherry-RMND5B and preparation of cells for fluorescence microscopy at 48 hours post-transfection. GFP-RMND5A and GFP-RMND5B displayed colocalisation in the nucleus and cytoplasm and in punctate cytoplasmic speckles (Magnification x1000). Experiments were performed twice and representative results are shown.
A
B GFP-RMND5A Cherry-RMND5B Hœchst 33258 Overlay
GFP-RMND5A Cherry-RMND5B Overlay Hœchst 33258
-ve control (488nm) Hœchst 33258 Overlay -ve control (546nm)
Chapter 6 Characterisation of RMND5 Protein Binding Partners
259
Cherry-RMND5B images were overlayed. These findings therefore demonstrated that
RMND5A and RMND5B colocalise in LNCaP cells.
6.2.6 Mass Spectrometric Identification of RMND5 Binding Partners
Both RMND5A and RMND5B were found in this thesis to possess E3 ubiquitin ligase
activity and to ubiquitinate the prostatic tumour suppressor NKX3.1, targeting it for
proteasomal degradation. As NKX3.1 expression is localised to the prostate and
RMND5 proteins are widely expressed, they are potentially able to ubiquitinate proteins
in the other tissue types in which they are expressed, and in all tissues would be able to
ubiquitinate multiple proteins. Furthermore, RMND5 proteins contain multiple protein-
protein interaction domains, suggesting that they possess cellular functions distinct from
their E3 ubiquitin ligase activity. The identification of RMND5 binding partners may
elucidate additional cellular roles of RMND5 proteins, for example by determining the
outcome(s) of the interaction. To commence this investigation, which may be continued
in future studies, RMND5 binding partners were identified using mass spectrometry.
For these studies 1D nano liquid chromatography electrospray ionisation-MS/MS was
used to determine proteins co-immunoprecipitating with either GFP-RMND5A or GFP-
RMND5B in LNCaP cells.
6.2.6.1 Identification of RMND5A Binding Proteins
To determine RMND5A binding partners, LNCaP cells growing in 4 x 10cm petri
dishes were transfected with pEGFP-RMND5A and at 48 hours post-transfection, the
cells were lysed, an aliquot was taken and the lysates were combined and
immunoprecipitated using GFP antibodies bound to Protein A beads (Section 3.1.4,
3.13). For the control, 4 petri dishes of untransfected cells were treated in the same
manner (Section 3.13). Samples were electrophoresed in 12% polyacrylamide gels and a
5µL aliquot was separately analysed by GFP western blotting, which identified a
~70kDa band corresponding in size to GFP-RMND5A in the total input and
immunoprecipitated sample, confirming successful GFP immunoprecipitation (Section
3.15, Figure 6.14A). A band corresponding to GFP-RMND5A was also present in the
unbound fraction, indicating that GFP-RMND5A immunoprecipitation was incomplete.
No bands were present in the mock immunoprecipitated samples. The remaining
immunoprecipitate, which was also electrophoresed in a 12% polyacrylamide gel, was
stained with Coomassie blue to visualise immunoprecipitated proteins (Section 3.15).
Chapter 6 Characterisation of RMND5 Protein Binding Partners
260
Eight bands present in the immunoprecipitate which were not present in the mock
immunoprecipitated control were excised from the gel, dried and sent to the Australian
Proteome Analysis Facility (APAF) for mass spectrometric analysis (Section 3.17,
Figure 6.14B). At APAF, all bands were cut into smaller pieces, destained, dried, then
trypsin digested from which the peptides were extracted using acetonitrile/formic acid
then concentrated ready for analysis by 1D nanoLC ESI-MS/MS (Section 3.17). Band 1
was analysed separately, and a small portion of each of bands 1-8 were combined and
analysed by 1D nanoLC ESI-MS/MS, resulting in the identification of multiple proteins
from the peptide fragments by analysis of the raw data using MASCOT, a program that
uses mass spectrometry data to identify proteins from primary sequence databases. To
screen the results, all peptide matches to a particular protein were individually analysed,
with those peptides having an individual ion score of >30 from the mass spectrometry
data indicating identity or extensive homology (p<0.05) to the matched protein.
Additionally, all proteins represented by two or more unique peptides with an individual
ion score >30 were considered to be positively identified in the screen.
The ~70kDa band 1, which corresponded in size to GFP-RMND5A, was found to
contain RMND5A (score = 369) when the raw mass spectrometry data was analysed
using MASCOT (Appendix IV, Figure 6.14C) (Perkins et al., 1999). RMND5A was the
seventh most abundant protein identified in this gel band and upon analysis, the peptide
coverage of RMND5A was 33% with 9 unique peptides (individual ion score >30)
(Appendix IV, Figure 6.14C, D). RMND5A was also identified in the combined
analysis of bands 1-8 (score = 113), with 4 unique peptides (individual ion score >30)
which were also present in band 1. The remaining list of identified proteins (from band
1 and bands 1-8) was then filtered against two contaminants databases, the Max Planck
Institute contaminants database (www.mpg.de/en) and the common repository of
adventitious proteins (cRAP) database (www.thegpm.org/crap/index.html), which
resulted in the removal of keratins and other proteins that are considered to be external
contaminating proteins and resulting in the identification of multiple putative RMND5A
binding partners (Table 6.2).
Chapter 6 Characterisation of RMND5 Protein Binding Partners
261
1 2 3 4 5
4 3
1 7
6 5 2 8
A
B
Lane 1: Total input (mock immunoprecipitated control) Lane 2: Total input (immunoprecipitated sample) Lane 3: Molecular weight marker Lane 4: Mock immunoprecipitation control Lane 5: Immunoprecipitated sample C
Chapter 6 Characterisation of RMND5 Protein Binding Partners
262
Figure 6.14: Immunoprecipitation of GFP-RMND5A and its binding partners. To identify RMND5A binding partners, LNCaP cells were transfected with plasmid encoding GFP-RMND5A and at 48 hours post-transfection, GFP immunoprecipitation was performed followed by electrophoresis of the immunoprecipitated proteins in 12% polyacrylamide gels. (A) GFP western blotting identified a ~70kDa band corresponding to GFP-RMND5A in both the total input and immunoprecipitated samples. (B) Coomassie blue staining identified multiple bands in the immunoprecipitated sample that were not present in the mock immunoprecipitated control. These bands (bands 1-8) were excised and analysed by 1D nano LC ESI/MS/MS to identify GFP-RMND5A and its binding partners (C) RMND5A was the seventh most abundant protein in band 1 with a score of 369 and 9 unique peptides corresponding to RMND5A identified. (D) Analysis of the RMND5A matches identified peptides distributed over the length of the protein and covering 33% of the protein sequence (red). Numbers show amino acid residues.
D
MDQCVTVERELEKVLHKFSGYGQLCERGLEELIDYTGGLKHEILQSHGQDAELSGTLSLVLTQCCKRIK
DTVQKLASDHKDIHSSVSRVGKAIDKNFDSDISSVGIDGCWQADSQRLLNEVMVEHFFRQGMLDVAEEL
CQESGLSVDPSQKEPFVELNRILEALKVRVLRPALEWAVSNREMLIAQNSSLEFKLHRLYFISLLMGGT
TNQREALQYAKNFQPFALNHQKDIQVLMGSLVYLRQGIENSPYVHLLDANQWADICDIFTRDACALLGL
SVESPLSVSFSAGCVALPALINIKAVIEQRQCTGVWNQKDELPIEVDLGKKCWYHSIFACPILRQQTTD
NNPPMKLVCGHIISRDALNKMFNGSKLKCPYCPMEQSPGDAKQIFF
1
391
Chapter 6 Characterisation of RMND5 Protein Binding Partners
263
Table 6.2 – Candidate RMND5A binding partners identified by mass spectrometry
Protein Abbreviation/Name No. of Unique Peptide Hits (band 1/all bands) *
Required for Meiotic Nuclear Division 5A RMND5A 10 and 4
Cytoskeletal
Tubulin TBA1A 5
TBA4A 10
TBB5 10
Actin ACTB 12
Lamin Lamin B1 1
Myosin Unconventional myosin VI 2
Myosin regulatory light chain 10
1
Microtubule Microtubule-actin cross linking factor 1 isoform 1/2/3/5
1
Merlin Merlin 3
Flotillin 2 FLOT2 1
Mitochondrial
ATP synthase subunit (Ox Phos) ATPα 4
ATPβ 8
Isocitrate dehydrogenase (NADP) ICD 1
Phosphoenolpyruvate carboxykinase PEPCK/ PCKGM 1
Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex
ODP2 0
Stress 70 protein (mitochondrial HSP) GRP75 9 and 1
RNA Processing
Heterogeneous nuclear ribonuclearprotein
HNRNPM 11
HNRNPQ 1
HNRNPC 8
HNRNPF 2
HNRNPH1 4
Probable ATP-dependent RNA-helicase DDX41 9 and 4
Chapter 6 Characterisation of RMND5 Protein Binding Partners
264
DDX5 3
DHX35 3
ATP-dependent RNA helicase DDX39A
Polyadenylate binding protein PABP1 8 and 2
PABP5 1
KH domain-containing RNA binding, signal transduction associated protein 1
KHDR1 1
THO complex subunit 3 THOR3 0
SNW domain containing protein 1 SNW1 6
Eukaryotic initiation factor IF4A3 9
IF4A1 1
Elongation factor 1α EF1A1 1
RNA binding Raly-like protein RALYL 1
Splicing factor SF3B3 2
SPF45 1
Poly(U)-binding splicing factor PUF60 18 and 11
Crooked neck-like protein 1 CRNL1 1
Poly (rC) binding protein 1 PCBP1 0
Heat Shock Proteins HSP71 9 and 4
HS71L 0
Heat shock cognate 71kDa HSPC7C 24 and 15
78kDa glucose regulated protein GRP78 9
Miscellaneous
X-ray repair cross complementing protein 6
XRCC6 4
Histone H3 1
Ubiquitin-40S ribosomal protein S27a RS27A 2 and 1
Exportin 2 XPO2 1
Interleukin enhancer binding factor 2 2
Inositol hexakisphosphate and diphosphoinositol-pentakisphosphate kinase 2
VIP2 1
Uncharacterised protein C14orf166B C14orf166B 0
POTE ankyrin domain family member E POTEE 1
Ubiquitin-40S ribosomal protein S27a RS27A 2
* Peptide individual ion score >30
Chapter 6 Characterisation of RMND5 Protein Binding Partners
265
The remaining proteins were filtered for proteins that bind sepharose and magnetic
beads, including cytoskeletal proteins, DEAD box proteins, hnRNPs, and eukaryotic
translation and initiation factors and those proteins that bind the GFP tag alone (Trinkle-
Mulcahy et al., 2008). The remaining proteins were then classified according to their
cellular localisation and/or role (Table 6.3), resulting in the identification of six
potential RMND5A binding partners, two of which are mitochondrial proteins, one of
which is a cytoplasmic protein and the remainder of which are nuclear proteins (Table
6.3). None of the identified proteins were CTLH complex components.
6.2.6.2 Investigation of a Putative RMND5A Binding Partner
Further investigation of the putative RMND5A binding partners identified by mass
spectrometry would involve confirmation of their interaction with RMND5A using
coimmunoprecipitation assays and specific antibodies to individual proteins. As the
number of significant peptide hits identified in the mass spectrometry screen for PUF60
were high, the peptide coverage of PUF60 from peptides identified in the mass
spectrometry screen was 44% and a PUF60 antibody was commercially available, it was
selected for further analysis (Figure 6.15A). Following purchase of the antibody, PUF60
western blotting was performed using LNCaP whole cell lysates and the optimum
primary antibody concentration was determined to be 1:1000 (Section 3.15, not shown).
To confirm the mass spectrometry results, LNCaP cells were transfected with plasmid
encoding GFP-RMND5A and at 48 hours post-transfection, the cells were lysed, an
aliquot taken (total input) and the remaining lysate was immunoprecipitated using GFP
antibodies (Section 3.1.4, 3.13). Samples were electrophoresed in 12% polyacrylamide
gels and analysed by GFP and PUF60 western blotting (Section 3.15). GFP western
blotting identified a ~70kDa band corresponding in size to GFP-RMND5A in the total
cellular input and immunoprecipitated fractions, indicating successful GFP-RMND5A
immunoprecipitation (Figure 6.15B). No GFP-immunoreactive bands were detected in
the mock immunoprecipitated control or the unbound fractions. Western blotting for
PUF60 identified a ~60kDa band in the total cellular input, the mock
immunoprecipitated control and immunoprecipitated sample fractions (Figure 6.15B).
These results indicated that PUF60 bound strongly to the bead matrix and was not a
specific GFP-RMND5A binding partner. During the time frame of this thesis, no
additional candidate RMND5A interactors have been investigated.
Chapter 6 Characterisation of RMND5 Protein Binding Partners
266
Table 6.3 – Function of RMND5A binding proteins identified by mass spectrometry
Protein No. of
Unique Peptide Hits*
Protein Coverage
Function Cellular Localisation
RMND5A 10 and 4 33% Member of the CTLH complex Nuclear/ cytoplasmic
Poly(U)-splicing factor PUF60
18 and 11
44% A splicing factor involved in splicing pre-mRNA that recognises 3’ splice sites. Together with another splicing factor U2AF65 enhances splicing. PUF60 can replace U2AF65 in vitro.
Nuclear
ATP synthase subunits α and β, mitochondrial
4 and 8 12.3% and 22%
Both form part of the catalytic core (F1) of the mitochondrial ATP synthase which synthesise ATP during oxidative phosphorylation.
Mitochondria
SNW domain containing protein 1 SNW1
6 17.4% SNW1/Ski interacting protein (SKIP), interacts with the Ski oncoprotein. SNW1 is a transcriptional coactivator or repressor. It binds the ligand binding domain of vitamin D and retinoic acid receptors, augmenting vitamin D, retinoic acid, oestrogen and glucocorticoid gene expression. SNW1 also functions as a splicing factor by interacting with U2AF65, recruiting it to p21 pre-mRNA. SNW1 controls BMP signalling in embryogenesis.
Mainly nuclear some light cytoplasmic
X-ray repair cross-complementing protein 6 XRCC6
4 13.6% XRCC6/ Ku70 plays a role in telomere maintenance by functioning as an ATP dependent DNA dependent helicase (together with Ku80 XRCC6 forms the Ku autoantigen nuclear complex), which is involved in DSB repair and antibody VDJ recombination, both by the NHEJ DNA repair pathway.
Nuclear
Ubiquitin-40S ribosomal protein S27a
2 21.1% Fusion protein between ubiquitin and ribosomal protein S27a. Post-translationally modified to produce ubiquitin and S27a, a component of the 40S ribosome.
Cytoplasmic
*Peptide individual ion score >30
Chapter 4 Characterisation of RMND5 Protein Binding Partners
267
Figure 6.15: Identification of putative GFP-RMND5A binding partners. PUF60 was the fourth most abundant protein present in band 1 (score=483) and the most abundant protein present when all bands in the immunoprecipitated sample were combined (bands 1-8, score=1245) for analysis. (A) Peptides matching PUF60 were located throughout the protein and covered 44% of the PUF60 amino acid sequence. (B) To assess whether PUF60 was a GFP-RMND5A binding partner, LNCaP cells were transfected with plasmid encoding GFP-RMND5A and harvested at 48 hours post-transfection for GFP immunoprecipitation. Electrophoresis of the immunoprecipitated proteins in 12% polyacrylamide gels and GFP western blotting identified a ~70kDa band corresponding in size to GFP-RMND5A in both the total input and immunoprecipitated sample. PUF60 western blotting identified a ~60kDa band in the total input, immunoprecipitated and mock immunoprecipitated control fractions indicating that PUF60 nonspecifically binds to the Protein A beads. Numbers indicate amino acid residues.
A PUF60
B
MATATIALQVNGQQGGGSEPAAAAAVVAAGDKWKPPQGTDSIKMENGQSTAAKLGLPPLTPEQQEALQKA
KKYAMEQSIKSVLVKQTIAHQQQQLTNLQMAAVTMGFGDPLSPLQSMAAQRQRALAIMCRVYVGSIYYEL
GEDTIRQAFAPFGPIKSIDMSWDSVTMKHKGFAFVEYEVPEAAQLALEQMNSVMLGGRNIKVGRPSNIGQ
AQPIIDQLAEEARAFNRIYVASVHQDLSDDDIKSVFEAFGKIKSCTLARDPTTGKHKGYGFIEYEKAQSS
QDAVSSMNLFDLGGQYLRVGKAVTPPMPLLTPATPGGLPPAAAVAAAAATAKITAQEAVAGAAVLGTLGT
PGLVSPALTLAQPLGTLPQAVMAAQAPGVITGVTPARPPIPVTIPSVGVVNPILASPPTLGLLEPKKEKE
EEELFPESERPEMLSEQEHMSISGSSARHMVMQKLLRKQESTVMVLRNMVDPKDIDDDLEGEVTEECGKF
GAVNRVIIYQEKQGEEEDAEIIVKIFVEFSIASETHKAIQALNGRWFAGRKVVAEVYDQERFDNSDLSA
1
559
Chapter 6 Characterisation of RMND5 Protein Binding Partners
268
6.2.6.3 Identification of RMND5B Binding Proteins
To identify RMND5B binding partners, LNCaP cells growing in 4 x 10cm petri dishes
were transfected with plasmid encoding GFP-RMND5B and at 48 hours post-
transfection, the cells were lysed, a total cellular input sample was taken and the
remaining lysate was immunoprecipitated using GFP antibodies (Section 3.1.4, 3.13).
For the mock immunoprecipitation controls, the same procedure was performed using
untransfected LNCaP cells (Section 3.13). A 5µL aliquot of each sample was
electrophoresed in a 12% polyacrylamide gel and analysed by GFP western blotting,
which identified a ~70kDa band corresponding in size to GFP-RMND5B in the total
input and immunoprecipitated samples, indicating successful immunoprecipitation
(Figure 6.16A). No bands were present in the mock immunoprecipitation control sample
or the unbound fraction. The remainder of the immunoprecipitated samples were
electrophoresed in a precast 12% polyacrylamide gel (to minimise the presence of
contaminating proteins) and stained with Coomassie blue to visualise the proteins
(Section 3.15, Figure 6.16B). The mock immunoprecipitated control contained a
prominent band at ~70kDa as well as a number of minor bands and the lane was divided
into 4 bands (Figure 6.16B). The immunoprecipitated sample, which was
electrophoresed in two lanes due to its higher volume (~60µL), was separated into 6
bands. Band 1 comprised a section of the gel at ~70kDa from both lanes and the
remainder of lane 3 was separated into 5 bands (bands 2-5), each of which was dried
and sent to APAF for mass spectrometric identification of the protein bands (Section
3.17, Figure 6.16B).
For mass spectrometry analysis at APAF, the mock immunoprecipitated control bands
(Mock bands 1-4, Figure 6.16B) and immunoprecipitated sample bands (IP bands 1-6,
Figure 6.16B) were cut into smaller fragments, destained, dried, trypsin digested and the
peptides extracted with acetonitrile/formic acid and then concentrated (Section 3.17).
Five µL of the mock immunoprecipitated bands (M1-4) were combined and 5µL of the
immunoprecipitated sample bands (IP 1-6) were combined and from each combined
sample, a 5µL aliquot was analysed separately by 1D nanoLC ESI-MS/MS. The raw
data from the mass spectrometry screen was analysed by MASCOT, resulting in the
identification of multiple peptides corresponding to numerous proteins (Appendix IV)
(Perkins et al., 1999). The peptide matches to a particular protein were analysed
individually. An ion score of >30 for a peptide from the mass spectrometry data
indicated identity or extensive homology (p<0.05) to the matched protein. Where two or
Chapter 6 Characterisation of RMND5 Protein Binding Partners
269
more unique peptides were identified with an ion score >30 for a particular protein, it
was considered a positive identification of that protein.
The mock immunoprecipitated control sample contained proteins known to be external
contaminants as well as proteins that have been reported to bind non-specifically to the
magnetic bead matrix used in the immunoprecipitation reaction (Trinkle-Mulcahy et al.,
2008). PUF60 was identified among these proteins (score = 1105) as were other splicing
factors, multiple keratins, heat shock proteins belonging to the hsp70 family, hnRNPs,
the cytoskeletal protein actin, eukaryotic transcription initiation and elongation factors
and DEAD box proteins (Appendix IV). Similarly, the immunoprecipitated sample also
yielded proteins that have been determined to be external contaminants and to bind the
bead matrix utilised. In the initial analysis, all of the proteins identified in the mass
spectrometric screens to present in both the immunoprecipitated sample and the mock
immunoprecipitated control were removed. The list of remaining that
immunoprecipitated with GFP-RMND5B was filtered against two contaminants
databases, the Max Planck Institute contaminants database (www.mpg.de/en) and the
common repository of adventitious proteins (cRAP) database
(www.thegpm.org/crap/index.html). The remaining proteins in the GFP-RMND5B
immunoprecipitated sample were classified according to their cellular localisation
and/or role (Table 6.4).
Of the proteins identified in the immunoprecipitated but not mock immunoprecipitated
samples, albumin and Elongation factor 1 alpha 1 have been identified as binding to
sepharose bead proteomes and therefore are considered unlikely to be specific binding
partners of GFP-RMND5B at this stage (Trinkle-Mulcahy et al., 2008). RMND5B
(score = 70) was identified in this screen from two unique peptides with individual ion
scores of >30, which gave a peptide coverage of 4.6% for RMND5B (Figure 6.16C, D).
One protein identified in this screen that obtained two unique peptide matches
(individual ion score >30), was ubiquitin-40s ribosomal protein S27a (score = 97) which
yielded a peptide coverage of 21.1% and also immunoprecipitated with GFP-RMND5A
(Figure 6.16E, F, Table 6.3). Other proteins identified obtained unique peptide matches
of 1, which is considered not to represent a positive identification for a particular
protein, however these results will be useful in the analysis of future mass spectrometric
investigations of RMND5B interacting partners. Although many proteins were removed
from the list of potential RMND5B (and RMND5A) interacting proteins due to their
Chapter 6 Characterisation of RMND5 Protein Binding Partners
270
inclusion in lists of known contaminants in this type of assay, both RMND5A and
RMND5B are multidomain proteins that may function in nucleocytoplasmic shuttling or
in other roles involving the cytoskeleton, and therefore specific interaction of RMND5
proteins with a subset of these proteins may be examined in future studies.
Table 6.4 – Putative RMND5B binding partners identified by mass spectrometry
Protein Number of Unique Peptide Hits*
Function Cellular Localisation
RMND5B 2 Unknown Nuclear/ cytoplasmic
Ubiquitin-40s ribosomal protein S27a
2 Ubiquitin-S27A fusion protein post-translationally processed to yield ubiquitin and the ribosomal 40S component S27A.
Cytoplasmic
POTE ankryin domain family member E
1 POTE proteins are expressed in prostate, testis, ovary and placenta. Ten homologues exist with a proposed signalling function.
Plasma membrane
Tubulin beta-4-A chain
1 A beta tubulin isoform, tubulins are a component of microtubules which comprise part of the cytoskeletal structure.
Cytoplasmic
Neprilysin 1 Membrane metallo-endopeptidase. Cytoplasmic
ATP synthase subunit beta
1 Forms part of a mitochondrial ATP synthase. Mitochondrial
Unconventional myosin-VI
1 Unconventional myosin VI is an ATPase that uses actin for the intracellular transport of vesicles and is involved in cell migration.
Nuclear/ cytoplasmic
Elongation factor 1-alpha 1
1 Protein biosynthesis/With PARP1 and TXK forms part of a transcription factor complex that regulates IFN-gamma expression.
Nuclear/ cytoplasmic
Serum albumin 3 Plasma carrier for steroid hormones, heme and fatty acids/Essential for the maintenance of osmotic pressure.
Plasma
* Peptide individual ion score >30
Chapter 6 Characterisation of RMND5 Protein Binding Partners
271
A
1 2 3 4 5
M1
M2
M3
M4
IP2 IP3 IP1 IP4
IP5
IP6
B
Lane 1: Molecular weight marker Lane 2: Mock immunoprecipitated control Lane 3: Molecular weight marker Lane 4: Immunoprecipitated sample 1 Lane 5: Immunoprecipitated sample 2
C
Chapter 6 Characterisation of RMND5 Protein Binding Partners
272
MEQCACVERELDKVLQKFLTYGQHCERSLEELLHYVGQLRAELASAALQGTPLSATLSLVMS
QCCRKIKDTVQKLASDHKDIHSSVSRVGKAIDRNFDSEICGVVSDAVWDAREQQQQILQMAI
VEHLYQQGMLSVAEELCQESTLNVDLDFKQPFLELNRILEALHEQDLGPALEWAVSHRQRLL
ELNSSLEFKLHRLHFIRLLAGGPAKQLEALSYARHFQPFARLHQREIQVMMGSLVYLRLGLE
KSPYCHLLDSSHWAEICETFTRDACSLLGLSVESPLSVSFASGCVALPVLMNIKAVIEQRQC
TGVWNHKDELPIEIELGMKCWYHSVFACPILRQQTSDSNPPIKLICGHVISRDALNKLINGG
KLKCPYCPMEQNPADGKRIIF
Figure 6.16: Immunoprecipitation of GFP-RMND5B and its binding partners. To identify RMND5B interacting proteins, LNCaP cells were transfected with pEGFP-RMND5B and at 48 hours post-transfection, GFP immunoprecipitation was performed followed by electrophoresis of the immunoprecipitated proteins in a precast 12% polyacrylamide gel. (A) GFP western blotting identified a ~70kDa band corresponding to GFP-RMND5B in both the total input (barely visible in image) and immunoprecipitated sample. (B) Coomassie blue staining identified multiple bands in the immunoprecipitated and mock immunoprecipitated sample. The mock immunoprecipitated control sample was divided into 4 bands (M1-4), and the immunoprecipitated sample, which was electrophoresed in two lanes, was separated into eleven bands, six (IP1-6) of which were analysed by mass spectrometry. (C) Two unique peptides were matched to RMND5B (score=70) using MASCOT. (D) Analysis of the peptide matches identified a protein coverage of 4.6%. (E) Ubiquitin-40S ribosomal protein S27a (score=97) obtained two unique peptide matches in the mass spectrometry screen for GFP-RMND5B binding partners. (F) The peptide matches for ubiquitin-40S ribosomal S27a produced a protein coverage of 21.1%. Numbers indicate amino acid residues.
D RMND5B
F Ubiquitin-40S ribosomal protein S27a
MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQ
KESTLHLVLRLRGGAKKRKKKSYTTPKKNKHKRKKVKLAVLKYYKVDENGKISRLRRECPSD
ECGAGVFMASHFDRHYCGKCCLTYCFNKPEDK
E
1
393
1
156
Chapter 6 Characterisation of RMND5 Protein Binding Partners
273
6.3 Discussion
In this chapter, expression of the CTLH complex members in prostate cancer cells was
evaluated using RT-PCR. The method was used to rapidly and qualitatively identify the
expression of the CTLH complex components, however since only mRNA levels were
examined, protein levels of the complex members in the prostate and breast cancer cells
lines used are unknown at this stage. Western blotting was not able to be used to
determine expression of each of the CTLH complex members for this thesis as suitable
antibodies against each complex component are not commercially available. However,
to continue investigation of the CTLH complex members in future studies, real time
PCR (RT-qPCR) may be used to quantitate mRNA levels (VanGuilder et al., 2008) and
the results can be augmented with western blotting as suitable antibodies are generated.
The finding that each complex component is expressed in two prostate and one breast
cancer cell line suggests that it is possible for the CTLH complex to form in these cell
types as has been demonstrated in HEK293 cells (Kobayashi et al., 2007). It is also
possible that the complex exists in many cell types as a large ~670kDa RanBPM
associated complex has been detected in a number of cell lines including HeLa cells
(Nishitani et al., 2001; Ideguchi et al., 2002; Umeda et al., 2003). Members of the
CTLH complex are also widely expressed with EMP, Twa1, muskelin and RanBPM
expression documented in a variety of human and mouse tissues and RanBPM
exhibiting highest expression in the prostate, ovaries and testes (Adams et al., 1998;
Rao et al., 2002; Umeda et al., 2003; Bala et al., 2006).
Interaction of RMND5A and RMND5B with RanBPM was investigated in this study as
a measure of whether the CTLH complex could form in prostate cancer cells using
either RMND5A or potentially RMND5B. These experiments were performed due to
the finding that the yeast orthologue of RanBPM, Vid30 functions as a core component
of the Vid30 complex, and, based on its multidomain structure RanBPM may fulfil a
similar role in the human CTLH complex (Pitre et al., 2006). The formation of the
CTLH complex was identified in initial reports using sucrose gradient sedimentation
and coimmunoprecipitation assays due to the association of the CTLH complex
components with RanBPM, further supporting the hypothesis that RanBPM is a core
component of the complex (Kobayashi et al., 2007). Similarly, RanBPM has been
hypothesised to function as a scaffolding protein, again supporting its role as an integral
member of the CTLH complex (Murrin and Talbot, 2007). Glycerol gradient
sedimentation has been used to demonstrate that yeast RMD5 forms part of a large
Chapter 6 Characterisation of RMND5 Protein Binding Partners
274
~600kDa protein complex, whilst in human HEK293 cells RMND5A was identified as
a CTLH complex member, with both of these findings supporting the inclusion of
RMND5 proteins in the CTLH complex (Regelmann et al., 2003; Kobayashi et al.,
2007). Both RMND5 proteins were confirmed to interact with RanBPM in prostate
cancer cells, providing additional evidence that the CTLH complex may form in this
cell type and further that RMND5B may join or replace RMND5A in the CTLH
complex. Thus, although RMND5 proteins were only identified in this thesis to interact
with a single core member of the CTLH complex, the results suggest that the CTLH
complex can form in prostate cancer cells and provide a starting point for additional
assays confirming the existence of the complex in these cells.
Additional methods for the detection of the CTLH complex in prostate cancer cells
include co-immunoprecipitation assays with each complex member, and although as
mentioned previously antibodies against endogenous members of the complex are not
commercially available, these studies may be performed following cloning and
transfection of the individual complex components into prostate cancer or other cell
types. Results may be confirmed by sucrose gradient centrifugation of the high
molecular weight CTLH complex from prostate cancer cells with mass spectrometry
used to identify complex components (Kobayashi et al., 2007). In the present study,
novel binding partners of RMND5 proteins were isolated by immunoprecipitation of
RMND5A/RMND5B and their interacting partners, with the coimmunoprecipitated
proteins identified using mass spectrometry. Although this is a commonly used method
for the identification of novel proteins, members of the CTLH complex were not were
not detected, which may have resulted from weak or transient interaction between the
complex components or the low levels of expression of complex members in this cell
type (Vasilescu et al., 2004). It is also feasible that the complex forms under specific
cellular conditions that remain to be identified, as has been shown for the yeast Vid30
complex, which is only activated under changing nutrient environments (Santt et al.,
2008).
The interaction of RMND5 proteins with RanBPM was used as an indicator of the
potential formation of the CTLH complex, however for these investigations, the larger,
90kDa isoform of RanBPM could not be isolated from three cell lines, although
multiple optimisation methods were employed. Thus, the smaller, 55kDa isoform of
RanBPM was cloned and used in immunoprecipitation and colocalisation assays with
Chapter 6 Characterisation of RMND5 Protein Binding Partners
275
RMND5 proteins. The 55kDa RanBPM isoform was the initial isoform reported, and in
the literature most studies that use the RanBPM (90kDa) isoform have obtained the
expression construct encoding the isoform from the group that originally described its
isolation (Nishitani et al., 2001). It is therefore possible that other groups have
experienced similar difficulty in amplifying the full length transcript, and indeed,
Nishitani et al. (2001) derived the original construct by amplifying the 5’ region of the
RanBPM (90kDa) isoform and ligating it with the RanBPM (55kDa) isoform,
supporting this hypothesis. PCR difficulties may be due to a stretch of repeating
nucleotides at the 5’ end of the transcript which encodes a string of proline and
glutamine residues in the amino-terminal region of the RanBPM (90kDa) protein
(Nishitani et al., 2001). The RanBPM (55kDa) isoform does not lack identifiable
protein domains and in this study the exogenous Cherry-tagged protein was found to
exhibit the same cellular distribution as that reported for the full length protein
(Kobayashi et al., 2007). Furthermore, RanBPM was able to interact and colocalise with
both RMND5 proteins in prostate cancer cells, which is consistent with previously
reported findings that RMND5A and RanBPM interact and colocalise in HEK293 cells
(Kobayashi et al., 2007). Interestingly, when coexpressed with either RMND5A or
RMND5B, the intracellular localisation of RanBPM was altered and it colocalised with
RMND5 proteins in punctate cytoplasmic speckles. Another CTLH complex
component, muskelin has been reported to exhibit a similar cytoplasmic punctate
appearance that is not associated with aggresomes and is dependent on its discoidin-like
domain and Kelch repeat domain (Prag et al., 2004). Although the localisation of the
three complex members in similar structures is interesting and supports formation of the
CTLH complex, the functional relevance of these cytoplasmic speckles is unknown but
may be investigated in future studies using antibodies or stains for intracellular
organelles such as endosomes or mitochondria.
The function of the CTLH complex has not yet been identified, and individually the
complex members are involved in diverse cellular processes, however there are several
lines of evidence supporting a role for the CTLH complex in protein degradation.
Firstly, as mentioned previously, the yeast Vid30 complex functions as an E3 ubiquitin
ligase complex, and this study has shown that both RMND5 proteins possess E3
ubiquitin ligase activity, which mirrors the function of their yeast orthologue RMD5
that provides the Vid30 complex with its enzymatic activity (Santt et al., 2008).
ARMC8α is reported to be associated with the proteasomal degradation of α-catenin and
Chapter 6 Characterisation of RMND5 Protein Binding Partners
276
enhances the interaction of the endosomal sorting protein HRS with ubiquitinated
proteins, potentially bringing ubiquitinated proteins in close proximity to HRS (Suzuki
et al., 2008; Tomaru et al., 2010), while RanBPM is also associated with the
deubiquitinating enzyme USP11 (Ideguchi et al., 2002). The protein domain
architecture of members of the CTLH complex also supports an E3 ubiquitin ligase
function, with RanBPM and muskelin containing SPRY and Kelch repeat domains,
respectively, both of which function as substrate recognition motifs in proteins forming
part of other E3 ubiquitin ligase complexes (Sun et al., 2009; Kuang et al., 2010; Lee et
al., 2010). Finally, the armadillo repeat domain is also present in proteins that are
associated with protein degradation, either those that possess intrinsic E3 ubiquitin
ligase activity or those that form part of E3 ubiquitin ligase complexes such as F-box
proteins (Tewari et al., 2010). In future studies, E3 ubiquitin ligase activity of the
CTLH complex could be investigated using in vitro ubiquitination assays by
reconstituting the complex either by coimmunoprecipitation of the entire complex or by
production of each of the components in bacterial cells or by other methods including in
vitro transcription/translation (as discussed in Section 4.3) (Skowyra et al., 1999; Chen
et al., 2006b; Ahn et al., 2011). In yeast, RMD5 and Gid9 function together to provide
the Vid30 complex with its E3 ubiquitin ligase activity, and collaboration of the human
orthologues of these factors, RMND5A and EMP may also be specifically investigated
in in vitro ubiquitination assays used to evaluate CTLH complex function (Braun et al.,
2011).
Another common feature of CTLH complex members is their role in cytoskeletal
organisation and cytoskeleton-based intracellular transport. The cytoskeleton consists of
microtubules, intermediate filaments and microfilaments (actin filaments) (Alberts et
al., 2004). Molecular motor proteins use these cytoskeletal elements for intracellular
transport, for example myosin molecules move cargo along actin filament “tracks”
whilst dyneins are microtubule associated proteins that among other functions carry
endosomal vesicles to the lysosome for degradation (Alberts et al., 2004; Nelson et al.,
2005). Actin is also essential for endocytosis and in particular, F-actin is important for
the transport of proteins such as caveolin from the plasma membrane to early
endosomes (Samaj et al., 2004). Members of the yeast Vid30 complex are not only
involved in the proteasomal degradation of FBPase, but under specific conditions,
FBPase is trafficked by members of the Vid30 complex to the endosomal system and
the yeast version of the lysosome, the vacuole for degradation (Brown et al., 2010;
Chapter 6 Characterisation of RMND5 Protein Binding Partners
277
Alibhoy et al., 2012). Specifically, Vid30 associates with Vid vesicles that contain
FBPase and with actin patches, thereby merging the Vid vesicles with the endocytic
pathway (Brown et al., 2010; Alibhoy et al., 2012). Muskelin, EMP, ARMC8α and
RanBPM are all involved in the intracellular transport of cargo or cytoskeletal
organisation, with muskelin mediating the intracellular cytoskeletal response to
thrombospondin and involved in both membrane bound receptor endocytosis and
endocytic transport (Adams et al., 1998; Ledee et al., 2005; Heisler et al., 2011). In a
similar role, muskelin connects F-actin and microtubule based transport of the
GABAAR α1 receptor by interacting with both myosin VI and dynein, thereby bridging
the two cytoskeletal based transport systems (Heisler et al., 2011), and together,
muskelin and RanBPM play a role in nucleocytoplasmic transport (Valiyaveettil et al.,
2008). EMP is associated with F-actin and due to this association, is vital for
enucleation and filopodia formation in erythroblasts and macrophages, respectively
(Soni et al., 2006). The LisH and Kelch repeat domains present in CTLH complex
members are also associated with intracellular transport of cargo, with the LisH domain
functioning as a dynein binding domain and the Kelch repeat an actin binding motif
(Adams et al., 2000; Emes and Ponting, 2001). In the initial screen for RMND5 binding
partners by mass spectrometry, multiple cytoskeletal proteins including actins, tubulins,
myosins, microtubule associated proteins, merlin, flotillin and lamins were identified,
suggesting that RMND5 proteins are associated with cytoskeletal organisation or
transport. However, as cytoskeletal proteins have been documented to non-specifically
bind the sepharose and magnetic bead matrix used, it will be particularly important to
confirm the interaction of individual cytoskeletal proteins with RMND5A or RMND5B
using additional assays (Trinkle-Mulcahy et al., 2008).
The CTLH complex components, RanBPM and RMND5A each have cellular
homologues, suggesting that RanBP10 and RMND5B may replace their paralogue in
the complex, thereby altering its function(s) or targets. Each of the homologous pairs of
proteins exhibits a high degree of amino acid identity, however RanBPM and RanBP10
have been documented to possess different functions while RMND5A and RMND5B
have not been characterised. For example, although both RanBPM and RanBP10 can
interact with the intracellular domain of the human growth factor receptor MET, only
the interaction of MET with RanBPM results in the activation of the Ras/ERK
signalling pathway (Wang et al., 2002a; Wang et al., 2004). RanBP10 has also been
reported as a tubulin binding protein that localises to microtubules in megakaryocytes
Chapter 6 Characterisation of RMND5 Protein Binding Partners
278
thereby playing a role in platelet shape and degranulation (Schulze et al., 2008; Kunert
et al., 2009). This function is consistent with that of other CTLH complex members
which are involved in microtubule dynamics and therefore cytoskeletal organisation.
Formation of the CTLH complex with either or both paralogues may extend the
function or substrates of the complex due to the proposed divergence in function of the
homologues and may be investigated in future studies.
Due to the presence of the LisH and RING dimerisation motifs in RMND5 proteins,
their association with each other was investigated. Many RING domain proteins use this
domain to form homodimers or heterodimers, which can enhance their enzymatic
activity (Section 4.2.1, 4.3), with the LisH domain additionally associated with the
formation of large protein complexes (Ahn et al., 2011). Coimmunoprecipitation and
colocalisation assays demonstrated that RMND5 proteins were able to interact and
colocalise in the nucleus and cytoplasm of prostate cancer cells. The finding suggests
that RMND5 proteins could function as heterodimers, however as RMND5 proteins
share a high degree of amino acid homology it may be that they are able to interact with
each other due to their similarity but that this interaction is not functionally relevant and
that they preferentially form homodimers in vivo. Whether RMND5 proteins
simultaneously form part of the CTLH complex and whether they function as single
subunit or multi-subunit E3 ubiquitin ligases or both remains to be addressed. Several
E3 ubiquitin ligases are reported to function in both modalities including Siah1 which
forms part of an SCF E3 ubiquitin ligase complex that ubiquitinates β-catenin or
alternatively functions as a single subunit that ubiquitinates a number of targets
including synphilin-1 (Liani et al., 2004; Dimitrova et al., 2010). Furthermore, it will be
interesting to determine whether RMND5A and EMP are able to function as E3
ubiquitin ligase heterodimers given the finding that yeast RMD5 and Gid9 function
together in the Vid30 complex (Santt et al., 2008; Braun et al., 2011). Typical E3
ubiquitin ligase complexes contain a single RING or HECT domain containing protein
with associated interchangeable complex members including adaptors and substrate
recognition subunits. For example, SCF and VHL E3 ubiquitin ligase complexes usually
contain the RING domain protein Rbx1, whilst the cullin and adaptor proteins may
change (Willems et al., 2004). Since it is the RING domain that interacts with E2
enzymes, substitution of the RING domain containing protein within an E3 complex
may alter the type of ubiquitination of the substrate proteins.
Chapter 6 Characterisation of RMND5 Protein Binding Partners
279
The cellular localisation of RMND5 proteins is interesting considering the identification
of multiple mitochondrial proteins as potential RMND5A and RMND5B binding
partners, including the α and β subunits of the mitochondrial ATP synthase involved in
oxidative phosphorylation, the TCA cycle enzyme isocitrate dehydrogenase and the
gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK). Although all
protein-protein interactions identified using mass spectrometry need to be confirmed by
further studies, the Vid30 complex ubiquitinates and targets for degradation the
gluconeogenic enzyme FBPase (Santt et al., 2008), and therefore it is feasible that
RMND5 proteins and potentially the CTLH complex, regulate levels of mitochondrial
enzymes by ubiquitination.
The mass spectrometric techniques employed in this study to identify putative RMND5
binding partners were based on the premise that by immunoprecipitating the protein of
interest, the endogenous binding partners of that protein may be coimmunoprecipitated
and therefore identified. Due to technical limitations of this method, it will important in
future studies to confirm protein-protein interactions of individual molecules using
additional methods such as co-immunoprecipitation, GST-pulldown and additionally
colocalisation (Guenther et al., 2000; Liang et al., 2008; Paul et al., 2011). In this study,
different controls were used including a mock reaction where untransfected cells were
immunoprecipitated with the same anti-GFP microbeads used to immunoprecipitate
GFP-RMND5 proteins. In this way, proteins that bound to the microbead matrix could
be identified. A preclearing step prior to immunoprecipitation with the micro beads
alone (unbound with GFP antibody) to remove proteins non-specifically bound to the
beads, was not included in these experiments and is not recommended by the GFP
microbead manufacturers for immunoprecipitation reactions (Vostal and Shulman,
1993; Falsone et al., 2008). Nonetheless this could be added in future experiments. In
the immunoprecipitation using GFP-RMND5A, the bands present in the mock control
were used to guide excision of bands for mass spectrometric analysis, with two
contaminants databases used to eliminate commonly contaminating proteins, including
external contaminants and proteins used in mass spectrometry quantitation (Keller et al.,
2008; Bell et al., 2009). A publication identifying the sepharose and magnetic bead
proteome was also used to eliminate proteins binding to the microbead matrix and GFP
tag (Trinkle-Mulcahy et al., 2008).
Chapter 6 Characterisation of RMND5 Protein Binding Partners
280
Mass spectrometric analysis of both immunoprecipitation assays identified many
common contaminating proteins including keratins and non-specific bead proteome
binding proteins, for example heat shock proteins (Trinkle-Mulcahy et al., 2008). These
proteins were also present in the mock immunoprecipitation control used for RMND5B
immunoprecipitation and thus were eliminated as external contaminants or non-specific
binding proteins. Although precautions were taken when preparing all reagents for
immunoprecipitation and mass spectrometry with ultrapure water, precast acrylamide
gels, nitrile gloves, a face mask and hair clips utilised, keratins were the predominant
proteins identified (Mann et al., 2001). As keratins are external contaminants they are
present on the outside of the gel, on the glass plates and may be added upon gel
handling and as such are exposed to trypsin during protein digestion and ionise
efficiently, resulting in their identification by mass spectrometry (Pandey et al., 2000;
Keller et al., 2008). In the future, mass spectrometry grade reagents and acrylamide gels
and tubes may be utilised to reduce the presence of external contaminants. The GFP tag
has been determined to bind multiple cellular proteins, and although a published
database was used to exclude GFP binding proteins, an additional control which may be
used in future experiments is the expression of the GFP tag alone (empty vector) in
mammalian cells followed by its immunoprecipitation with anti-GFP microbeads
(Trinkle-Mulcahy et al., 2008; Paul et al., 2011).
As a result of using contaminants databases, many proteins were eliminated from the
original mass spectrometry screen, some of which may have been real RMND5 binding
partners. Alternative approaches that may be employed to diminish interference by
non-specific binding proteins involve the use of quantitative-mass spectrometry and
isotope labelling such as stable isotope labelling with amino acids in culture (SILAC),
which includes a negative control to account for proteins non-specifically binding the
affinity matrix or protein tag (Ong et al., 2002). In these studies, the negative control
culture is labelled with light 12C medium and the experimental sample is labelled with
heavy 13C medium (Trinkle-Mulcahy et al., 2008). Following cell lysis the samples are
mixed, resulting in both negative control and experimental samples being
immunoprecipitated simultaneously and analysed by mass spectrometry in the same run,
reducing variability between experiments (Trinkle-Mulcahy et al., 2008). This mass
spectrometry based technique is now widely used to identify novel protein-protein
interactions and its use has been expanded to study post-translational modifications and
Chapter 6 Characterisation of RMND5 Protein Binding Partners
281
to identify cell proteomes under various conditions (Dammer et al., 2012; Deeb et al.,
2012; Udeshi et al., 2012; Yin et al., 2012).
Proteins with significant unique peptide matches of 2 or more were considered a match
for the presence of that protein in this study and therefore among the list of putative
RMND5 binding partners there were a range of proteins with significant hits from 2 to
18 (Higdon and Kolker, 2007). These results will be further analysed in future studies as
the top peptide matches do not necessarily reflect the strength of the protein-protein
interaction and any of the proteins may be genuine binding partners. For the methods
used, differences in binding affinity, the proportion of each protein that interacts with
the protein of interest in the cell at any given time, the abundance of the protein, the
efficiency of trypsin digestion and peptide ionisation may all account for differences in
the number of significant peptide matches identified (Nesvizhskii and Aebersold, 2005;
Higdon and Kolker, 2007; Trinkle-Mulcahy et al., 2008).
Another important element to consider in protein identification by mass spectrometry is
the peptide coverage of the identified proteins as a more complete protein coverage
lends more weight to the presence of that protein in the immunoprecipitate. Although
the entire protein may be present as trypsin digested peptides in the sample, usually only
between 20-30% are identified by mass spectrometry and there are multiple proposed
reasons for this. Some trypsin digested peptides do not ionise well, or due to unknown
mechanisms, the intensity of the peptide may be suppressed in a mixture of peptides,
long stretches of arginine and lysine lacking peptides may fall outside of the measured
m/z interval or the protein may be present at a low abundance (Nesvizhskii and
Aebersold, 2005; Hjerno and Hojrup, 2006). Therefore, whilst the presence of only a
portion of the protein represented by peptide matches does not rule out the presence of
that protein due to the above-mentioned reasons, it is also possible for example that a
degradation product of the protein has non-specifically bound to the protein of interest
or the affinity matrix. When RMND5A was immunoprecipitated, 33% protein coverage
was obtained, and the peptide matches were found throughout the RMND5A protein
sequence, with 9 unique peptide matches identified when protein band 1 was analysed
alone and 4 unique significant peptide matches when all protein bands were combined
and analysed together. In the literature, protein sequence coverage of 14% representing
eight unique peptides has been used as a positive protein identification (Block et al.,
Chapter 6 Characterisation of RMND5 Protein Binding Partners
282
2011). For RMND5B, 2 unique peptides obtained a protein coverage of 4.6% which is
low, however 2 unique peptides is considered a positive protein identification.
The top ranked putative RMND5A binding partner identified was PUF60, which had 18
significant peptide matches accounting for 46% of the PUF60 protein sequence. PUF60
was identified when the band containing only RMND5A was analysed and also when
all 8 protein bands of interest were combined and analysed, and importantly, different
peptides were identified when only band 1 was analysed compared to when all of the
protein bands were combined and analysed. As PUF60 (~60kDa) corresponded in size
to the region excised from the gel (~60-70kDa), the overall findings provided good
support of the further investigation of PUF60 as an RMND5A binding partner. While
other splicing factors were identified and eliminated in this screen due to their reported
ability to bind the affinity matrix (Trinkle-Mulcahy et al., 2008), PUF60 was not
eliminated as it did not appear in the published databases and the number of peptide
matches to PUF60 were high compared to other splicing factors identified (Trinkle-
Mulcahy et al., 2008). Subsequent mass spectrometry analysis of GFP-RMND5B
binding partners including a mock immunoprecipitated control confirmed PUF60 as
binding to the magnetic bead matrix. This experiment therefore highlights the
importance of analysis of the mock immunoprecipitation control by mass spectrometry,
as visual analysis of bands present in the immunoprecipitated sample that are not
present in the mock control is not sufficient.
Proteins identified as putative RMND5A and RMND5B binding partners, with at least 2
unique peptides andbetween 12.3% - 33% protein sequence coverage included
mitochondrial, cytoplasmic protein and nuclear proteins, a splicing factor, a
transcription factor and a protein involved in DNA repair. The transcription factor
SNW1, identified as a putative RMND5A binding partner has been demonstrated to
interact with the 3’ splicing factor U2AF65, in particular in relation to p21 pre-mRNA
splicing, and is therefore hypothesised to couple transcription and RNA splicing (Zhang
et al., 2003; Chen et al., 2011b). PUF60, which was also identified in this screen, is
homologous to and interacts with U2AF65, and PUF60 can substitute for U2AF65 in
vitro (Hastings et al., 2007; Corsini et al., 2009). Therefore, whether SNW1 specifically
interacts with RMND5A or whether SNW1 was coimmunoprecipitated with PUF60,
remains to be determined. However, SNW1 was not identified in the mock
immunoprecipitated control or GFP-RMND5B immunoprecipitated sample mass
Chapter 6 Characterisation of RMND5 Protein Binding Partners
283
spectrometry results, therefore an interaction between RMND5A and SNW1 will be
ascertained by future coimmunoprecipitation reactions.
The peptide hits and sequence coverage identified in the GFP-RMND5B mass
spectrometry screen were low compared to that of the peptide matches obtained when
GFP-RMND5A and its binding partners were analysed by mass spectrometry, which
resulted in the identification of RMND5B and ubiquitin-40s ribosomal protein S27A as
the only two proteins represented by two unique peptides (individual ion score >30).
This may have been due to division of the GFP-RMND5B immunoprecipitation sample
between two lanes for electrophoresis, with only one of the lanes analysed (thereby
diluting the amount of sample used). Additionally, in the GFP-RMND5A mass
spectrometry analysis, individual bands were excised in comparison to the GFP-
RMND5B mass spectrometry screen where large sections of the gel were excised for
analysis. Thus, to concentrate the proteins present, the removal of smaller gel bands
from the gel which may be analysed individually may yield results with higher
scores/peptide matches to each protein. In addition, identification of low abundance
proteins present in each band may be possible, which are not identified when large
sections of gels are combined and analysed.
Although several of the proteins identified in the GFP-RMND5A and GFP-RMND5B
mass spectrometry screens had only one unique peptide (individual ion score >30)
where two or more are generally considered to be a positive identification for a protein,
some of these proteins were identified in both GFP-RMND5A and GFP-RMND5B
mass spectrometry screens. These proteins were ubiquitin-40s ribosomal protein S27A,
mitochondrial ATP synthase subunit β, POTE ankyrin domain family member E,
tubulin β-4A and unconventional myosin-VI. As the proteins were not present in the
mock immunoprecipitation control or in the contaminants databases, the proteins are
potential RMND5 protein binding partners, which may be investigated in future studies
using additional analyses including coimmunoprecipitation assays and colocalisation
microscopy (Ciruela et al., 2010). RMND5A contains a putative GAT-like domain, a
ubiquitin-binding domain (Section 4.2.1, 4.3), therefore if RMND5A/RMND5B is
confirmed to interact with ubiquitin-40S ribosomal protein S27A it will be interesting to
identify the protein domain responsible for this interaction. Additionally, as ubiquitin-
40S ribosomal protein S27A is a fusion protein of ubiquitin and ribosomal protein S27A
prior to its processing, it will be important to determine results of the outcome of this
Chapter 6 Characterisation of RMND5 Protein Binding Partners
284
interaction (Wong et al., 1993). Thus, at the close of this project, RMND5A and
RMND5B have been demonstrated to bind NKX3.1 and RanBPM, with mass
spectrometric analysis of RMND5A and RMND5B interacting proteins identifying
several candidate binding partners. These may be further investigated in conjunction
with additional mass spectrometric screens to delineate other RMND5 protein
interactors including CTLH complex members.
Chapter 7 General Discussion
Chapter 7: General Discussion
Chapter 7 General Discussion
285
7.1 General Discussion – The Role of E3 Ubiquitin Ligases in
Transcriptional Regulation Gene expression is a tightly controlled process and as such transcription, the initial step
in this process, is regulated by a number of mechanisms, in particular post-translational
modifications including acetylation, phosphorylation and ubiquitination. Ubiquitin and
the proteasome participate in the regulation of transcription by modifying the function
of histones, RNA Polymerase II (RNA Pol II) and transcription factors (Davie and
Murphy, 1990; Li et al., 1993; Fuchs et al., 1997; Mitsui and Sharp, 1999; Starita et al.,
2005). Histone ubiquitination alters chromatin structure, affecting the accessibility of
the transcriptional machinery to sites of transcription and also acts as a signal to recruit
regulators of transcription (Fierz et al., 2011; Hammond-Martel et al., 2012). By
regulating RNA Pol II and the transcription factor machinery directly, ubiquitination
affects their protein levels and activity via proteolytic and non-proteolytic mechanisms
(Mitsui and Sharp, 1999; Akiyama et al., 2005; Yan et al., 2009; Dao et al., 2012).
Together with other post-translational modifications, this multi-levelled approach to
transcriptional regulation exerts fine control over gene expression, ensuring that genes
will only be expressed in response to the correct cellular signals.
Since transcription factors, which possess activator and/or repressor functions, play a
significant role in determining which genes are expressed at any given time, they form
an important point of transcriptional regulation. Perhaps the best known and most
comprehensively characterised outcome of transcription factor ubiquitination by E3
ubiquitin ligases is the proteasome dependent degradation of the transcription factor. In
the present study, the transcription factor NKX3.1 was determined to be ubiquitinated
by the E3 ubiquitin ligases RMND5A and RMND5B, thus targeting NKX3.1 for
degradation by the proteasome. Along with the ability of polyubiquitination to promote
the degradation of transcription factors, there is an established role of ubiquitination in
modulating the activity of the transcription factor prior to its degradation. The potency
of transcriptional activation domains (TADs), present in activators and coactivators, has
been shown to inversely correlate with their half-life, particularly in the case of acidic
TADs (Molinari et al., 1999). Molinari and colleagues demonstrated that TADs from
various transcription factors fused to the GAL4 DNA binding domain were able to
induce transcription and that their half-lives were dependent on the strength with which
these chimeric transcription factors activated transcription (Molinari et al., 1999). This
Chapter 7 General Discussion
286
rapid protein turnover was also dependent on the presence of the GAL4 DNA-binding
domain, suggesting that the transcription factor must be bound to DNA and activated to
trigger ubiquitination (Molinari et al., 1999). A further example of this mechanism was
demonstrated in yeast where yeast transcription factors containing the VP16 TAD are
ubiquitinated by the SCFMET30 E3 ubiquitin ligase complex, however in cells lacking
Met30, the LED-VP16 transcription factor does not undergo ubiquitination and
subsequent degradation and loses its transactivation ability (Salghetti et al., 2001).
These findings suggest that in addition to its role in transactivation, the TAD acts as a
destabilisation domain and that ubiquitination of this domain modulates LED-VP16
degradation and transactivation activity. Therefore, as the activation and destruction of
the transcription factor are linked though the TAD, when the transcription factor has
fulfilled its transcriptional activity and is no longer needed, its degradation ensures that
it can no longer affect transcription. NKX3.1, which has a short half-life of ~25 minutes
contains an acidic domain and is able to activate the transcription of genes such as
PCAN1, although it is better known as a transcriptional repressor (Olsson et al., 2001,
Liu et al., 2000, Zhang et al., 2008a, Chen et al., 2002, Thomas et al., 2006). Further
investigation of the role NKX3.1 ubiquitination in the regulation of both its degradation
and its transcriptional activity, will identify the involvement of RMND5 proteins in this
process.
Transcription factor degradation can also be regulated by multiple E3s in response to
different cellular pathways, thereby allowing fine-tuned regulation of transcription
factor expression. Similarly, NKX3.1 protein levels are likely to be regulated by
multiple E3 ubiquitin ligases including TOPORS, which has been established to
ubiquitinate NKX3.1 resulting in its proteasome dependent degradation (Guan et al.,
2008). The present study has demonstrated that two additional E3 ubiquitin ligases,
RMND5A and RMND5B also regulate NKX3.1 protein levels by ubiquitination,
although their contribution in the normal maintenance of NKX3.1 levels is yet to be
determined. As NKX3.1 is involved in multiple cellular functions including
transcription and the DNA damage response, it is feasible that each E3 ubiquitin ligase
ubiquitinates NKX3.1 in response to different environmental conditions and in relation
to its different biological activities (Gelmann et al., 2003; Guan et al., 2008; Erbaykent-
Tepedelen et al., 2011). While individual transcription factors may be regulated by
multiple E3 ubiquitin ligases, single E3 ubiquitin ligases may target multiple
transcription factors, playing a central role in transcriptional regulation. For example,
Chapter 7 General Discussion
287
TOPORS is able to ubiquitinate both NKX3.1 and p53, and therefore deregulation of
TOPORS expression in prostate tumours may affect a number of transcriptional
pathways (Rajendra et al., 2004; Guan et al., 2008). In addition to the findings in this
thesis that RMND5 proteins ubiquitinate NKX3.1 and target it for proteasomal
degradation, overexpression of either RMND5A or RMND5B were also found to result
in a dose-dependent reduction in AR levels (not shown). The transcription factor SNW1
was additionally identified as a candidate RMND5A binding partner, and when
overexpressed with NKX3.1, RMND5 proteins were predominantly located in the
nucleus of prostate cancer cells, implicating both RMND5 proteins in the regulation of
several transcription factors.
The regulation of transcription factors by E3 ubiquitin ligases is not solely associated
with protein degradation, and both monoubiquitination and polyubiquitination of
transcription factors has been linked to alterations in other aspects of their cellular
activity or function. While proteolytic degradation is the ultimate means of halting the
activity of a transcription factor, the cellular localisation of transcription factors is also
an important factor in the regulation of their activity. Ubiquitination of a number of
transcription factors has been reported to alter their intracellular localisation and in the
present study, overexpression of RMND5 proteins resulted in reduced levels of nuclear
NKX3.1 and its predominant cytoplasmic localisation. A similar mechanism occurs
following ubiquitination of p53 by MDM2, with high levels of MDM2 resulting in p53
polyubiquitination and degradation, however when MDM2 levels are low, MDM2
monoubiquitinates p53 resulting in its nuclear export where it cannot directly regulate
transcription (Section 5.3) (Boyd et al., 2000; Geyer et al., 2000; Li et al., 2003). In the
cytoplasm, p53 plays roles in apoptosis and autophagy, indicating that nuclear export
does not always abrogate the activity of the transcription factor protein, and it is
possible that NKX3.1 similarly performs alternative functions when relocated to the
cytoplasm following overexpression of RMND5 proteins (Mihara et al., 2003; Tasdemir
et al., 2008). Conversely, the proteolytic cleavage of transcription factors triggered by
ubiquitination can promote their nuclear localisation to fulfil their transcriptional
regulatory role and well known examples of this include the activation of the nuclear
factor kappa B1 (NF-κB1) subunits p52 and p50 (Lin et al., 1998, Orian et al., 1999,
Orian et al., 2000). This occurs following cleavage of their precursors p105 and p100
via ubiquitination by the SCFβ-TRCP E3 ubiquitin ligase complex, leading to the
Chapter 7 General Discussion
288
degradation of all but the amino-terminal transcriptionally active p52 and p50 domains
(Lin et al., 1998; Orian et al., 1999; Orian et al., 2000).
Monoubiquitination of transcription factors is another non-proteolytic mechanism of
transcription factor modulation, but although well-established, the mechanism by which
monoubiquitination regulates transcription factor function remains incompletely
understood. An unusual example of this mechanism is the ubiquitination of SRC3, a
transcriptional coactivator. The phosphorylation-dependent monoubiquitination of
SRC3 activates its transcriptional regulatory activity, however, this initial ubiquitination
is extended and progressively results in the formation of a polyubiquitin chain, which
triggers the proteasomal degradation of SRC3, thereby allowing a specific interval
during which SRC3 can function as transcriptional coactivator (Wu et al., 2007a). In
addition to its effects on transcription factor activity, non-proteolytic ubiquitination can
also affect cofactor binding. The activity of the Met4 transcription factor is regulated in
this manner, and ubiquitination of Met4 by the SCFMET30 E3 ubiquitin ligase does not
alter its promoter binding capacity but interferes with its ability to interact with the Cbf1
transcription factor, thereby resulting in the failure to form an active Met4
transcriptional complex (Kaiser et al., 2000).
In addition to their ability to regulate transcription factors by ubiquitination, it is evident
that a subset of E3 ubiquitin ligases are able to function directly in transcription
themselves by forming part of the transcriptional machinery. One of the well-
characterised E3 ubiquitin ligases with this dual function in transcription is BRCA1,
with the BRCA1/BARD1 E3 ubiquitin ligase heterodimer proteins forming part of the
RNA Pol II holoenzyme (Chiba and Parvin, 2002). This complex consists of RNA Pol
II and general transcription factor members as well as suppressor of RNA Pol B (SRB),
which are all required for the initiation of transcription (Koleske and Young, 1995;
Scully et al., 1997; Chiba and Parvin, 2002). BRCA1 associates with the RNA Pol II
holoenzyme complex by interacting with RNA Helicase, and with its amino-terminal
which contains the RING domain, BRCA1 is hypothesised to associate with the
complex via BARD1 (Anderson et al., 1998; Chiba and Parvin, 2002). In response to
DNA damage, the BRCA1/BARD1 heterodimer ubiquitinates RNA Pol II, targeting it
for proteasomal degradation (Kleiman et al., 2005; Starita et al., 2005). However, in
unstressed cells BRCA1 functions as a co-activator of a subset of genes including p53
by interacting with enhancers, thereby linking the enhancers to the RNA Pol II
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289
holoenzyme to promote transcription (Ouchi et al., 2000; MacLachlan et al., 2002).
Other examples of E3 ubiquitin ligases that participate in transcription are the yeast E3
ubiquitin ligase Rsp5 and its human orthologue hPRF1 as well as the HECT E3
ubiquitin ligase E6-AP which both function as coactivators for steroid hormone
receptors (Imhof and McDonnell, 1996; Nawaz et al., 1999). In a previous study in the
laboratory, RMND5B was identified to possess transcriptional repressor activity,
implying that RMND5B and perhaps RMND5A play additional as yet uncharacterised
roles in transcriptional regulation (Dawson, 2006). Although RMND5 proteins do not
contain defined DNA binding domains, the presence of multiple protein-protein
interaction domains implies that they are able to interact with a number of proteins
including transcription factors, or form part of transcription factor complexes, thereby
exerting transcriptional activator or repressor activities in a similar manner to other
LisH domain containing proteins (Section 4.1.3.1) (Li et al., 2000; Zhang et al., 2002;
Yoon et al., 2003, Choi et al., 2008). This role for RMND5 proteins is further supported
by the presence of putative steroid hormone receptor binding domains, nuclear receptor
boxes (NRBs) present in both proteins and the finding in this study that both proteins
accumulate in the nucleus following overexpression with the transcription factor,
NKX3.1. Additionally, the DNA damage repair factor, XRR5C and the transcription
factor SNW1 were identified in this study as potential RMND5A binding partners.
The ability of BRCA1 and BARD1 to participate in a variety of cellular processes
including DNA repair, cell cycle control and apoptosis is due to their multi-domain
architecture (Jin et al., 1997; Westermark et al., 2003; Fabbro et al., 2004; Schuchner et
al., 2005). Multi-functional E3 ubiquitin ligases possess a similar protein domain
architecture, with the RING domain located at the far amino- or carboxy-terminal and
additional protein domains located in the remaining length of the protein, suggesting
that in many cases the RING domain activity is separate from that of the residual
protein domains. However, the remaining protein domains may also function as
substrate recognition elements in single subunit E3 ubiquitin ligases or as association
domains for those E3 ubiquitin ligases forming part of multi-subunit E3 ubiquitin ligase
complexes. Although BARD1 interacts with BRCA1 through its RING domain, thereby
enhancing BRCA1 E3 ubiquitin ligase activity, BARD1 contains carboxy-terminal
ankyrin repeats and two BRCT domains which mediate BRCA1 independent cellular
roles in cell cycle regulation, p53 stabilisation and apoptosis (Wu et al., 1996; Xia et al.,
2003). By interacting with the DNA-PK subunit Ku-70 and p53, BARD1 induces p53
Chapter 7 General Discussion
290
phosphorylation and stabilisation, and in the cytoplasm, BARD1 associates with the
mitochondria, triggering apoptosis by stimulating Bax oligomerisation (Feki et al.,
2005; Tembe and Henderson, 2007). In comparison, the proteins investigated in the
present study, RMND5A and RMND5B possess an analogous protein domain
architecture, a carboxy-terminal RING domain and three additional protein-protein
interaction domains, the LisH, CTLH and CRA domains located amino-terminally to
the RING domain, suggesting that they too are multi-functional proteins. This is further
supported by the findings in this study that both proteins exhibit diffuse nuclear and
cytoplasmic localisation, with accumulation in cytoplasmic vesicles in a proportion of
cells that potentially signifies multiple functions of RMND5 proteins in distinct cellular
compartments. This hypothesis is supported by the identification of mitochondrial
proteins as putative RMND5A and RMND5B binding partners.
The ubiquitination of transcription factors, which has the ability to regulate transcription
factor activity and function, can be carried out either by single subunit or multi-subunit
E3 ubiquitin ligases. Single subunit E3 ubiquitin ligases typically contain a
HECT/RING/U-box domain and substrate recognition domains and tend to be multi-
functional proteins. One such example is the U-box E3 ubiquitin ligase CHIP which
functions as a co-chaperone and as an E3 ubiquitin ligase, interacting with the
chaperones Hsc70-Hsp70 and Hsp90 through its amino terminal tetricopeptide repeat,
whilst retaining its ubiquitin ligase activity mediated by its carboxy-terminal U-box
domain (Ballinger et al., 1999; Jiang et al., 2001). By interacting with molecular
chaperones, CHIP tips the balance of the protein folding machinery towards protein
ubiquitination and degradation, thereby connecting these two integral systems
responsible for the regulation of protein integrity. In its role as a transcriptional
regulator, CHIP ubiquitinates p53 and c-Myc resulting in their proteasomal degradation
(Esser et al., 2005; Naito et al., 2010; Paul et al., 2012). An interesting aspect of single
subunit E3s is their ability to form homodimers or heterodimers, most often resulting in
enhancement of their enzymatic activity. This is perhaps best typified by the
MDM2/MDMX heterodimer which targets p53 for proteasomal degradation, although
individually neither MDM2 nor MDMX are able to polyubiquitinate p53 in vitro,
demonstrating the importance of both proteins in the activity of the heterodimer (Wang
et al., 2011). In the absence of heterodimerisation, MDM2 and MDMX perform
distinct cellular roles that are independent of each other. MDM2 is able to ubiquitinate a
number of proteins including the transcription factor p73 and is a coactivator of the
Chapter 7 General Discussion
291
ApoCIII promoter, while MDMX contributes to p53 induced apoptosis by interacting
with BCL-2 in the mitochondria, facilitating the interaction between p53 and BCL2 and
thereby inducing cytochrome C release and apoptosis (Mancini et al., 2009; Kubo et al.,
2010; Yang et al., 2012). Similarly, in this study, the structurally related RMND5
proteins were demonstrated to interact and colocalise, implicating the proteins in
homodimer or heterodimer formation, conceivably with respect to the coregulation of
specific cellular targets as is the case for MDM2 and MDMX. As each RMND5 protein
was identified to possess E3 ubiquitin ligase activity in vitro it is also possible that they
have individual cellular targets and due to their multi-domain architecture that they
mediate alternative cellular functions similar to that of other heterodimeric E3 ubiquitin
ligases such as BRCA1/BARD1.
Proteins possessing E3 ubiquitin ligase activity, particularly those containing RING
domains are also capable of forming part of large, multi-protein E3 ubiquitin ligase
complexes, thus broadening the list of potential substrates of the E3 by extending the
range of available substrate recognition domains. E3 ubiquitin ligase complexes
involved in the regulation of transcription factors tend to be of the SCF (Skp-Cullin-F-
box) family, which all contain the RBX1 RING domain protein, cullin scaffolding
protein and a Skp 1 linker protein which binds a number of substrate recognition
components, including F-box proteins (Willems et al., 2004, Sun et al., 2007). In this
manner, the invariant core E3 complex is able to ubiquitinate and thereby regulate a
range of target proteins depending on the F-box protein with which it is associated.
Upon formation of the SCF complex with the F-box protein Fbw7, the KLF5
transcription factor is rapidly degraded whilst replacement of Fbw7 for β-TrCP results
in the ubiquitination and degradation of the transcription factor β-catenin (Latres et al.,
1999; Zhao et al., 2010). As a proposed E3 ubiquitin ligase complex, the CTLH
complex investigated in the present study is comprised of proteins of similar protein
domain architecture. This complex includes RMND5A, an E3 ubiquitin ligase, the
ARMC8α putative adaptor protein and RanBPM and muskelin, potential substrate
recognition components that contain SPRY and Kelch repeat domains present in F-box
and SOCS box proteins which function as substrate recognition components in other E3
ubiquitin ligase complexes (Kobayashi et al., 2007; Kuang et al., 2010; Lee et al.,
2010). Although the function of the CTLH complex as a multiprotein E3 ubiquitin
ligase complex is supported by the characterised function of several of its components
and the reported activity of its yeast orthologue as an E3 ubiquitin ligase complex (Santt
Chapter 7 General Discussion
292
et al., 2008), the transcriptional regulatory activity of the CTLH complex is more
speculative. However, RanBPM is a binding partner and transcriptional activator of
steroid hormone receptors and multiple transcription factors, while RMND5A contains
putative NRBs and regulates the transcription factor NKX3.1 (Rao et al., 2002;
Brunkhorst et al., 2005; Dawson, 2006; Poirier et al., 2006). Therefore, the
transcriptional regulatory function of RMND5 proteins and the CTLH complex may be
further investigated in future studies.
Due to their important roles as regulators of gene transcription, it is unsurprising that the
dysregulation of multiple E3 ubiquitin ligases is associated with a variety of cancers.
Furthermore, as many E3 enzymes, not limited to those with transcriptional regulatory
roles, are involved in multiple cellular processes including apoptosis, cell cycle
regulation and genome stability, their disruption not only affects their transcriptional
regulatory roles but their broader cellular functions, thereby associating the abnormal
functions of E3 ubiquitin ligase with all stages of malignancy. The number of E3
ubiquitin ligases implicated in disease is constantly increasing and includes well-
characterised proteins such as BRCA1, MDM2, SCFFbw7 and WWP1, which have been
classified as oncogenes (e.g. MDM2) or tumour suppressor genes (e.g. BRCA1),
depending upon the cancer-associated abnormality involved (Oliner et al., 1992;
Hashizume et al., 2001; Chen et al., 2007; O'Neil et al., 2007). This classification is not
invariant but depending upon the cellular role(s) of the substrate(s), the E3 enzyme may
function in either capacity. In many cases, due to their pervasive role in cellular
pathways, it is the E3 ubiquitin ligase that balances opposing oncogenic and tumour
suppressor functions of abnormal or aberrantly regulated genes, and due to their defined
substrate specificity, it is not surprising that multiple E3s are being investigated as
possible therapeutic targets (Chen et al., 2008; Edelmann et al., 2011; Buckley et al.,
2012; McCormack et al., 2012). As E3 ubiquitin ligases and regulators of the
transcription factor NKX3.1 as well as other unidentified cellular targets, RMND5
proteins are themselves dysregulated in a number of cancers. Particularly of interest is
the RMND5B chromosomal locus, 5q35.5 which is located in a region associated with
prostate cancer heritability (Xu et al., 2005; Christensen et al., 2010). Additionally, the
RMND5A and RMND5B gene loci are disrupted in a number of cancers including
ovarian carcinoma, mantle cell lymphoma, pilocytic astrocytoma, non-small lung cell
carcinoma, neuroblastoma and breast carcinoma implying that RMND5 proteins
perform important cellular functions in a number of different cell types (Section 4.1.4)
Chapter 7 General Discussion
293
(Mendes-da-Silva et al., 2000; Mosse et al., 2005; Camps et al., 2006; Johannsdottir et
al., 2006; Li et al., 2008; Belirgen et al., 2012). The identification of these cellular roles
and additional target proteins of RMND5 ubiquitination will aid in the understanding of
the consequences of RMND5 disruption in cancers and if appropriate, may add to the
many E3 ubiquitin ligases targeted for cancer therapeutics.
7.2 Future Directions The investigations in this thesis, that RMND5 proteins function as E3 ubiquitin ligases
and ubiquitinate the transcription factor, NKX3.1 leading to its proteasomal
degradation, have yielded results which may be expanded in future studies
characterising the cellular functions of RMND5 proteins, particularly with regard to
their ubiquitination targets, including NKX3.1.
The cellular functions of proteins are in part mediated by their intracellular localisation,
expression levels and binding partners, elucidation of which requires the availability of
specific antibodies against the protein of interest. Although for the short term,
exogenous tagged protein may be used in in vivo or in vitro functional assays, and
indeed in some case may be more suitable than use of the endogenous protein, it is
ultimately essential that the endogenous protein should be investigated. In this
laboratory, previous attempts to generate monoclonal or polyclonal antibodies against
RMND5B have not been successful (not shown), and while two commercial RMND5A
antibodies are currently available, neither is validated and where western blotting is
reported, the molecular size of the protein band does not correspond to that of
RMND5A. The RMND5A and RMND5B amino acid sequences are highly similar in
mouse, rat, rabbit and other species commonly used to generate antibodies, potentially
reducing the antigenicity of the proteins, however it will be important for future studies
to develop antibodies against RMND5A and RMND5B in order to investigate the
cellular localisation and protein levels of endogenous RMND5 proteins both in
mammalian cell lines and in normal and malignant tissues. These antibodies may also
be used to assess whether endogenous RMND5 proteins interact in mammalian cells
and to provide evidence supporting their heterodimer formation or their inclusion in the
CTLH complex.
Chapter 7 General Discussion
294
In this study, transient overexpression of RMND5 proteins was used to determine their
effects on substrate proteins as the endogenous levels of either RMND5A or RMND5B
were not known (although the expression of both genes was verified using RT-PCR).
Following determination of endogenous RMND5A and RMND5B levels, future in vitro
studies to determine alternative functions or additional substrates of RMND5 proteins
can include the use of siRNA knockdown of either or both RMND5A and RMND5B.
Due to the high degree of similarity between RMND5A and RMND5B, it is likely that
they share cellular targets or that one may compensate for loss of the other, therefore the
function of both RMND5 proteins should be investigated together. Knockdown of E3
ubiquitin ligase expression by siRNA is commonly used to examine E3 function and
should result in increased expression of substrate proteins where ubiquitination by the
E3 ubiquitin ligase (e.g. RMND5A or RMND5B) results in proteasomal degradation
(Zhong et al., 2005; Nishitani et al., 2006). These experiments will therefore be critical
for confirmation of the identity of endogenous RMND5 substrate proteins such as
NKX3.1.
Investigation of the in vitro function of both RMND5A and RMND5B RING domains
confirmed that, like their yeast orthologue, both proteins possess E3 ubiquitin ligase
activity (Santt et al., 2008). Future studies expanding on these findings would confirm
the activity of full length RMND5 proteins in in vitro ubiquitination assays by
producing RMND5A/RMND5B using alternative protein tags in bacteria or in vitro
transcription/translation methods as discussed previously (Section 4.3). The presence of
all protein domains including substrate recognition domains following full length
protein production, would allow the inclusion of putative substrate proteins in in vitro
ubiquitination assays. In the presence of particular substrates or in auto-ubiquitination
assays, it will be useful if both RMND5 proteins are assayed simultaneously to assess
whether they are able to function as heterodimers and whether this enhances their
ubiquitination activity (Hashizume et al., 2001). The soluble full length RMND5
proteins may also be used to obtain crystal structures of both proteins which could be
used to assess whether RMND5 proteins form homodimers or heterodimers, and the
mechanism by which this activity may enhance their individual activity or allow them to
ubiquitinate distinct proteins. Crystal structures may also aid in the characterisation of
RMND5 protein interactions with other CTLH complex members and delineate
RMND5 protein interactions with different E2 conjugating enzymes, a finding of this
study.
Chapter 7 General Discussion
295
Both RMND5 proteins were determined to interact with a number of E2 conjugating
enzymes in in vitro ubiquitination assays performed for this thesis. As some E2
enzymes are chain initiating enzymes while others appear to function predominantly as
chain elongating E2 enzymes, the E2 enzymes that require prior monoubiquitination of
the substrate would not be identified using these methods (Windheim et al., 2008; Ye
and Rape, 2009; Williamson et al., 2011). Thus, it will be important to determine more
comprehensively which E2 conjugating enzymes are able to interact with RMND5
proteins in vivo, as the range of E2 enzymes may differ between the two proteins, in
vivo results may differ compared to in vitro findings, and interactions may depend on
specific culture (environmental) conditions. Identification of the E2 enzymes with
which RMND5 proteins interact to mediate ubiquitin transfer will be important as it is
the E2 enzyme that determines the ubiquitin chain topology attached to the substrate
and therefore the ultimate fate of the substrate, although the E3 ubiquitin ligase also
plays an important role in this process (Windheim et al., 2008; David et al., 2011).
Once the E2 conjugating enzymes have been identified, the outcome of substrate
ubiquitination by RMND5 proteins can be predicted. These predictions can then be
assessed biochemically using linkage specific antibodies or ubiquitin mutants to
determine the ubiquitin linkages of specific RMND5 substrate proteins, for example
NKX3.1 (Wu-Baer et al., 2003; Newton et al., 2008; Matsumoto et al., 2010). As
NKX3.1 is targeted for degradation following RMND5 overexpression, this implies that
together with the relevant E2 enzyme, RMND5 proteins are involved in the formation
of lysine 11, lysine 29 or lysine 48 polyubiquitin chains, therefore the type of ubiquitin
chains attached to NKX3.1 under these conditions can be verified experimentally (Chau
et al., 1989; Gregori et al., 1990; Johnson et al., 1995; Matsumoto et al., 2010).
Similarly, following the identification of additional substrates of RMND5
ubiquitination, the types of ubiquitin chains attached by the E2/E3 enzyme pair can be
determined as at this stage it is unknown whether RMND5 proteins always target their
substrate proteins for proteasomal degradation. Due to the inclusion of RMND5A and
possibly RMND5B in the human CTLH complex, and the association of members of
the complex with endosomal protein degradation (where monoubiquitination serves as
an internalisation trigger for membrane bound proteins and receptors), it will be
interesting to determine the outcomes of ubiquitination of substrates of RMND5
proteins and the CTLH complex. These include whether the outcomes of the substrate
proteins differ depending on the function of RMND5A or RMND5B as single subunit
Chapter 7 General Discussion
296
E3 ubiquitin ligases or as part of the CTLH complex (Haglund et al., 2003a; Haglund et
al., 2003b). The studies will be assisted in part by use of the above mentioned linkage
specific antibodies (see also Sections 4.3, 5.3).
In the present study, the prostatic tumour suppressor, NKX3.1 was investigated as a
substrate of RMND5 mediated ubiquitination. In the future, the RMND5 mediated
degradation of NKX3.1 should be further examined including the specific
environmental or cellular signals under which RMND5 proteins mediate NKX3.1
ubiquitination, in particular as another E3 ubiquitin ligase, TOPORS is also involved in
NKX3.1 degradation by the proteasome (Guan et al., 2008). NKX3.1 expression is
androgen (AR) regulated and although this is in part mediated by androgen regulation of
NKX3.1 gene transcription (Prescott et al., 1998), it is apparent that NKX3.1 protein is
also androgen regulated as androgen withdrawal induces proteasome mediated
degradation of NKX3.1. The effects of androgen addition or depletion on NKX3.1
ubiquitination could be investigated in future studies, including the identification of the
E3 ubiquitin ligases involved. Since NKX3.1 is regulated by phosphorylation and
phosphorylation by CK2 is reported to protect NKX3.1 from ubiquitin-mediated
degradation, it is likely that there is an interplay between these two types of post-
translational modifications, which could be further elucidated by identification of the E3
ubiquitin ligase(s) whose activity is modified by CK2 phosphorylation of NKX3.1 (Li et
al., 2006; Markowski et al., 2008). Under conditions where NKX3.1 ubiquitination is
induced, it may be determined whether phosphorylation of particular NKX3.1 residues
is a signal for its ubiquitination as it is for other proteins (Punga et al., 2006; Scaglioni
et al., 2008). Furthermore, if RMND5 proteins either alone or as part of the CTLH
complex are able to mediate phospho-NKX3.1 ubiquitination, the means by which they
are able to recognise phosphorylated NKX3.1 will require investigation as neither
protein contains a canonical phospho-recognition motif. Of additional interest would be
the identification of specific lysine residues of NKX3.1 which are ubiquitinated by
RMND5 proteins and/or TOPORS, with ubiquitination of lysine residues within the
homeodomain hypothesised to target NKX3.1 for proteasomal degradation (Ju et al.,
2009).
NKX3.1 is a DNA binding transcription factor, and it is feasible that RMND5 proteins
ubiquitinate DNA-bound NKX3.1, implying that it is active NKX3.1 which is
ubiquitinated and hence targeted for degradation once it is “spent”, thereby allowing its
Chapter 7 General Discussion
297
removal from the DNA and replacement with a fresh transcription factor. Another
aspect of the interaction between RMND5A/RMND5B and NKX3.1 that would
increase understanding of its biological consequences would be to determine the
domains of each protein required for this interaction. Using yeast two-hybrid assays, it
has been determined in this laboratory that the interaction between RMND5B and
NKX3.1 may be mediated in part by the CTLH and CRA domains of RMND5B,
although the result requires confirmation, and RMND5A-NKX3.1 interaction domains
have not been reported (Lau, 2008). The CTLH domain and part of the CRA domain are
also required for the interaction of RMND5A with ARMC8α (Kobayashi et al., 2007),
suggesting that if RMND5 proteins use these domains to form part of the CTLH
complex, they cannot simultaneously interact with NKX3.1. As such, RMND5 protein
interaction with NKX3.1 may not occur in conjunction with the CTLH complex but
may be mediated by RMND5 proteins functioning as single subunit E3 ubiquitin
ligases.
In this study, mass spectrometry was used to identify putative RMND5 binding partners
and the candidate binding partners isolated could be further investigated and confirmed
using additional functional studies such as co-immunoprecipitation assays, GST-
pulldown assays and colocalisation microscopy before additional experiments can be
performed assessing the functional outcomes of the interactions. Characterisation of the
interacting proteins will facilitate identification of additional functions of RMND5
proteins potentially mediated by the RING domain or by the additional protein domains
present in the proteins. Alternatively, individual RMND5 protein interacting factors
may function to regulate RMND5 activity. Although the proteins identified in these co-
immunoprecipitation experiments should contain RMND5 binding partners, the proteins
are not expected to be substrates of RMND5 ubiquitination as the interaction between
an E3 ubiquitin ligase and its substrate is usually transient and may be weak, especially
if the E3 ubiquitin ligase is a multimeric complex (Burande et al., 2009; Andrews et al.,
2010). In the future, experiments involving mass spectrometric identification of
RMND5 protein interacting factors may be performed using either wild-type
RMND5A/RMND5B or mutant RMND5A/RMND5B, including RING domain
mutants. Mutation of the RING domain may prolong the interaction between the E3
ubiquitin ligase and its substrate as the substrate can no longer be ubiquitinated by the
E3 enzyme (Hu and Fearon, 1999). Comparison of the proteins binding to wild-type or
mutant RMND5A/RMND5B may therefore identify proteins which are more likely to
Chapter 7 General Discussion
298
be substrate proteins (Hu and Fearon, 1999). Alternatively, for large scale screening of
potential RMND5 substrate proteins, protein microarrays may be used employing a
microarray based system and in vitro ubiquitination assays which are each carried out
using the E3 ubiquitin ligase of interest and different potential substrate proteins (Gupta
et al., 2007; Persaud et al., 2009; Andrews et al., 2010). In this way, multiple
potentially relevant substrates of RMND5 protein ubiquitination could be identified
which may then be confirmed individually using in vitro and in vivo assays.
Although RMND5A and RMND5B are widely expressed, factors that regulate their
expression have not been investigated and these studies may be commenced using
transcription factor binding bioinformatics tools to predict potential transcription factor
binding sites in the promoter or untranslated regions (UTR) of the RMND5A and
RMND5B genes. Following the identification of potential transcription factor binding
sites, chromatin immunoprecipitation (ChIP), DNase I footprinting and electrophoretic
mobility shift assays (EMSA) can be performed to specifically detect protein-DNA
interactions, and luciferase assays may be used to assess transcriptional activation or
repressor functions of the transcription factors on the putative binding elements in the
regulatory regions of the RMND5A and RMND5B genes (Liu et al., 2010; Kerschner
and Harris, 2012). Finally, site directed mutagenesis of putative transcription factor
binding sites may be included in EMSA and luciferase assays to confirm the function of
the elements and associated transcription factors in the regulation of RMND5A and/or
RMND5B expression.
Previous studies in the laboratory identified that RMND5B exerted a transcriptional
repressor activity on an NKX3.1 consensus binding element (Dawson, 2006) and to
further characterise the transcriptional regulatory role of RMND5B and the potential
transcriptional function of RMND5A, luciferase assays may be used to determine
whether RMND5A is also able to exert transcriptional repressor activity upon an
NKX3.1 responsive element, with results confirmed using ChIP assays and EMSAs. If
RMND5 proteins are shown from mass spectrometry screens to bind transcription
factors or mediators of transcription factor complexes, this may be further investigated
using immunoprecipitation and ChIP assays, which will assist in characterising the
mechanisms of transcriptional regulatory activity, as RMND5 proteins do not contain
identifiable DNA binding domains (Shang et al., 2002; Perissi et al., 2004). In
conjunction with these studies, microarray technology may be used to identify potential
Chapter 7 General Discussion
299
RMND5 target genes following overexpression or knockdown of the expression of each
protein (Yang et al., 2008; Gorte et al., 2011).
Both RMND5A and RMND5B were found to exhibit a diffuse nuclear and cytoplasmic
localisation, however a punctate cytoplasmic distribution was also evident in a
proportion of cells. The characterisation of these punctate bodies and whether they are
of functional significance may be ascertained in future studies (Section 4.3) in particular
to follow up results of mass spectrometric identification of RMND5A and RMND5B
binding partners which included mitochondrial proteins. This finding is interesting
given that gluconeogenic and TCA cycle enzymes have been identified as substrates of
the yeast Vid30 complex (Santt et al., 2008). In relation to the broad intracellular
distribution of RMND5 proteins, it will be important to determine where the CTLH
complex forms, whether it functions as an E3 ubiquitin ligase complex, and whether the
CTLH and Vid30 complexes retain overlapping substrates (Kobayashi et al., 2007;
Santt et al., 2008). To assess its function, the CTLH complex may be reconstituted in
vitro, as has been successfully performed for other E3 ubiquitin ligase complexes
(Skowyra et al., 1999; Chen et al., 2006b). These studies may then be used to determine
whether either or both RMND5A and RMND5B can form part of the CTLH complex,
and whether the inclusion of RMND5A and/or RMND5B in the CTLH complex alters
the substrates or type of ubiquitination of the substrates. In yeast, Gid9 functions with
RMD5 to provide the Vid30 complex with its E3 ubiquitin ligase activity and a similar
function of human EMP, orthologue of Gid9, may be investigated in relation to the
CTLH complex in future studies (Braun et al., 2011). The functional consequences of
an additional E3 ubiquitin ligase in the CTLH complex can also be determined,
including augmentation of the E3 activity of RMND5A/RMND5B and alternative types
of ubiquitination of substrate proteins caused by interaction with additional E2
conjugating enzymes. If the CTLH complex can be reconstituted in vitro it is possible
that all or part of the complex could be crystallised, providing information related to the
roles of each complex member that would complement mapping of the protein-protein
interaction domains to delineate CTLH complex topology (Menssen et al., 2012).
Accumulation of this information may also resolve the substrate recognition
components of the CTLH complex, which are currently hypothesised to be RanBPM
and muskelin due to the presence of their SPRY and Kelch repeat domains (Kuang et
al., 2010; Lee et al., 2010), as well as substrates of the CTLH complex.
Chapter 7 General Discussion
300
Since the expression, tissue distribution and biological activities of RMND5 proteins
are incompletely characterised, the generation of RMND5A and RMND5B transgenic
or knockout mice may aid in the elucidation of their normal physiological roles. Based
on the similar activities and high amino acid sequence homology of RMND5A and
RMND5B, it is feasible that RMND5A and/or RMND5B knockout mice may not
exhibit a distinctive phenotype as loss of expression of one RMND5 protein may be
compensated by the function of the other, therefore knockout of both RMND5A and
RMND5B may need to be generated. Conversely, if RMND5 knockout or transgenic
mice display embryonic lethality or die shortly after birth, conditional knockout or
transgenic mice may be an option for the investigation of RMND5 protein functions.
These animals may be observed for changes in development, growth, fertility and
behaviour and their tissues may be analysed using, for example, immunohistochemistry
and gene expression analysis to determine the effects of the abnormal expression of
RMND5 proteins (Crawley, 2007).
The ultimate aim of this research would be to determine the expression and function of
each RMND5 protein in normal human tissues as well as in cancer or other pathological
states. For these studies, mRNA expression, protein levels and the tissue distribution of
each of RMND5A and RMND5B may be investigated if appropriate tissues are
available. As RMND5 proteins are highly homologous they may have similar or
overlapping targets, however their tissue distribution may differ resulting in distinct
expression profiles of RMND5 regulated proteins which include a spectrum of
commonly regulated proteins amongst proteins whose expression is specifically
regulated by RMND5A or RMND5B. Tissue microarrays would also allow the
comparison of RMND5 protein expression in normal versus tumour tissue samples for
different cancers types, with protein analysis and real-time RT-PCR of these tissues
potentially identifying concordant changes in the expression of specific RMND5
substrates in cancer types where RMND5 proteins function as important determinants of
gene or protein expression. Analysis of the chromosomal loci of RMND5A and
RMND5B, 2p11.2 and 5q35.3, respectively using FISH or other technologies would
enable determination of abnormalities involving the RMND5 gene loci (e.g. gene copy
number), and sequencing of each gene would enable the assessment of mutations in the
RMND5 coding regions in malignant tissues that may affect their protein function and
therefore the regulation of their substrates (Bartlett, 2004).
Chapter 7 General Discussion
301
7.3 Concluding Remarks This study has demonstrated that human RMND5 proteins function as E3 ubiquitin
ligases in prostate cancer cells, with RMND5A interacting with UbcH2, UbcH5b and
UbcH5c, and RMND5B interacting with UbcH5b and UbcH5c to mediate ubiquitin
transfer. Both RMND5A and RMND5B are able to ubiquitinate the prostate specific
tumour suppressor, NKX3.1 resulting in its proteasome-dependent degradation. It is
possible that RMND5A and RMND5B form part of the multiprotein CTLH complex,
and that the additional LisH, CTLH and CRA domains of each protein are able to
mediate alternative, as yet uncharacterised functions. As both RMND5 chromosomal
loci are disrupted in a number of cancers, the identification of RMND5 substrates of
ubiquitination and their additional cellular functions may increase knowledge of their
contributions to normal physiology and to cancer initiation or progression.
Chapter 8 References
Chapter 8: References
Chapter 8 References
302
Abate-Shen, C. and M. M. Shen (2000). "Molecular genetics of prostate cancer." Genes Dev 14(19): 2410-2434.
Abate-Shen, C., W. A. Banach-Petrosky, X. Sun, K. D. Economides, N. Desai, J. P. Gregg, A. D. Borowsky, R. D. Cardiff and M. M. Shen (2003). "Nkx3.1; Pten mutant mice develop invasive prostate adenocarcinoma and lymph node metastases." Cancer Res 63(14): 3886-3890.
Abate-Shen, C., M. M. Shen and E. Gelmann (2008). "Integrating differentiation and cancer: the Nkx3.1 homeobox gene in prostate organogenesis and carcinogenesis." Differentiation 76(6): 717-727.
Abdulkadir, S. A., J. A. Magee, T. J. Peters, Z. Kaleem, C. K. Naughton, P. A. Humphrey and J.
Milbrandt (2002). "Conditional loss of Nkx3.1 in adult mice induces prostatic intraepithelial neoplasia." Mol Cell Biol 22(5): 1495-1503.
Abdulkadir, S. A. (2005). "Mechanisms of prostate tumorigenesis: roles for transcription factors Nkx3.1 and Egr1." Ann N Y Acad Sci 1059: 33-40.
Adams, J. C., B. Seed and J. Lawler (1998). "Muskelin, a novel intracellular mediator of cell adhesive and cytoskeletal responses to thrombospondin-1." EMBO J 17(17): 4964-4974.
Adams, J., R. Kelso and L. Cooley (2000). "The kelch repeat superfamily of proteins: propellers of cell function." Trends Cell Biol 10(1): 17-24.
Adhikari, A. and Z. J. Chen (2009). "Diversity of polyubiquitin chains." Dev Cell 16(4): 485-486.
Adler, D., N. Kanji, K. Trpkov, G. Fick and R. M. Hughes (2003). "HPC2/ELAC2 gene variants associated with incident prostate cancer." J Hum Genet 48(12): 634-638.
Ahn, J., Z. Novince, J. Concel, C. H. Byeon, A. M. Makhov, I. J. Byeon, P. Zhang and A. M. Gronenborn (2011). "The Cullin-RING E3 ubiquitin ligase CRL4-DCAF1 complex dimerizes via a short helical region in DCAF1." Biochemistry 50(8): 1359-1367.
Akamatsu, S., R. Takata, K. Ashikawa, N. Hosono, N. Kamatani, T. Fujioka, O. Ogawa, M. Kubo, Y. Nakamura and H. Nakagawa (2010). "A functional variant in NKX3.1 associated with prostate cancer susceptibility down-regulates NKX3.1 expression." Hum Mol Genet 19(21): 4265-4272.
Akana, J., A. A. Fedorov, E. Fedorov, W. R. Novak, P. C. Babbitt, S. C. Almo and J. A. Gerlt (2006). "D-Ribulose 5-phosphate 3-epimerase: functional and structural relationships to members of the ribulose-phosphate binding (beta/alpha)8-barrel superfamily." Biochemistry 45(8): 2493-2503.
Akiyama, H., T. Kamitani, X. Yang, R. Kandyil, L. C. Bridgewater, M. Fellous, Y. Mori-Akiyama and B. de Crombrugghe (2005). "The transcription factor Sox9 is degraded by the ubiquitin-proteasome system and stabilized by a mutation in a ubiquitin-target site." Matrix Biol 23(8): 499-505.
Alberts, B., D. Bray, K. Hopkin, A. Johnson, J. Lewis, M. Raff, K. Roberts and P. Walter, Eds. (2004). "Essential Cell Biology ". 2nd Ed. New York, Garland Science, Taylor and Francis Group pp 573-600.
Alibhoy, A. A., B. J. Giardina, D. D. Dunton and H. L. Chiang (2012). "Vid30 is required for the association of Vid vesicles and actin patches in the vacuole import and degradation pathway." Autophagy 8(1): 29-46.
Chapter 8 References
303
Alpi, A. F., P. E. Pace, M. M. Babu and K. J. Patel (2008). "Mechanistic insight into site-restricted monoubiquitination of FANCD2 by Ube2t, FANCL, and FANCI." Mol Cell 32(6): 767-777.
Amerik, A., S. Swaminathan, B. A. Krantz, K. D. Wilkinson and M. Hochstrasser (1997). "In vivo disassembly of free polyubiquitin chains by yeast Ubp14 modulates rates of protein degradation by the proteasome." EMBO J 16(16): 4826-4838.
Anderson, S. F., B. P. Schlegel, T. Nakajima, E. S. Wolpin and J. D. Parvin (1998). "BRCA1 protein is linked to the RNA polymerase II holoenzyme complex via RNA helicase A." Nat Genet 19(3): 254-256.
Anderson, P. D., S. A. McKissic, M. Logan, M. Roh, O. E. Franco, J. Wang, I. Doubinskaia, R. van der Meer, S. W. Hayward, C. M. Eischen, I. E. Eltoum and S. A. Abdulkadir (2012). "Nkx3.1 and Myc crossregulate shared target genes in mouse and human prostate tumorigenesis." J Clin Invest 122(5): 1907-1919.
Andrews, P. S., S. Schneider, E. Yang, M. Michaels, H. Chen, J. Tang and R. Emkey (2010). "Identification of substrates of SMURF1 ubiquitin ligase activity utilizing protein microarrays." Assay Drug Dev Technol 8(4): 471-487.
Anglesio, M. S., V. Evdokimova, N. Melnyk, L. Zhang, C. V. Fernandez, P. E. Grundy, S.
Leach, M. A. Marra, A. R. Brooks-Wilson, J. Penninger and P. H. Sorensen (2004). "Differential expression of a novel ankyrin containing E3 ubiquitin-protein ligase, Hace1, in sporadic Wilms' tumor versus normal kidney." Hum Mol Genet 13(18): 2061-2074.
Arakawa, A., M. Matsuo-Takasaki, A. Takai, H. Inomata, M. Matsumura, M. Ikeya, K. Takahashi, Y. Miyachi, N. Sasai and Y. Sasai (2007). "The secreted EGF-Discoidin factor xDel1 is essential for dorsal development of the Xenopus embryo." Dev Biol 306(1): 160-169.
Asatiani, E., W. X. Huang, A. Wang, E. Rodriguez Ortner, L. R. Cavalli, B. R. Haddad and E. P. Gelmann (2005). "Deletion, methylation, and expression of the NKX3.1 suppressor gene in primary human prostate cancer." Cancer Res 65(4): 1164-1173.
Aslan, G., B. Irer, B. Tuna, K. Yorukoglu, F. Saatcioglu and I. Celebi (2006). "Analysis of NKX3.1 expression in prostate cancer tissues and correlation with clinicopathologic features." Pathol Res Pract 202(2): 93-98.
Bai, D., H. Chen and B. R. Huang (2003). "RanBPM is a novel binding protein for p75NTR." Biochem Biophys Res Commun 309(3): 552-557.
Bailey, S. J. and S. F. Brewster (2011). "Prostate cancer: to screen or not to screen." Arch Esp Urol 64(5): 406-418.
Bailly, V., S. Prakash and L. Prakash (1997). "Domains required for dimerization of yeast Rad6 ubiquitin-conjugating enzyme and Rad18 DNA binding protein." Mol Cell Biol 17(8): 4536-4543.
Bala, S., A. Kumar, S. Soni, S. Sinha and M. Hanspal (2006). "Emp is a component of the nuclear matrix of mammalian cells and undergoes dynamic rearrangements during cell division." Biochem Biophys Res Commun 342(4): 1040-1048.
Ballinger, C. A., P. Connell, Y. Wu, Z. Hu, L. J. Thompson, L. Y. Yin and C. Patterson (1999). "Identification of CHIP, a novel tetratricopeptide repeat-containing protein that interacts with heat shock proteins and negatively regulates chaperone functions." Mol Cell Biol 19(6): 4535-4545.
Barbieri, C. E., S. C. Baca, M. S. Lawrence, F. Demichelis, M. Blattner, J. P. Theurillat, T. A. White, P. Stojanov, E. Van Allen, N. Stransky, E. Nickerson, S. S. Chae, G. Boysen, D. Auclair, R. C. Onofrio, K. Park, N. Kitabayashi, T. Y. MacDonald, K. Sheikh, T.
Chapter 8 References
304
Vuong, C. Guiducci, K. Cibulskis, A. Sivachenko, S. L. Carter, G. Saksena, D. Voet, W. M. Hussain, A. H. Ramos, W. Winckler, M. C. Redman, K. Ardlie, A. K. Tewari, J. M. Mosquera, N. Rupp, P. J. Wild, H. Moch, C. Morrissey, P. S. Nelson, P. W. Kantoff, S. B. Gabriel, T. R. Golub, M. Meyerson, E. S. Lander, G. Getz, M. A. Rubin and L. A. Garraway (2012). "Exome sequencing identifies recurrent SPOP, FOXA1 and MED12 mutations in prostate cancer." Nat Genet 44(6): 685-689.
Barford, D. (2011). "Structural insights into anaphase-promoting complex function and mechanism." Philos Trans R Soc Lond B Biol Sci 366(1584): 3605-3624.
Barnabas, N., L. Xu, A. Savera, Z. Hou and E. R. Barrack (2011). "Chromosome 8 markers of metastatic prostate cancer in African American men: gain of the MIR151 gene and loss of the NKX3-1 gene." Prostate 71(8): 857-871.
Bartlett, J. M. (2004). "Fluorescence in situ hybridization: technical overview." Methods Mol Med 97: 77-87.
Baumgartner, S., K. Hofmann, R. Chiquet-Ehrismann and P. Bucher (1998). "The discoidin domain family revisited: new members from prokaryotes and a homology-based fold prediction." Protein Sci 7(7): 1626-1631.
Behrends, C. and J. W. Harper (2011). "Constructing and decoding unconventional ubiquitin chains." Nat Struct Mol Biol 18(5): 520-528.
Belirgen, M., S. G. Berrak, H. Ozdag, S. U. Bozkurt, E. Eksioglu-Demiralp and M. M. Ozek (2012). "Biologic tumor behavior in pilocytic astrocytomas." Childs Nerv Syst 28(3): 375-389.
Bell, A. W., E. W. Deutsch, C. E. Au, R. E. Kearney, R. Beavis, S. Sechi, T. Nilsson and J. J. Bergeron (2009). "A HUPO test sample study reveals common problems in mass spectrometry-based proteomics." Nat Methods 6(6): 423-430.
Berleth, E. S. and C. M. Pickart (1996). "Mechanism of ubiquitin conjugating enzyme E2-230K: catalysis involving a thiol relay?" Biochemistry 35(5): 1664-1671.
Berry, R., J. J. Schroeder, A. J. French, S. K. McDonnell, B. J. Peterson, J. M. Cunningham, S. N. Thibodeau and D. J. Schaid (2000). "Evidence for a prostate cancer-susceptibility locus on chromosome 20." Am J Hum Genet 67(1): 82-91.
Best, C. J., J. W. Gillespie, Y. Yi, G. V. Chandramouli, M. A. Perlmutter, Y. Gathright, H. S. Erickson, L. Georgevich, M. A. Tangrea, P. H. Duray, S. Gonzalez, A. Velasco, W. M. Linehan, R. J. Matusik, D. K. Price, W. D. Figg, M. R. Emmert-Buck and R. F. Chuaqui (2005). "Molecular alterations in primary prostate cancer after androgen ablation therapy." Clin Cancer Res 11(19 Pt 1): 6823-6834.
Bethel, C. R., D. Faith, X. Li, B. Guan, J. L. Hicks, F. Lan, R. B. Jenkins, C. J. Bieberich and A. M. De Marzo (2006). "Decreased NKX3.1 protein expression in focal prostatic atrophy, prostatic intraepithelial neoplasia, and adenocarcinoma: association with gleason score and chromosome 8p deletion." Cancer Res 66(22): 10683-10690.
Bethel, C. R. and C. J. Bieberich (2007). "Loss of Nkx3.1 expression in the transgenic adenocarcinoma of mouse prostate model." Prostate 67(16): 1740-1750.
Bhatia-Gaur, R., A. A. Donjacour, P. J. Sciavolino, M. Kim, N. Desai, P. Young, C. R. Norton, T. Gridley, R. D. Cardiff, G. R. Cunha, C. Abate-Shen and M. M. Shen (1999). "Roles for Nkx3.1 in prostate development and cancer." Genes Dev 13(8): 966-977.
Bieberich, C. J., K. Fujita, W. W. He and G. Jay (1996). "Prostate-specific and androgen-dependent expression of a novel homeobox gene." J Biol Chem 271(50): 31779-31782.
Bienko, M., C. M. Green, N. Crosetto, F. Rudolf, G. Zapart, B. Coull, P. Kannouche, G. Wider, M. Peter, A. R. Lehmann, K. Hofmann and I. Dikic (2005). "Ubiquitin-binding domains
Chapter 8 References
305
in Y-family polymerases regulate translesion synthesis." Science 310(5755): 1821-1824.
Bill-Axelson, A., L. Holmberg, M. Ruutu, M. Haggman, S. O. Andersson, S. Bratell, A. Spangberg, C. Busch, S. Nordling, H. Garmo, J. Palmgren, H. O. Adami, B. J. Norlen and J. E. Johansson (2005). "Radical prostatectomy versus watchful waiting in early prostate cancer." N Engl J Med 352(19): 1977-1984.
Block, A. S., S. Saraswati, C. F. Lichti, M. Mahadevan and A. B. Diekman (2011). "Co-purification of Mac-2 binding protein with galectin-3 and association with prostasomes in human semen." Prostate 71(7): 711-721.
Bluemn, E. G. and P. S. Nelson (2012). "The androgen/androgen receptor axis in prostate cancer." Curr Opin Oncol 24(3): 251-257.
Blume-Jensen, P. and T. Hunter (2001). "Oncogenic kinase signalling." Nature 411(6835): 355-365.
Boname, J. M., M. Thomas, H. R. Stagg, P. Xu, J. Peng and P. J. Lehner (2010). "Efficient internalization of MHC I requires lysine-11 and lysine-63 mixed linkage polyubiquitin chains." Traffic 11(2): 210-220.
Bonita, D. P., S. Miyake, M. L. Lupher, Jr., W. Y. Langdon and H. Band (1997). "Phosphotyrosine binding domain-dependent upregulation of the platelet-derived growth factor receptor alpha signaling cascade by transforming mutants of Cbl: implications for Cbl's function and oncogenicity." Mol Cell Biol 17(8): 4597-4610.
Bowen, C., L. Bubendorf, H. J. Voeller, R. Slack, N. Willi, G. Sauter, T. C. Gasser, P. Koivisto, E. E. Lack, J. Kononen, O. P. Kallioniemi and E. P. Gelmann (2000). "Loss of NKX3.1 expression in human prostate cancers correlates with tumor progression." Cancer Res 60(21): 6111-6115.
Bowen, C., A. Stuart, J. H. Ju, J. Tuan, J. Blonder, T. P. Conrads, T. D. Veenstra and E. P. Gelmann (2007). "NKX3.1 homeodomain protein binds to topoisomerase I and enhances its activity." Cancer Res 67(2): 455-464.
Bowen, C. and E. P. Gelmann (2010). "NKX3.1 activates cellular response to DNA damage." Cancer Res 70(8): 3089-3097.
Boyd, S. D., K. Y. Tsai and T. Jacks (2000). "An intact HDM2 RING-finger domain is required for nuclear exclusion of p53." Nat Cell Biol 2(9): 563-568.
Braun, B., T. Pfirrmann, R. Menssen, K. Hofmann, H. Scheel and D. H. Wolf (2011). "Gid9, a second RING finger protein contributes to the ubiquitin ligase activity of the Gid complex required for catabolite degradation." FEBS Lett 585(24): 3856-3861.
Brawley, O. W. (2012). "Prostate cancer epidemiology in the United States." World J Urol 30(2): 195-200.
Brooks, C. L. and W. Gu (2006). "p53 ubiquitination: Mdm2 and beyond." Mol Cell 21(3): 307-315.
Brown, C. R., A. B. Wolfe, D. Cui and H. L. Chiang (2008). "The vacuolar import and degradation pathway merges with the endocytic pathway to deliver fructose-1,6-bisphosphatase to the vacuole for degradation." J Biol Chem 283(38): 26116-26127.
Brown, C. R., D. Dunton and H. L. Chiang (2010). "The vacuole import and degradation pathway utilizes early steps of endocytosis and actin polymerization to deliver cargo proteins to the vacuole for degradation." J Biol Chem 285(2): 1516-1528.
Chapter 8 References
306
Bruce, M. C., V. Kanelis, F. Fouladkou, A. Debonneville, O. Staub and D. Rotin (2008). "Regulation of Nedd4-2 self-ubiquitination and stability by a PY motif located within its HECT-domain." Biochem J 415(1): 155-163.
Brunkhorst, A., M. Karlen, J. Shi, M. Mikolajczyk, M. A. Nelson, M. Metsis and O. Hermanson (2005). "A specific role for the TFIID subunit TAF4 and RanBPM in neural progenitor differentiation." Mol Cell Neurosci 29(2): 250-258.
Brzovic, P. S., P. Rajagopal, D. W. Hoyt, M. C. King and R. E. Klevit (2001). "Structure of a BRCA1-BARD1 heterodimeric RING-RING complex." Nat Struct Biol 8(10): 833-837.
Brzovic, P. S., A. Lissounov, D. E. Christensen, D. W. Hoyt and R. E. Klevit (2006). "A UbcH5/ubiquitin noncovalent complex is required for processive BRCA1-directed ubiquitination." Mol Cell 21(6): 873-880.
Buckley, D. L., I. Van Molle, P. C. Gareiss, H. S. Tae, J. Michel, D. J. Noblin, W. L. Jorgensen, A. Ciulli and C. M. Crews (2012). "Targeting the von Hippel-Lindau E3 ubiquitin ligase using small molecules to disrupt the VHL/HIF-1alpha interaction." J Am Chem Soc 134(10): 4465-4468.
Burande, C. F., M. L. Heuze, I. Lamsoul, B. Monsarrat, S. Uttenweiler-Joseph and P. G. Lutz (2009). "A label-free quantitative proteomics strategy to identify E3 ubiquitin ligase substrates targeted to proteasome degradation." Mol Cell Proteomics 8(7): 1719-1727.
Burger, A. M., Y. Gao, Y. Amemiya, H. J. Kahn, R. Kitching, Y. Yang, P. Sun, S. A. Narod, W. M. Hanna and A. K. Seth (2005). "A novel RING-type ubiquitin ligase breast cancer-associated gene 2 correlates with outcome in invasive breast cancer." Cancer Res 65(22): 10401-10412.
Burroughs, A. M., M. Jaffee, L. M. Iyer and L. Aravind (2008). "Anatomy of the E2 ligase fold: implications for enzymology and evolution of ubiquitin/Ub-like protein conjugation." J Struct Biol 162(2): 205-218.
Cadwell, K. and L. Coscoy (2005). "Ubiquitination on nonlysine residues by a viral E3 ubiquitin ligase." Science 309(5731): 127-130.
Cahana, A., T. Escamez, R. S. Nowakowski, N. L. Hayes, M. Giacobini, A. von Holst, O. Shmueli, T. Sapir, S. K. McConnell, W. Wurst, S. Martinez and O. Reiner (2001). "Targeted mutagenesis of Lis1 disrupts cortical development and LIS1 homodimerization." Proc Natl Acad Sci U S A 98(11): 6429-6434.
Cai, C., S. Chen, P. Ng, G. J. Bubley, P. S. Nelson, E. A. Mostaghel, B. Marck, A. M. Matsumoto, N. I. Simon, H. Wang and S. P. Balk (2011). "Intratumoral de novo steroid synthesis activates androgen receptor in castration-resistant prostate cancer and is upregulated by treatment with CYP17A1 inhibitors." Cancer Res 71(20): 6503-6513.
Camps, J., I. Salaverria, M. J. Garcia, E. Prat, S. Bea, J. C. Pole, L. Hernandez, J. Del Rey, J. C. Cigudosa, M. Bernues, C. Caldas, D. Colomer, R. Miro and E. Campo (2006). "Genomic imbalances and patterns of karyotypic variability in mantle-cell lymphoma cell lines." Leuk Res 30(8): 923-934.
Cantor, S. B., D. W. Bell, S. Ganesan, E. M. Kass, R. Drapkin, S. Grossman, D. C. Wahrer, D. C. Sgroi, W. S. Lane, D. A. Haber and D. M. Livingston (2001). "BACH1, a novel helicase-like protein, interacts directly with BRCA1 and contributes to its DNA repair function." Cell 105(1): 149-160.
Cardoso, C., R. J. Leventer, N. Matsumoto, J. A. Kuc, M. B. Ramocki, S. K. Mewborn, L. L. Dudlicek, L. F. May, P. L. Mills, S. Das, D. T. Pilz, W. B. Dobyns and D. H. Ledbetter (2000). "The location and type of mutation predict malformation severity in isolated lissencephaly caused by abnormalities within the LIS1 gene." Hum Mol Genet 9(20): 3019-3028
Chapter 8 References
307
Carson, J. A., R. A. Fillmore, R. J. Schwartz and W. E. Zimmer (2000). "The smooth muscle gamma-actin gene promoter is a molecular target for the mouse bagpipe homologue, mNkx3-1, and serum response factor." J Biol Chem 275(50): 39061-39072.
Carver, B. S., J. Tran, A. Gopalan, Z. Chen, S. Shaikh, A. Carracedo, A. Alimonti, C. Nardella, S. Varmeh, P. T. Scardino, C. Cordon-Cardo, W. Gerald and P. P. Pandolfi (2009). "Aberrant ERG expression cooperates with loss of PTEN to promote cancer progression in the prostate." Nat Genet 41(5): 619-624.
Castro, A., C. Bernis, S. Vigneron, J. C. Labbe and T. Lorca (2005). "The anaphase-promoting complex: a key factor in the regulation of cell cycle." Oncogene 24(3): 314-325.
Center, M. M., A. Jemal, J. Lortet-Tieulent, E. Ward, J. Ferlay, O. Brawley and F. Bray (2012). "International variation in prostate cancer incidence and mortality rates." Eur Urol 61(6): 1079-1092.
Cerna, D. and D. K. Wilson (2005). "The structure of Sif2p, a WD repeat protein functioning in the SET3 corepressor complex." J Mol Biol 351(4): 923-935.
Chastagner, P., A. Israel and C. Brou (2006). "Itch/AIP4 mediates Deltex degradation through the formation of K29-linked polyubiquitin chains." EMBO Rep 7(11): 1147-1153.
Chau, V., J. W. Tobias, A. Bachmair, D. Marriott, D. J. Ecker, D. K. Gonda and A. Varshavsky (1989). "A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein." Science 243(4898): 1576-1583.
Chen, Z. and C. M. Pickart (1990). "A 25-kilodalton ubiquitin carrier protein (E2) catalyzes multi-ubiquitin chain synthesis via lysine 48 of ubiquitin." J Biol Chem 265(35): 21835-21842.
Chen, Z. J., L. Parent and T. Maniatis (1996). "Site-specific phosphorylation of IkappaBalpha by a novel ubiquitination-dependent protein kinase activity." Cell 84(6): 853-862.
Chen, C. Y. and R. J. Schwartz (1996). "Recruitment of the tinman homolog Nkx-2.5 by serum response factor activates cardiac alpha-actin gene transcription." Mol Cell Biol 16(11): 6372-6384.
Chen, H., A. K. Nandi, X. Li and C. J. Bieberich (2002a). "NKX-3.1 interacts with prostate-derived Ets factor and regulates the activity of the PSA promoter." Cancer Res 62(2): 338-340.
Chen, Y., R. Derin, R. S. Petralia and M. Li (2002b). "Actinfilin, a brain-specific actin-binding protein in postsynaptic density." J Biol Chem 277(34): 30495-30501.
Chen, C. D., D. S. Welsbie, C. Tran, S. H. Baek, R. Chen, R. Vessella, M. G. Rosenfeld and C. L. Sawyers (2004). "Molecular determinants of resistance to antiandrogen therapy." Nat Med 10(1): 33-39.
Chen, H. and C. J. Bieberich (2005). "Structural and functional analysis of domains mediating interaction between NKX-3.1 and PDEF." J Cell Biochem 94(1): 168-177.
Chen, B., J. Mariano, Y. C. Tsai, A. H. Chan, M. Cohen and A. M. Weissman (2006a). "The activity of a human endoplasmic reticulum-associated degradation E3, gp78, requires its Cue domain, RING finger, and an E2-binding site." Proc Natl Acad Sci U S A 103(2): 341-346.
Chen, H., Y. Shen, X. Tang, L. Yu, J. Wang, L. Guo, Y. Zhang, H. Zhang, S. Feng, E. Strickland, N. Zheng and X. W. Deng (2006b). "Arabidopsis CULLIN4 Forms an E3 Ubiquitin Ligase with RBX1 and the CDD Complex in Mediating Light Control of Development." Plant Cell 18(8): 1991-2004.
Chapter 8 References
308
Chen, C., X. Sun, P. Guo, X. Y. Dong, P. Sethi, W. Zhou, Z. Zhou, J. Petros, H. F. Frierson, Jr., R. L. Vessella, A. Atfi and J. T. Dong (2007). "Ubiquitin E3 ligase WWP1 as an oncogenic factor in human prostate cancer." Oncogene 26(16): 2386-2394.
Chen, Q., W. Xie, D. J. Kuhn, P. M. Voorhees, A. Lopez-Girona, D. Mendy, L. G. Corral, V. P. Krenitsky, W. Xu, L. Moutouh-de Parseval, D. R. Webb, F. Mercurio, K. I. Nakayama, K. Nakayama and R. Z. Orlowski (2008). "Targeting the p27 E3 ligase SCF(Skp2) results in p27- and Skp2-mediated cell-cycle arrest and activation of autophagy." Blood 111(9): 4690-4699.
Chen, M., Y. Ye, H. Yang, P. Tamboli, S. Matin, N. M. Tannir, C. G. Wood, J. Gu and X. Wu (2009). "Genome-wide profiling of chromosomal alterations in renal cell carcinoma using high-density single nucleotide polymorphism arrays." Int J Cancer 125(10): 2342-2348.
Chen, J., K. V. Giridhar, L. Zhang, S. Xu and Q. J. Wang (2011a). "A protein kinase C/protein kinase D pathway protects LNCaP prostate cancer cells from phorbol ester-induced apoptosis by promoting ERK1/2 and NF-{kappa}B activities." Carcinogenesis 32(8): 1198-1206.
Chen, Y., L. Zhang and K. A. Jones (2011b). "SKIP counteracts p53-mediated apoptosis via selective regulation of p21Cip1 mRNA splicing." Genes Dev 25(7): 701-716.
Cher, M. L., D. MacGrogan, R. Bookstein, J. A. Brown, R. B. Jenkins and R. H. Jensen (1994). "Comparative genomic hybridization, allelic imbalance, and fluorescence in situ hybridization on chromosome 8 in prostate cancer." Genes Chromosomes Cancer 11(3): 153-162.
Cheng, L., S. Lemmon and V. Lemmon (2005). "RanBPM is an L1-interacting protein that regulates L1-mediated mitogen-activated protein kinase activation." J Neurochem 94(4): 1102-1110.
Chiang, M. C. and H. L. Chiang (1998). "Vid24p, a novel protein localized to the fructose-1, 6-bisphosphatase-containing vesicles, regulates targeting of fructose-1,6-bisphosphatase from the vesicles to the vacuole for degradation." J Cell Biol 140(6): 1347-1356.
Chiba, N. and J. D. Parvin (2002). "The BRCA1 and BARD1 association with the RNA polymerase II holoenzyme." Cancer Res 62(15): 4222-4228.
Chiu, Y. H., Q. Sun and Z. J. Chen (2007). "E1-L2 activates both ubiquitin and FAT10." Mol Cell 27(6): 1014-1023.
Choi, H. K., K. C. Choi, H. B. Kang, H. C. Kim, Y. H. Lee, S. Haam, H. G. Park and H. G. Yoon (2008). "Function of multiple Lis-Homology domain/WD-40 repeat-containing proteins in feed-forward transcriptional repression by silencing mediator for retinoic and thyroid receptor/nuclear receptor corepressor complexes." Mol Endocrinol 22(5): 1093-1104.
Chornokur, G., K. Dalton, M. E. Borysova and N. B. Kumar (2011). "Disparities at presentation, diagnosis, treatment, and survival in African American men, affected by prostate cancer." Prostate 71(9): 985-997.
Christensen, D. E., P. S. Brzovic and R. E. Klevit (2007). "E2-BRCA1 RING interactions dictate synthesis of mono- or specific polyubiquitin chain linkages." Nat Struct Mol Biol 14(10): 941-948.
Christensen, G. B., A. B. Baffoe-Bonnie, A. George, I. Powell, J. E. Bailey-Wilson, J. D. Carpten, G. G. Giles, J. L. Hopper, G. Severi, D. R. English, W. D. Foulkes, L. Maehle, P. Moller, R. Eeles, D. Easton, M. D. Badzioch, A. S. Whittemore, I. Oakley-Girvan, C. L. Hsieh, L. Dimitrov, J. Xu, J. L. Stanford, B. Johanneson, K. Deutsch, L. McIntosh, E. A. Ostrander, K. E. Wiley, S. D. Isaacs, P. C. Walsh, W. B. Isaacs, S. N. Thibodeau,
Chapter 8 References
309
S. K. McDonnell, S. Hebbring, D. J. Schaid, E. M. Lange, K. A. Cooney, T. L. Tammela, J. Schleutker, T. Paiss, C. Maier, H. Gronberg, F. Wiklund, M. Emanuelsson, J. M. Farnham, L. A. Cannon-Albright and N. J. Camp (2010). "Genome-wide linkage analysis of 1,233 prostate cancer pedigrees from the International Consortium for Prostate Cancer Genetics using novel sumLINK and sumLOD analyses." Prostate 70(7): 735-744.
Ciruela, F., J. P. Vilardaga and V. Fernandez-Duenas (2010). "Lighting up multiprotein complexes: lessons from GPCR oligomerization." Trends Biotechnol 28(8): 407-415.
Ciechanover, A., H. Heller, S. Elias, A. L. Haas and A. Hershko (1980). "ATP-dependent conjugation of reticulocyte proteins with the polypeptide required for protein degradation." Proc Natl Acad Sci U S A 77(3): 1365-1368.
Ciechanover, A., H. Heller, R. Katz-Etzion and A. Hershko (1981). "Activation of the heat-stable polypeptide of the ATP-dependent proteolytic system." Proc Natl Acad Sci U S A 78(2): 761-765.
Ciechanover, A., S. Elias, H. Heller and A. Hershko (1982). ""Covalent affinity" purification of ubiquitin-activating enzyme." J Biol Chem 257(5): 2537-2542.
Ciechanover, A. and A. L. Schwartz (1989). "The ubiquitin-dependent proteolytic pathway: specificity of recognition of the proteolytic substrates." Revis Biol Celular 20: 217-234.
Ciechanover, A. and R. Ben-Saadon (2004). "N-terminal ubiquitination: more protein substrates join in." Trends Cell Biol 14(3): 103-106.
Ciechanover, A. (2012). "Intracellular protein degradation: from a vague idea thru the lysosome and the ubiquitin-proteasome system and onto human diseases and drug targeting." Biochim Biophys Acta 1824(1): 3-13.
Clark, J. P. and C. S. Cooper (2009). "ETS gene fusions in prostate cancer." Nat Rev Urol 6(8): 429-439.
Connolly, R. M., M. A. Carducci and E. S. Antonarakis (2012). "Use of androgen deprivation therapy in prostate cancer: indications and prevalence." Asian J Androl 14(2): 177-186.
Cook, J. C. and P. B. Chock (1992). "Isoforms of mammalian ubiquitin-activating enzyme." J Biol Chem 267(34): 24315-24321.
Cook, J. C. and P. B. Chock (1995). "Phosphorylation of ubiquitin-activating enzyme in cultured cells." Proc Natl Acad Sci U S A 92(8): 3454-3457.
Cookson, M. R., P. J. Lockhart, C. McLendon, C. O'Farrell, M. Schlossmacher and M. J. Farrer (2003). "RING finger 1 mutations in Parkin produce altered localization of the protein." Hum Mol Genet 12(22): 2957-2965.
Corsini, L., M. Hothorn, G. Stier, V. Rybin, K. Scheffzek, T. J. Gibson and M. Sattler (2009). "Dimerization and protein binding specificity of the U2AF homology motif of the splicing factor Puf60." J Biol Chem 284(1): 630-639.
Cottee, P. A., E. L. O. Y. G. Abs, A. J. Nisbet and R. B. Gasser (2006). "Ubiquitin-conjugating enzyme genes in Oesophagostomum dentatum." Parasitol Res 99(2): 119-125.
Cox, M. M. (2012). "Recombinant protein vaccines produced in insect cells." Vaccine 30(10): 1759-1766.
Craft, N., Y. Shostak, M. Carey and C. L. Sawyers (1999). "A mechanism for hormone-independent prostate cancer through modulation of androgen receptor signaling by the HER-2/neu tyrosine kinase." Nat Med 5(3): 280-285.
Craiu, A., M. Gaczynska, T. Akopian, C. F. Gramm, G. Fenteany, A. L. Goldberg and K. L. Rock (1997). "Lactacystin and clasto-lactacystin beta-lactone modify multiple
Chapter 8 References
310
proteasome beta-subunits and inhibit intracellular protein degradation and major histocompatibility complex class I antigen presentation." J Biol Chem 272(20): 13437-13445.
Crawley, J. N. (2007). "What's Wrong With My Mouse?: Behavioral Phenotyping of Transgenic and Knockout Mice". New Jersey, John Wiley and Sons.
Cruz, C., F. Ventura, R. Bartrons and J. L. Rosa (2001). "HERC3 binding to and regulation by ubiquitin." FEBS Lett 488(1-2): 74-80.
Cummins, J. M., C. Rago, M. Kohli, K. W. Kinzler, C. Lengauer and B. Vogelstein (2004). "Tumour suppression: disruption of HAUSP gene stabilizes p53." Nature 428(6982): 1 p following 486.
Cummins, J. M. and B. Vogelstein (2004). "HAUSP is required for p53 destabilization." Cell Cycle 3(6): 689-692.
Damber, J. E. and G. Aus (2008). "Prostate cancer." Lancet 371(9625): 1710-1721.
Dammer, E. B., C. Fallini, Y. M. Gozal, D. M. Duong, W. Rossoll, P. Xu, J. J. Lah, A. I. Levey, J. Peng, G. J. Bassell and N. T. Seyfried (2012). "Coaggregation of RNA-Binding Proteins in a Model of TDP-43 Proteinopathy with Selective RGG Motif Methylation and a Role for RRM1 Ubiquitination." PLoS One 7(6): e38658.
Dao, K. H., M. D. Rotelli, C. L. Petersen, S. Kaech, W. D. Nelson, J. E. Yates, A. E. Hanlon Newell, S. B. Olson, B. J. Druker and G. C. Bagby (2012). "FANCL ubiquitinates beta-catenin and enhances its nuclear function." Blood 120(2): 323-334.
Das, R., J. Mariano, Y. C. Tsai, R. C. Kalathur, Z. Kostova, J. Li, S. G. Tarasov, R. L. McFeeters, A. S. Altieri, X. Ji, R. A. Byrd and A. M. Weissman (2009). "Allosteric activation of E2-RING finger-mediated ubiquitylation by a structurally defined specific E2-binding region of gp78." Mol Cell 34(6): 674-685.
David, Y., N. Ternette, M. J. Edelmann, T. Ziv, B. Gayer, R. Sertchook, Y. Dadon, B. M. Kessler and A. Navon (2011). "E3 ligases determine ubiquitination site and conjugate type by enforcing specificity on E2 enzymes." J Biol Chem 286(51): 44104-44115.
Davie, J. R. and L. C. Murphy (1990). "Level of ubiquitinated histone H2B in chromatin is coupled to ongoing transcription." Biochemistry 29(20): 4752-4757.
Dawson, L. (2006). FLJ22318: a novel binding partner of the NKX3-1 homeodomain protein in prostate cancer cells. PhD Thesis. Murdoch University, Perth, Western Australia.
de Bono, J. S., C. J. Logothetis, A. Molina, K. Fizazi, S. North, L. Chu, K. N. Chi, R. J. Jones, O. B. Goodman, Jr., F. Saad, J. N. Staffurth, P. Mainwaring, S. Harland, T. W. Flaig, T. E. Hutson, T. Cheng, H. Patterson, J. D. Hainsworth, C. J. Ryan, C. N. Sternberg, S. L. Ellard, A. Flechon, M. Saleh, M. Scholz, E. Efstathiou, A. Zivi, D. Bianchini, Y. Loriot, N. Chieffo, T. Kheoh, C. M. Haqq and H. I. Scher (2011). "Abiraterone and increased survival in metastatic prostate cancer." N Engl J Med 364(21): 1995-2005.
Deeb, S. J., R. C. D'Souza, J. Cox, M. Schmidt-Supprian and M. Mann (2012). "Super-SILAC allows classification of diffuse large B-cell lymphoma subtypes by their protein expression profiles." Mol Cell Proteomics 11(5): 77-89.
De Marzo, A. M., E. A. Platz, S. Sutcliffe, J. Xu, H. Gronberg, C. G. Drake, Y. Nakai, W. B. Isaacs and W. G. Nelson (2007). "Inflammation in prostate carcinogenesis." Nat Rev Cancer 7(4): 256-269.
Deng, L., C. Wang, E. Spencer, L. Yang, A. Braun, J. You, C. Slaughter, C. Pickart and Z. J. Chen (2000). "Activation of the IkappaB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain." Cell 103(2): 351-361.
Chapter 8 References
311
Denti, S., A. Sirri, A. Cheli, L. Rogge, G. Innamorati, S. Putignano, M. Fabbri, R. Pardi and E. Bianchi (2004). "RanBPM is a phosphoprotein that associates with the plasma membrane and interacts with the integrin LFA-1." J Biol Chem 279(13): 13027-13034.
Deribe, Y. L., T. Pawson and I. Dikic (2010). "Post-translational modifications in signal integration." Nat Struct Mol Biol 17(6): 666-672.
Deshaies, R. J. and C. A. Joazeiro (2009). "RING domain E3 ubiquitin ligases." Annu Rev Biochem 78: 399-434.
Dhavan, R., P. L. Greer, M. A. Morabito, L. R. Orlando and L. H. Tsai (2002). "The cyclin-
dependent kinase 5 activators p35 and p39 interact with the alpha-subunit of Ca2+/calmodulin-dependent protein kinase II and alpha-actinin-1 in a calcium-dependent manner." J Neurosci 22(18): 7879-7891.
Dikic, I., S. Wakatsuki and K. J. Walters (2009). "Ubiquitin-binding domains - from structures to functions." Nat Rev Mol Cell Biol 10(10): 659-671.
Dimitrova, Y. N., J. Li, Y. T. Lee, J. Rios-Esteves, D. B. Friedman, H. J. Choi, W. I. Weis, C. Y. Wang and W. J. Chazin (2010). "Direct ubiquitination of beta-catenin by Siah-1 and regulation by the exchange factor TBL1." J Biol Chem 285(18): 13507-13516.
Doetzlhofer, A., H. Rotheneder, G. Lagger, M. Koranda, V. Kurtev, G. Brosch, E. Wintersberger and C. Seiser (1999). "Histone deacetylase 1 can repress transcription by binding to Sp1." Mol Cell Biol 19(8): 5504-5511.
Dong, J. T. (2006). "Prevalent mutations in prostate cancer." J Cell Biochem 97(3): 433-447.
Dunn, R., D. A. Klos, A. S. Adler and L. Hicke (2004). "The C2 domain of the Rsp5 ubiquitin ligase binds membrane phosphoinositides and directs ubiquitination of endosomal cargo." J Cell Biol 165(1): 135-144.
Dynek, J. N., T. Goncharov, E. C. Dueber, A. V. Fedorova, A. Izrael-Tomasevic, L. Phu, E. Helgason, W. J. Fairbrother, K. Deshayes, D. S. Kirkpatrick and D. Vucic (2010). "c-IAP1 and UbcH5 promote K11-linked polyubiquitination of RIP1 in TNF signalling." EMBO J 29(24): 4198-4209.
Eddins, M. J., C. M. Carlile, K. M. Gomez, C. M. Pickart and C. Wolberger (2006). "Mms2-Ubc13 covalently bound to ubiquitin reveals the structural basis of linkage-specific polyubiquitin chain formation." Nat Struct Mol Biol 13(10): 915-920.
Edelmann, M. J., B. Nicholson and B. M. Kessler (2011). "Pharmacological targets in the ubiquitin system offer new ways of treating cancer, neurodegenerative disorders and infectious diseases." Expert Rev Mol Med 13: e35.
Edge, S. B., D. R. Byrd, C. C. Compton, A. G. Fritz, F. L. Greene and A. Trotti, Eds. (2010). "AJCC Cancer Staging Handbook". New York, Springer, pp525-538.
Edwin, F., K. Anderson and T. B. Patel (2010). "HECT domain-containing E3 ubiquitin ligase Nedd4 interacts with and ubiquitinates Sprouty2." J Biol Chem 285(1): 255-264.
Eletr, Z. M., D. T. Huang, D. M. Duda, B. A. Schulman and B. Kuhlman (2005). "E2 conjugating enzymes must disengage from their E1 enzymes before E3-dependent ubiquitin and ubiquitin-like transfer." Nat Struct Mol Biol 12(10): 933-934.
Emes, R. D. and C. P. Ponting (2001). "A new sequence motif linking lissencephaly, Treacher Collins and oral-facial-digital type 1 syndromes, microtubule dynamics and cell migration." Hum Mol Genet 10(24): 2813-2820.
Emmert-Buck, M. R., C. D. Vocke, R. O. Pozzatti, P. H. Duray, S. B. Jennings, C. D. Florence, Z. Zhuang, D. G. Bostwick, L. A. Liotta and W. M. Linehan (1995). "Allelic loss on
Chapter 8 References
312
chromosome 8p12-21 in microdissected prostatic intraepithelial neoplasia." Cancer Res 55(14): 2959-2962.
Enyenihi, A. H. and W. S. Saunders (2003). "Large-scale functional genomic analysis of sporulation and meiosis in Saccharomyces cerevisiae." Genetics 163(1): 47-54.
Erbaykent-Tepedelen, B., B. Ozmen, L. Varisli, C. Gonen-Korkmaz, B. Debelec-Butuner, H. Muhammed Syed, O. Yilmazer-Cakmak and K. S. Korkmaz (2011). "NKX3.1 contributes to S phase entry and regulates DNA damage response (DDR) in prostate cancer cell lines." Biochem Biophys Res Commun 414(1): 123-128.
Esposito, D. and D. K. Chatterjee (2006). "Enhancement of soluble protein expression through the use of fusion tags." Curr Opin Biotechnol 17(4): 353-358.
Esser, C., M. Scheffner and J. Hohfeld (2005). "The chaperone-associated ubiquitin ligase CHIP is able to target p53 for proteasomal degradation." J Biol Chem 280(29): 27443-27448.
Ettenberg, S. A., Y. R. Rubinstein, P. Banerjee, M. M. Nau, M. M. Keane and S. Lipkowitz (1999). "cbl-b inhibits EGF-receptor-induced apoptosis by enhancing ubiquitination and degradation of activated receptors." Mol Cell Biol Res Commun 2(2): 111-118.
Eylert, M. F. and R. Persad (2012). "Management of prostate cancer." Br J Hosp Med (Lond) 73(2): 95-99.
Fabbro, M., S. Schuechner, W. W. Au and B. R. Henderson (2004). "BARD1 regulates BRCA1 apoptotic function by a mechanism involving nuclear retention." Exp Cell Res 298(2): 661-673.
Falsone, S. F., B. Gesslbauer and A. J. Kungl (2008). "Coimmunoprecipitation and proteomic
analyses." Methods Mol Biol 439: 291-308. Fang, S., J. P. Jensen, R. L. Ludwig, K. H. Vousden and A. M. Weissman (2000). "Mdm2 is a
RING finger-dependent ubiquitin protein ligase for itself and p53." J Biol Chem 275(12): 8945-8951.
Fang, S., K. L. Lorick, J. P. Jensen and A. M. Weissman (2003). "RING finger ubiquitin protein ligases: implications for tumorigenesis, metastasis and for molecular targets in cancer." Semin Cancer Biol 13(1): 5-14.
Fang, S. and A. M. Weissman (2004). "A field guide to ubiquitylation." Cell Mol Life Sci 61(13): 1546-1561.
Feki, A., C. E. Jefford, P. Berardi, J. Y. Wu, L. Cartier, K. H. Krause and I. Irminger-Finger (2005). "BARD1 induces apoptosis by catalysing phosphorylation of p53 by DNA-damage response kinase." Oncogene 24(23): 3726-3736.
Feng, Y., C. Zhang, Q. Luo, X. Wei, B. Jiang, H. Zhu, L. Zhang, L. Jiang, M. Liu and X. Xiao (2012). "A novel WD-repeat protein, WDR26, inhibits apoptosis of cardiomyocytes induced by oxidative stress." Free Radic Res 46(6): 777-784.
Ferlay, J., H. Shin, F. Bray, D. Forman, C. Mathers and D. Parkin (2010) "GLOBOCAN 2008 v1.2, Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 10 ".
Fierz, B., C. Chatterjee, R. K. McGinty, M. Bar-Dagan, D. P. Raleigh and T. W. Muir (2011). "Histone H2B ubiquitylation disrupts local and higher-order chromatin compaction." Nat Chem Biol 7(2): 113-119.
Finley, D., A. Ciechanover and A. Varshavsky (1984). "Thermolability of ubiquitin-activating enzyme from the mammalian cell cycle mutant ts85." Cell 37(1): 43-55.
Fisher, R. D., B. Wang, S. L. Alam, D. S. Higginson, H. Robinson, W. I. Sundquist and C. P. Hill (2003). "Structure and ubiquitin binding of the ubiquitin-interacting motif." J Biol Chem 278(31): 28976-28984.
Chapter 8 References
313
Francis, D. M. and R. Page (2010). "Strategies to optimize protein expression in E. coli." Curr Protoc Protein Sci Chapter 5: Unit 5 24 21-29.
Franke, W. W., M. D. Goldschmidt, R. Zimbelmann, H. M. Mueller, D. L. Schiller and P. Cowin (1989). "Molecular cloning and amino acid sequence of human plakoglobin, the common junctional plaque protein." Proc Natl Acad Sci U S A 86(11): 4027-4031.
Freemont, P. S. (1993). "The RING finger. A novel protein sequence motif related to the zinc finger." Ann N Y Acad Sci 684: 174-192.
Fu, Q. S., C. J. Zhou, H. C. Gao, Y. J. Jiang, Z. R. Zhou, J. Hong, W. M. Yao, A. X. Song, D. H. Lin and H. Y. Hu (2009). "Structural basis for ubiquitin recognition by a novel domain from human phospholipase A2-activating protein." J Biol Chem 284(28): 19043-19052.
Fuchs, S. Y., B. Xie, V. Adler, V. A. Fried, R. J. Davis and Z. Ronai (1997). "c-Jun NH2-terminal kinases target the ubiquitination of their associated transcription factors." J Biol Chem 272(51): 32163-32168.
Fuks, F., P. J. Hurd, R. Deplus and T. Kouzarides (2003). "The DNA methyltransferases associate with HP1 and the SUV39H1 histone methyltransferase." Nucleic Acids Res 31(9): 2305-2312.
Funayama, S., J. M. Gancedo and C. Gancedo (1980). "Turnover of yeast fructose-bisphosphatase in different metabolic conditions." Eur J Biochem 109(1): 61-66.
Furukawa, M., T. Ohta and Y. Xiong (2002). "Activation of UBC5 ubiquitin-conjugating enzyme by the RING finger of ROC1 and assembly of active ubiquitin ligases by all cullins." J Biol Chem 277(18): 15758-15765.
Furukawa, M., P. S. Andrews and Y. Xiong (2005). "Assays for RING family ubiquitin ligases." Methods Mol Biol 301: 37-46.
Gallagher, E., M. Gao, Y. C. Liu and M. Karin (2006). "Activation of the E3 ubiquitin ligase Itch through a phosphorylation-induced conformational change." Proc Natl Acad Sci U S A 103(6): 1717-1722.
Gancedo, C. (1971). "Inactivation of fructose-1,6-diphosphatase by glucose in yeast." J Bacteriol 107(2): 401-405.
Gao, R., C. J. McCormick, M. J. Arthur, R. Ruddell, F. Oakley, D. E. Smart, F. R. Murphy, M. P. Harris and D. A. Mann (2002). "High efficiency gene transfer into cultured primary rat and human hepatic stellate cells using baculovirus vectors." Liver 22(1): 15-22.
Gao, D., Y. K. Yang, R. P. Wang, X. Zhou, F. C. Diao, M. D. Li, Z. H. Zhai, Z. F. Jiang and D. Y. Chen (2009). "REUL is a novel E3 ubiquitin ligase and stimulator of retinoic-acid-inducible gene-I." PLoS One 4(6): e5760.
Garcia-Gonzalo, F. R., R. Bartrons, F. Ventura and J. L. Rosa (2005). "Requirement of phosphatidylinositol-4,5-bisphosphate for HERC1-mediated guanine nucleotide release from ARF proteins." FEBS Lett 579(2): 343-348.
Garcia-Guzman, M., E. Larsen and K. Vuori (2000). "The proto-oncogene c-Cbl is a positive regulator of Met-induced MAP kinase activation: a role for the adaptor protein Crk." Oncogene 19(35): 4058-4065.
Garcia-Higuera, I., E. Manchado, P. Dubus, M. Canamero, J. Mendez, S. Moreno and M. Malumbres (2008). "Genomic stability and tumour suppression by the APC/C cofactor Cdh1." Nat Cell Biol 10(7): 802-811.
Chapter 8 References
314
Gary, B., R. Azuero, G. S. Mohanty, W. C. Bell, I. E. Eltoum and S. A. Abdulkadir (2004). "Interaction of Nkx3.1 and p27kip1 in prostate tumor initiation." Am J Pathol 164(5): 1607-1614.
Gelmann, E. P., D. J. Steadman, J. Ma, N. Ahronovitz, H. J. Voeller, S. Swope, M. Abbaszadegan, K. M. Brown, K. Strand, R. B. Hayes and M. J. Stampfer (2002). "Occurrence of NKX3.1 C154T polymorphism in men with and without prostate cancer and studies of its effect on protein function." Cancer Res 62(9): 2654-2659.
Gelmann, E. P., C. Bowen and L. Bubendorf (2003). "Expression of NKX3.1 in normal and malignant tissues." Prostate 55(2): 111-117.
Gerritsen, W. R. (2012). "The evolving role of immunotherapy in prostate cancer." Ann Oncol 23 Suppl 8: viii22-viii27.
Gerlitz, G., E. Darhin, G. Giorgio, B. Franco and O. Reiner (2005). "Novel functional features of the Lis-H domain: role in protein dimerization, half-life and cellular localization." Cell Cycle 4(11): 1632-1640.
Geyer, R. K., Z. K. Yu and C. G. Maki (2000). "The MDM2 RING-finger domain is required to promote p53 nuclear export." Nat Cell Biol 2(9): 569-573.
Giannini, A. L., Y. Gao and M. J. Bijlmakers (2008). "T-cell regulator RNF125/TRAC-1 belongs to a novel family of ubiquitin ligases with zinc fingers and a ubiquitin-binding domain." Biochem J 410(1): 101-111.
Gleason, D. F. and G. T. Mellinger (1974). "Prediction of prognosis for prostatic adenocarcinoma by combined histological grading and clinical staging." J Urol 111(1): 58-64.
Gorte, M., A. Horstman, R. B. Page, R. Heidstra, A. Stromberg and K. Boutilier (2011). "Microarray-based identification of transcription factor target genes." Methods Mol Biol 754: 119-141.
Greenbaum, L., D. J. Katcoff, H. Dou, Y. Gozlan and Z. Malik (2003). "A porphobilinogen deaminase (PBGD) Ran-binding protein interaction is implicated in nuclear trafficking of PBGD in differentiating glioma cells." Oncogene 22(34): 5221-5228.
Greene, F. L. and L. H. Sobin (2002). "The TNM system: our language for cancer care." J Surg Oncol 80(3): 119-120.
Greene, K. L., P. C. Albertsen, R. J. Babaian, H. B. Carter, P. H. Gann, M. Han, D. A. Kuban, A. O. Sartor, J. L. Stanford, A. Zietman and P. Carroll (2009). "Prostate specific antigen best practice statement: 2009 update." J Urol 182(5): 2232-2241.
Gregori, L., M. S. Poosch, G. Cousins and V. Chau (1990). "A uniform isopeptide-linked multiubiquitin chain is sufficient to target substrate for degradation in ubiquitin-mediated proteolysis." J Biol Chem 265(15): 8354-8357.
Gregory, C. W., B. He, R. T. Johnson, O. H. Ford, J. L. Mohler, F. S. French and E. M. Wilson (2001a). "A mechanism for androgen receptor-mediated prostate cancer recurrence after androgen deprivation therapy." Cancer Res 61(11): 4315-4319.
Gregory, C. W., R. T. Johnson, Jr., J. L. Mohler, F. S. French and E. M. Wilson (2001b). "Androgen receptor stabilization in recurrent prostate cancer is associated with hypersensitivity to low androgen." Cancer Res 61(7): 2892-2898.
Gong, X., W. Ye, H. Zhou, X. Ren, Z. Li, W. Zhou, J. Wu, Y. Gong, Q. Ouyang, X. Zhao and X. Zhang (2009). "RanBPM is an acetylcholinesterase-interacting protein that translocates into the nucleus during apoptosis." Acta Biochim Biophys Sin (Shanghai) 41(11): 883-891.
Chapter 8 References
315
Guan, B., P. Pungaliya, X. Li, C. Uquillas, L. N. Mutton, E. H. Rubin and C. J. Bieberich (2008). "Ubiquitination by TOPORS regulates the prostate tumor suppressor NKX3.1." J Biol Chem 283(8): 4834-4840.
Guenther, M. G., W. S. Lane, W. Fischle, E. Verdin, M. A. Lazar and R. Shiekhattar (2000). "A core SMRT corepressor complex containing HDAC3 and TBL1, a WD40-repeat protein linked to deafness." Genes Dev 14(9): 1048-1057.
Gupta, M., P. Kogut, F. J. Davis, N. S. Belaguli, R. J. Schwartz and M. P. Gupta (2001). "Physical interaction between the MADS box of serum response factor and the TEA/ATTS DNA-binding domain of transcription enhancer factor-1." J Biol Chem 276(13): 10413-10422.
Gupta, R., B. Kus, C. Fladd, J. Wasmuth, R. Tonikian, S. Sidhu, N. J. Krogan, J. Parkinson and D. Rotin (2007). "Ubiquitination screen using protein microarrays for comprehensive identification of Rsp5 substrates in yeast." Mol Syst Biol 3: 116.
Haas, A. L. and I. A. Rose (1982). "The mechanism of ubiquitin activating enzyme. A kinetic
and equilibrium analysis." J Biol Chem 257(17): 10329-10337.
Haas, A. L., J. V. Warms, A. Hershko and I. A. Rose (1982). "Ubiquitin-activating enzyme. Mechanism and role in protein-ubiquitin conjugation." J Biol Chem 257(5): 2543-2548.
Haas, A. L., P. B. Reback and V. Chau (1991). "Ubiquitin conjugation by the yeast RAD6 and CDC34 gene products. Comparison to their putative rabbit homologs, E2(20K) AND E2(32K)." J Biol Chem 266(8): 5104-5112.
Hafizi, S., A. Gustafsson, J. Stenhoff and B. Dahlback (2005). "The Ran binding protein RanBPM interacts with Axl and Sky receptor tyrosine kinases." Int J Biochem Cell Biol 37(11): 2344-2356.
Haggman, M. J., K. J. Wojno, C. P. Pearsall and J. A. Macoska (1997). "Allelic loss of 8p sequences in prostatic intraepithelial neoplasia and carcinoma." Urology 50(4): 643-647.
Haglund, K., P. P. Di Fiore and I. Dikic (2003a). "Distinct monoubiquitin signals in receptor endocytosis." Trends Biochem Sci 28(11): 598-603.
Haglund, K., S. Sigismund, S. Polo, I. Szymkiewicz, P. P. Di Fiore and I. Dikic (2003b). "Multiple monoubiquitination of RTKs is sufficient for their endocytosis and degradation." Nat Cell Biol 5(5): 461-466.
Hakli, M., K. L. Lorick, A. M. Weissman, O. A. Janne and J. J. Palvimo (2004). "Transcriptional coregulator SNURF (RNF4) possesses ubiquitin E3 ligase activity." FEBS Lett 560(1-3): 56-62.
Haluska, P., Jr., A. Saleem, Z. Rasheed, F. Ahmed, E. W. Su, L. F. Liu and E. H. Rubin (1999). "Interaction between human topoisomerase I and a novel RING finger/arginine-serine protein." Nucleic Acids Res 27(12): 2538-2544.
Haldeman, M. T., G. Xia, E. M. Kasperek and C. M. Pickart (1997). "Structure and function of ubiquitin conjugating enzyme E2-25K: the tail is a core-dependent activity element." Biochemistry 36(34): 10526-10537.
Hammer, E., R. Heilbronn and S. Weger (2007). "The E3 ligase Topors induces the accumulation of polysumoylated forms of DNA topoisomerase I in vitro and in vivo." FEBS Lett 581(28): 5418-5424.
Hammerle, M., J. Bauer, M. Rose, A. Szallies, M. Thumm, S. Dusterhus, D. Mecke, K. D. Entian and D. H. Wolf (1998). "Proteins of newly isolated mutants and the amino-terminal proline are essential for ubiquitin-proteasome-catalyzed catabolite degradation
Chapter 8 References
316
of fructose-1,6-bisphosphatase of Saccharomyces cerevisiae." J Biol Chem 273(39): 25000-25005.
Hammond-Martel, I., H. Yu and B. Affar el (2012). "Roles of ubiquitin signaling in transcription regulation." Cell Signal 24(2): 410-421.
Handley, P. M., M. Mueckler, N. R. Siegel, A. Ciechanover and A. L. Schwartz (1991). "Molecular cloning, sequence, and tissue distribution of the human ubiquitin-activating enzyme E1." Proc Natl Acad Sci U S A 88(1): 258-262.
Hanspal, M. and J. S. Hanspal (1994). "The association of erythroblasts with macrophages promotes erythroid proliferation and maturation: a 30-kD heparin-binding protein is involved in this contact." Blood 84(10): 3494-3504.
Hanspal, M., Y. Smockova and Q. Uong (1998). "Molecular identification and functional characterization of a novel protein that mediates the attachment of erythroblasts to macrophages." Blood 92(8): 2940-2950.
Hanzelmann, P., A. Schafer, D. Voller and H. Schindelin (2012). "Structural insights into functional modes of proteins involved in ubiquitin family pathways." Methods Mol Biol 832: 547-576.
Hao, Y., K. Sekine, A. Kawabata, H. Nakamura, T. Ishioka, H. Ohata, R. Katayama, C. Hashimoto, X. Zhang, T. Noda, T. Tsuruo and M. Naito (2004). "Apollon ubiquitinates SMAC and caspase-9, and has an essential cytoprotection function." Nat Cell Biol 6(9): 849-860.
Hao, B., N. Zheng, B. A. Schulman, G. Wu, J. J. Miller, M. Pagano and N. P. Pavletich (2005). "Structural basis of the Cks1-dependent recognition of p27(Kip1) by the SCF(Skp2) ubiquitin ligase." Mol Cell 20(1): 9-19.
Hao, B., S. Oehlmann, M. E. Sowa, J. W. Harper and N. P. Pavletich (2007). "Structure of a Fbw7-Skp1-cyclin E complex: multisite-phosphorylated substrate recognition by SCF ubiquitin ligases." Mol Cell 26(1): 131-143.
Harper, S. and D. W. Speicher (2011). "Purification of proteins fused to glutathione S-transferase." Methods Mol Biol 681: 259-280.
Harris, W. P., E. A. Mostaghel, P. S. Nelson and B. Montgomery (2009). "Androgen deprivation therapy: progress in understanding mechanisms of resistance and optimizing androgen depletion." Nat Clin Pract Urol 6(2): 76-85.
Harvey, K. F., A. Dinudom, P. Komwatana, C. N. Jolliffe, M. L. Day, G. Parasivam, D. I. Cook and S. Kumar (1999). "All three WW domains of murine Nedd4 are involved in the regulation of epithelial sodium channels by intracellular Na+." J Biol Chem 274(18): 12525-12530.
Hasegawa, H., H. Katoh, H. Fujita, K. Mori and M. Negishi (2000). "Receptor isoform-specific interaction of prostaglandin EP3 receptor with muskelin." Biochem Biophys Res Commun 276(1): 350-354.
Hashizume, R., M. Fukuda, I. Maeda, H. Nishikawa, D. Oyake, Y. Yabuki, H. Ogata and T. Ohta (2001). "The RING heterodimer BRCA1-BARD1 is a ubiquitin ligase inactivated by a breast cancer-derived mutation." J Biol Chem 276(18): 14537-14540.
Hastings, M. L., E. Allemand, D. M. Duelli, M. P. Myers and A. R. Krainer (2007). "Control of pre-mRNA splicing by the general splicing factors PUF60 and U2AF(65)." PLoS One 2(6): e538.
Haupt, Y., R. Maya, A. Kazaz and M. Oren (1997). "Mdm2 promotes the rapid degradation of p53." Nature 387(6630): 296-299.
Chapter 8 References
317
He, W. W., P. J. Sciavolino, J. Wing, M. Augustus, P. Hudson, P. S. Meissner, R. T. Curtis, B. K. Shell, D. G. Bostwick, D. J. Tindall, E. P. Gelmann, C. Abate-Shen and K. C. Carter (1997). "A novel human prostate-specific, androgen-regulated homeobox gene (NKX3.1) that maps to 8p21, a region frequently deleted in prostate cancer." Genomics 43(1): 69-77.
Heidenreich, A., G. Aus, M. Bolla, S. Joniau, V. B. Matveev, H. P. Schmid and F. Zattoni (2008). "EAU guidelines on prostate cancer." Eur Urol 53(1): 68-80.
Heisler, F. F., S. Loebrich, Y. Pechmann, N. Maier, A. R. Zivkovic, M. Tokito, T. J. Hausrat, M. Schweizer, R. Bahring, E. L. Holzbaur, D. Schmitz and M. Kneussel (2011). "Muskelin regulates actin filament- and microtubule-based GABA(A) receptor transport in neurons." Neuron 70(1): 66-81.
Hershko, A., A. Ciechanover, H. Heller, A. L. Haas and I. A. Rose (1980). "Proposed role of ATP in protein breakdown: conjugation of protein with multiple chains of the polypeptide of ATP-dependent proteolysis." Proc Natl Acad Sci U S A 77(4): 1783-1786.
Hershko, A., A. Ciechanover and I. A. Rose (1981). "Identification of the active amino acid residue of the polypeptide of ATP-dependent protein breakdown." J Biol Chem 256(4): 1525-1528.
Hershko, A., H. Heller, S. Elias and A. Ciechanover (1983). "Components of ubiquitin-protein ligase system. Resolution, affinity purification, and role in protein breakdown." J Biol Chem 258(13): 8206-8214.
Hershko, D., G. Bornstein, O. Ben-Izhak, A. Carrano, M. Pagano, M. M. Krausz and A. Hershko (2001). "Inverse relation between levels of p27(Kip1) and of its ubiquitin ligase subunit Skp2 in colorectal carcinomas." Cancer 91(9): 1745-1751.
Hicke, L. (1999). "Gettin' down with ubiquitin: turning off cell-surface receptors, transporters and channels." Trends Cell Biol 9(3): 107-112.
Higdon, R. and E. Kolker (2007). "A predictive model for identifying proteins by a single peptide match." Bioinformatics 23(3): 277-280.
Hjerno, K. and P. Hojrup (2006). "Protein Identification by MALDI-TOF MS and Database Searching". Odense, Denmark, University of Southern Denmark.
Ho, Y., A. Gruhler, A. Heilbut, G. D. Bader, L. Moore, S. L. Adams, A. Millar, P. Taylor, K. Bennett, K. Boutilier, L. Yang, C. Wolting, I. Donaldson, S. Schandorff, J. Shewnarane, M. Vo, J. Taggart, M. Goudreault, B. Muskat, C. Alfarano, D. Dewar, Z. Lin, K. Michalickova, A. R. Willems, H. Sassi, P. A. Nielsen, K. J. Rasmussen, J. R. Andersen, L. E. Johansen, L. H. Hansen, H. Jespersen, A. Podtelejnikov, E. Nielsen, J. Crawford, V. Poulsen, B. D. Sorensen, J. Matthiesen, R. C. Hendrickson, F. Gleeson, T. Pawson, M. F. Moran, D. Durocher, M. Mann, C. W. Hogue, D. Figeys and M. Tyers (2002). "Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry." Nature 415(6868): 180-183.
Hochreiter, W. W. (2008). "The issue of prostate cancer evaluation in men with elevated prostate-specific antigen and chronic prostatitis." Andrologia 40(2): 130-133.
Hochstrasser, M. (2009). "Origin and function of ubiquitin-like proteins." Nature 458(7237): 422-429.
Hoffman, M. and H. L. Chiang (1996). "Isolation of degradation-deficient mutants defective in the targeting of fructose-1,6-bisphosphatase into the vacuole for degradation in Saccharomyces cerevisiae." Genetics 143(4): 1555-1566.
Chapter 8 References
318
Hofmann, R. M. and C. M. Pickart (1999). "Noncanonical MMS2-encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for DNA repair." Cell 96(5): 645-653.
Hofmann, K. and L. Falquet (2001). "A ubiquitin-interacting motif conserved in components of the proteasomal and lysosomal protein degradation systems." Trends Biochem Sci 26(6): 347-350.
Hollister, J., E. Grabenhorst, M. Nimtz, H. Conradt and D. L. Jarvis (2002). "Engineering the protein N-glycosylation pathway in insect cells for production of biantennary, complex N-glycans." Biochemistry 41(50): 15093-15104.
Holmberg, L., A. Bill-Axelson, G. Steineck, H. Garmo, J. Palmgren, E. Johansson, H. O. Adami
and J. E. Johansson (2012). "Results from the Scandinavian Prostate Cancer Group Trial Number 4: a randomized controlled trial of radical prostatectomy versus watchful waiting." J Natl Cancer Inst Monogr 2012(45): 230-233.
Holmes, K. A., J. S. Song, X. S. Liu, M. Brown and J. S. Carroll (2008). "Nkx3-1 and LEF-1 function as transcriptional inhibitors of estrogen receptor activity." Cancer Res 68(18): 7380-7385.
Honda, R., H. Tanaka and H. Yasuda (1997). "Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53." FEBS Lett 420(1): 25-27.
Honda, R. and H. Yasuda (2000). "Activity of MDM2, a ubiquitin ligase, toward p53 or itself is dependent on the RING finger domain of the ligase." Oncogene 19(11): 1473-1476.
Hoppe, T. (2005). "Multiubiquitylation by E4 enzymes: 'one size' doesn't fit all." Trends Biochem Sci 30(4): 183-187.
Hu, G. and E. R. Fearon (1999). "Siah-1 N-terminal RING domain is required for proteolysis function, and C-terminal sequences regulate oligomerization and binding to target proteins." Mol Cell Biol 19(1): 724-732.
Huang, L., E. Kinnucan, G. Wang, S. Beaudenon, P. M. Howley, J. M. Huibregtse and N. P. Pavletich (1999). "Structure of an E6AP-UbcH7 complex: insights into ubiquitination by the E2-E3 enzyme cascade." Science 286(5443): 1321-1326.
Huang, D. T., A. Paydar, M. Zhuang, M. B. Waddell, J. M. Holton and B. A. Schulman (2005a). "Structural basis for recruitment of Ubc12 by an E2 binding domain in NEDD8's E1." Mol Cell 17(3): 341-350.
Huang, H., K. M. Regan, F. Wang, D. Wang, D. I. Smith, J. M. van Deursen and D. J. Tindall (2005b). "Skp2 inhibits FOXO1 in tumor suppression through ubiquitin-mediated degradation." Proc Natl Acad Sci U S A 102(5): 1649-1654.
Huang, D. T., H. W. Hunt, M. Zhuang, M. D. Ohi, J. M. Holton and B. A. Schulman (2007). "Basis for a ubiquitin-like protein thioester switch toggling E1-E2 affinity." Nature 445(7126): 394-398.
Huang, A., R. N. de Jong, H. Wienk, G. S. Winkler, H. T. Timmers and R. Boelens (2009). "E2-c-Cbl recognition is necessary but not sufficient for ubiquitination activity." J Mol Biol 385(2): 507-519.
Huang, H., M. S. Jeon, L. Liao, C. Yang, C. Elly, J. R. Yates, 3rd and Y. C. Liu (2010). "K33-linked polyubiquitination of T cell receptor-zeta regulates proteolysis-independent T cell signaling." Immunity 33(1): 60-70.
Hudson, A. M. and L. Cooley (2008). "Phylogenetic, structural and functional relationships between WD- and Kelch-repeat proteins." Subcell Biochem 48: 6-19.
Chapter 8 References
319
Hugosson, J., J. Stranne and S. V. Carlsson (2011). "Radical retropubic prostatectomy: a review of outcomes and side-effects." Acta Oncol 50 Suppl 1: 92-97.
Huibregtse, J. M., M. Scheffner and P. M. Howley (1994). "E6-AP directs the HPV E6-dependent inactivation of p53 and is representative of a family of structurally and functionally related proteins." Cold Spring Harb Symp Quant Biol 59: 237-245.
Huibregtse, J. M., M. Scheffner, S. Beaudenon and P. M. Howley (1995). "A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase." Proc Natl Acad Sci U S A 92(7): 2563-2567.
Hurley, J. H., S. Lee and G. Prag (2006). "Ubiquitin-binding domains." Biochem J 399(3): 361-372.
Ideguchi, H., A. Ueda, M. Tanaka, J. Yang, T. Tsuji, S. Ohno, E. Hagiwara, A. Aoki and Y. Ishigatsubo (2002). "Structural and functional characterization of the USP11 deubiquitinating enzyme, which interacts with the RanGTP-associated protein RanBPM." Biochem J 367(Pt 1): 87-95.
Ikeda, F. and I. Dikic (2008). "Atypical ubiquitin chains: new molecular signals. 'Protein Modifications: Beyond the Usual Suspects' review series." EMBO Rep 9(6): 536-542.
Ikeda, H. and T. K. Kerppola (2008). "Lysosomal localization of ubiquitinated Jun requires multiple determinants in a lysine-27-linked polyubiquitin conjugate." Mol Biol Cell 19(11): 4588-4601.
Ikeda, F., N. Crosetto and I. Dikic (2010). "What determines the specificity and outcomes of ubiquitin signaling?" Cell 143(5): 677-681.
Imhof, M. O. and D. P. McDonnell (1996). "Yeast RSP5 and its human homolog hRPF1 potentiate hormone-dependent activation of transcription by human progesterone and glucocorticoid receptors." Mol Cell Biol 16(6): 2594-2605.
Ingham, R. J., G. Gish and T. Pawson (2004). "The Nedd4 family of E3 ubiquitin ligases: functional diversity within a common modular architecture." Oncogene 23(11): 1972-1984.
Irminger-Finger, I. and C. E. Jefford (2006). "Is there more to BARD1 than BRCA1?" Nat Rev Cancer 6(5): 382-391.
Iskakova, M. B., W. Szaflarski, M. Dreyfus, J. Remme and K. H. Nierhaus (2006). "Troubleshooting coupled in vitro transcription-translation system derived from Escherichia coli cells: synthesis of high-yield fully active proteins." Nucleic Acids Res 34(19): e135.
Ito, A., Y. Kawaguchi, C. H. Lai, J. J. Kovacs, Y. Higashimoto, E. Appella and T. P. Yao (2002). "MDM2-HDAC1-mediated deacetylation of p53 is required for its degradation." EMBO J 21(22): 6236-6245.
Iwata, T., D. Schultz, J. Hicks, G. K. Hubbard, L. N. Mutton, T. L. Lotan, C. Bethel, M. T. Lotz, S. Yegnasubramanian, W. G. Nelson, C. V. Dang, M. Xu, U. Anele, C. M. Koh, C. J. Bieberich and A. M. De Marzo (2010). "MYC overexpression induces prostatic intraepithelial neoplasia and loss of Nkx3.1 in mouse luminal epithelial cells." PLoS One 5(2): e9427.
Jiang, J., C. A. Ballinger, Y. Wu, Q. Dai, D. M. Cyr, J. Hohfeld and C. Patterson (2001). "CHIP is a U-box-dependent E3 ubiquitin ligase: identification of Hsc70 as a target for ubiquitylation." J Biol Chem 276(46): 42938-42944.
Jiang, A., C. Yu, P. Zhang, W. Chen, W. Liu, X. Hu and J. Zhang (2006a). "p53 overexpression represses androgen-mediated induction of NKX3.1 in a prostate cancer cell line." Exp Mol Med 38(6): 625-633.
Chapter 8 References
320
Jiang, A. L., P. J. Zhang, W. W. Chen, W. W. Liu, C. X. Yu, X. Y. Hu, X. Q. Zhang and J. Y. Zhang (2006b). "Effects of 9-cis retinoic acid on human homeobox gene NKX3.1 expression in prostate cancer cell line LNCaP." Asian J Androl 8(4): 435-441.
Jin, Y., X. L. Xu, M. C. Yang, F. Wei, T. C. Ayi, A. M. Bowcock and R. Baer (1997). "Cell cycle-dependent colocalization of BARD1 and BRCA1 proteins in discrete nuclear domains." Proc Natl Acad Sci U S A 94(22): 12075-12080.
Jin, J., X. Li, S. P. Gygi and J. W. Harper (2007). "Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging." Nature 447(7148): 1135-1138.
Jin, L., A. Williamson, S. Banerjee, I. Philipp and M. Rape (2008). "Mechanism of ubiquitin-chain formation by the human anaphase-promoting complex." Cell 133(4): 653-665.
Jin, Z., Y. Li, R. Pitti, D. Lawrence, V. C. Pham, J. R. Lill and A. Ashkenazi (2009). "Cullin3-based polyubiquitination and p62-dependent aggregation of caspase-8 mediate extrinsic apoptosis signaling." Cell 137(4): 721-735.
Jin, G., J. Sun, W. Liu, Z. Zhang, L. W. Chu, S. T. Kim, J. Feng, D. Duggan, J. D. Carpten, F. Wiklund, H. Gronberg, W. B. Isaacs, S. L. Zheng and J. Xu (2011). "Genome-wide copy-number variation analysis identifies common genetic variants at 20p13 associated with aggressiveness of prostate cancer." Carcinogenesis 32(7): 1057-1062.
Joazeiro, C. A., S. S. Wing, H. Huang, J. D. Leverson, T. Hunter and Y. C. Liu (1999). "The tyrosine kinase negative regulator c-Cbl as a RING-type, E2-dependent ubiquitin-protein ligase." Science 286(5438): 309-312.
Jones, S. N., A. E. Roe, L. A. Donehower and A. Bradley (1995). "Rescue of embryonic lethality in Mdm2-deficient mice by absence of p53." Nature 378(6553): 206-208.
Johannsdottir, H. K., G. Jonsson, G. Johannesdottir, B. A. Agnarsson, H. Eerola, A. Arason, P. Heikkila, V. Egilsson, H. Olsson, O. T. Johannsson, H. Nevanlinna, A. Borg and R. B. Barkardottir (2006). "Chromosome 5 imbalance mapping in breast tumors from BRCA1 and BRCA2 mutation carriers and sporadic breast tumors." Int J Cancer 119(5): 1052-1060.
Johnson, E. S., P. C. Ma, I. M. Ota and A. Varshavsky (1995). "A proteolytic pathway that recognizes ubiquitin as a degradation signal." J Biol Chem 270(29): 17442-17456.
Johnson, A. E., S. E. Collier, M. D. Ohi and K. L. Gould (2012). "Fission Yeast Dma1 Requires RING Domain Dimerization for Its Ubiquitin Ligase Activity and Mitotic Checkpoint Function." J Biol Chem 287(31): 25741-25748.
Joos, S., U. S. Bergerheim, Y. Pan, H. Matsuyama, M. Bentz, S. du Manoir and P. Lichter (1995). "Mapping of chromosomal gains and losses in prostate cancer by comparative genomic hybridization." Genes Chromosomes Cancer 14(4): 267-276.
Ju, J. H., J. S. Maeng, M. Zemedkun, N. Ahronovitz, J. W. Mack, J. A. Ferretti, E. P. Gelmann and J. M. Gruschus (2006). "Physical and functional interactions between the prostate suppressor homeoprotein NKX3.1 and serum response factor." J Mol Biol 360(5): 989-999.
Ju, J. H., J. S. Maeng, D. Y. Lee, G. Piszczek, E. P. Gelmann and J. M. Gruschus (2009). "Interactions of the acidic domain and SRF interacting motifs with the NKX3.1 homeodomain." Biochemistry 48(44): 10601-10607.
July, L. V., M. Akbari, T. Zellweger, E. C. Jones, S. L. Goldenberg and M. E. Gleave (2002). "Clusterin expression is significantly enhanced in prostate cancer cells following androgen withdrawal therapy." Prostate 50(3): 179-188.
Chapter 8 References
321
Kaiser, P., W. Seufert, L. Hofferer, B. Kofler, C. Sachsenmaier, H. Herzog, S. Jentsch, M. Schweiger and R. Schneider (1994). "A human ubiquitin-conjugating enzyme homologous to yeast UBC8." J Biol Chem 269(12): 8797-8802.
Kaiser, P., K. Flick, C. Wittenberg and S. I. Reed (2000). "Regulation of transcription by ubiquitination without proteolysis: Cdc34/SCF(Met30)-mediated inactivation of the transcription factor Met4." Cell 102(3): 303-314.
Kales, S. C., P. E. Ryan, M. M. Nau and S. Lipkowitz (2010). "Cbl and human myeloid neoplasms: the Cbl oncogene comes of age." Cancer Res 70(12): 4789-4794.
Kamitani, T., H. P. Nguyen and E. T. Yeh (1997). "Preferential modification of nuclear proteins by a novel ubiquitin-like molecule." J Biol Chem 272(22): 14001-14004.
Kassenbrock, C. K. and S. M. Anderson (2004). "Regulation of ubiquitin protein ligase activity in c-Cbl by phosphorylation-induced conformational change and constitutive activation by tyrosine to glutamate point mutations." J Biol Chem 279(27): 28017-28027.
Katzen, F., G. Chang and W. Kudlicki (2005). "The past, present and future of cell-free protein synthesis." Trends Biotechnol 23(3): 150-156.
Kawakami, T., T. Chiba, T. Suzuki, K. Iwai, K. Yamanaka, N. Minato, H. Suzuki, N. Shimbara, Y. Hidaka, F. Osaka, M. Omata and K. Tanaka (2001). "NEDD8 recruits E2-ubiquitin to SCF E3 ligase." EMBO J 20(15): 4003-4012.
Kay, G. F., A. Ashworth, G. D. Penny, M. Dunlop, S. Swift, N. Brockdorff and S. Rastan (1991). "A candidate spermatogenesis gene on the mouse Y chromosome is homologous to ubiquitin-activating enzyme E1." Nature 354(6353): 486-489.
Kee, Y. and J. M. Huibregtse (2007). "Regulation of catalytic activities of HECT ubiquitin ligases." Biochem Biophys Res Commun 354(2): 329-333.
Keller, B. O., J. Sui, A. B. Young and R. M. Whittal (2008). "Interferences and contaminants encountered in modern mass spectrometry." Anal Chim Acta 627(1): 71-81.
Kerscher, O., R. Felberbaum and M. Hochstrasser (2006). "Modification of proteins by ubiquitin and ubiquitin-like proteins." Annu Rev Cell Dev Biol 22: 159-180.
Kerschner, J. L. and A. Harris (2012). "Transcriptional networks driving enhancer function in the CFTR gene." Biochem J.
Khalili, M., L. N. Mutton, B. Gurel, J. L. Hicks, A. M. De Marzo and C. J. Bieberich (2010). "Loss of Nkx3.1 expression in bacterial prostatitis: a potential link between inflammation and neoplasia." Am J Pathol 176(5): 2259-2268.
Khoury, C. M., Z. Yang, X. Y. Li, M. Vignali, S. Fields and M. T. Greenwood (2008). "A TSC22-like motif defines a novel antiapoptotic protein family." FEMS Yeast Res 8(4): 540-563.
Kim, M. J., R. Bhatia-Gaur, W. A. Banach-Petrosky, N. Desai, Y. Wang, S. W. Hayward, G. R. Cunha, R. D. Cardiff, M. M. Shen and C. Abate-Shen (2002a). "Nkx3.1 mutant mice recapitulate early stages of prostate carcinogenesis." Cancer Res 62(11): 2999-3004.
Kim, M. J., R. D. Cardiff, N. Desai, W. A. Banach-Petrosky, R. Parsons, M. M. Shen and C. Abate-Shen (2002b). "Cooperativity of Nkx3.1 and Pten loss of function in a mouse model of prostate carcinogenesis." Proc Natl Acad Sci U S A 99(5): 2884-2889.
Kim, M. H., D. R. Cooper, A. Oleksy, Y. Devedjiev, U. Derewenda, O. Reiner, J. Otlewski and Z. S. Derewenda (2004). "The structure of the N-terminal domain of the product of the lissencephaly gene Lis1 and its functional implications." Structure 12(6): 987-998.
Kim, H., J. Huang and J. Chen (2007). "CCDC98 is a BRCA1-BRCT domain-binding protein involved in the DNA damage response." Nat Struct Mol Biol 14(8): 710-715.
Chapter 8 References
322
Kim, T., S. Kim, H. M. Yun, K. C. Chung, Y. S. Han, H. S. Shin and H. Rhim (2009). "Modulation of Ca(v)3.1 T-type Ca2+ channels by the ran binding protein RanBPM." Biochem Biophys Res Commun 378(1): 15-20.
Kim, H. C. and J. M. Huibregtse (2009). "Polyubiquitination by HECT E3s and the determinants of chain type specificity." Mol Cell Biol 29(12): 3307-3318.
Kim, H. C., A. M. Steffen, M. L. Oldham, J. Chen and J. M. Huibregtse (2011). "Structure and function of a HECT domain ubiquitin-binding site." EMBO Rep 12(4): 334-341.
King, J. C., J. Xu, J. Wongvipat, H. Hieronymus, B. S. Carver, D. H. Leung, B. S. Taylor, C. Sander, R. D. Cardiff, S. S. Couto, W. L. Gerald and C. L. Sawyers (2009). "Cooperativity of TMPRSS2-ERG with PI3-kinase pathway activation in prostate oncogenesis." Nat Genet 41(5): 524-526.
Kirisako, T., K. Kamei, S. Murata, M. Kato, H. Fukumoto, M. Kanie, S. Sano, F. Tokunaga, K. Tanaka and K. Iwai (2006). "A ubiquitin ligase complex assembles linear polyubiquitin chains." EMBO J 25(20): 4877-4887.
Kirkpatrick, D. S., N. A. Hathaway, J. Hanna, S. Elsasser, J. Rush, D. Finley, R. W. King and S. P. Gygi (2006). "Quantitative analysis of in vitro ubiquitinated cyclin B1 reveals complex chain topology." Nat Cell Biol 8(7): 700-710.
Kitagawa, K., Y. Kotake and M. Kitagawa (2009). "Ubiquitin-mediated control of oncogene and tumor suppressor gene products." Cancer Sci 100(8): 1374-1381.
Kleiman, F. E. and J. L. Manley (1999). "Functional interaction of BRCA1-associated BARD1 with polyadenylation factor CstF-50." Science 285(5433): 1576-1579.
Kleiman, F. E., F. Wu-Baer, D. Fonseca, S. Kaneko, R. Baer and J. L. Manley (2005). "BRCA1/BARD1 inhibition of mRNA 3' processing involves targeted degradation of RNA polymerase II." Genes Dev 19(10): 1227-1237.
Klemperer, N. S., E. S. Berleth and C. M. Pickart (1989). "A novel, arsenite-sensitive E2 of the ubiquitin pathway: purification and properties." Biochemistry 28(14): 6035-6041.
Kocan, M., M. B. Dalrymple, R. M. Seeber, B. J. Feldman and K. D. Pfleger (2010). "Enhanced BRET Technology for the Monitoring of Agonist-Induced and Agonist-Independent Interactions between GPCRs and beta-Arrestins." Front Endocrinol (Lausanne) 1: 12.
Kobayashi, N., J. Yang, A. Ueda, T. Suzuki, K. Tomaru, M. Takeno, K. Okuda and Y. Ishigatsubo (2007). "RanBPM, Muskelin, p48EMLP, p44CTLH, and the armadillo-repeat proteins ARMC8alpha and ARMC8beta are components of the CTLH complex." Gene 396(2): 236-247.
Koegl, M., T. Hoppe, S. Schlenker, H. D. Ulrich, T. U. Mayer and S. Jentsch (1999). "A novel ubiquitination factor, E4, is involved in multiubiquitin chain assembly." Cell 96(5): 635-644.
Koleske, A. J. and R. A. Young (1995). "The RNA polymerase II holoenzyme and its implications for gene regulation." Trends Biochem Sci 20(3): 113-116.
Kolman, C. J., J. Toth and D. K. Gonda (1992). "Identification of a portable determinant of cell cycle function within the carboxyl-terminal domain of the yeast CDC34 (UBC3) ubiquitin conjugating (E2) enzyme." EMBO J 11(8): 3081-3090.
Kost, T. A., J. P. Condreay and D. L. Jarvis (2005). "Baculovirus as versatile vectors for protein expression in insect and mammalian cells." Nat Biotechnol 23(5): 567-575.
Kozlov, G., P. Peschard, B. Zimmerman, T. Lin, T. Moldoveanu, N. Mansur-Azzam, K. Gehring and M. Park (2007). "Structural basis for UBA-mediated dimerization of c-Cbl ubiquitin ligase." J Biol Chem 282(37): 27547-27555.
Chapter 8 References
323
Kramer, S., T. Ozaki, K. Miyazaki, C. Kato, T. Hanamoto and A. Nakagawara (2005). "Protein stability and function of p73 are modulated by a physical interaction with RanBPM in mammalian cultured cells." Oncogene 24(5): 938-944.
Krogan, N. J., G. Cagney, H. Yu, G. Zhong, X. Guo, A. Ignatchenko, J. Li, S. Pu, N. Datta, A. P. Tikuisis, T. Punna, J. M. Peregrin-Alvarez, M. Shales, X. Zhang, M. Davey, M. D. Robinson, A. Paccanaro, J. E. Bray, A. Sheung, B. Beattie, D. P. Richards, V. Canadien, A. Lalev, F. Mena, P. Wong, A. Starostine, M. M. Canete, J. Vlasblom, S. Wu, C. Orsi, S. R. Collins, S. Chandran, R. Haw, J. J. Rilstone, K. Gandi, N. J. Thompson, G. Musso, P. St Onge, S. Ghanny, M. H. Lam, G. Butland, A. M. Altaf-Ul, S. Kanaya, A. Shilatifard, E. O'Shea, J. S. Weissman, C. J. Ingles, T. R. Hughes, J. Parkinson, M. Gerstein, S. J. Wodak, A. Emili and J. F. Greenblatt (2006). "Global landscape of protein complexes in the yeast Saccharomyces cerevisiae." Nature 440(7084): 637-643.
Kuang, Z., R. S. Lewis, J. M. Curtis, Y. Zhan, B. M. Saunders, J. J. Babon, T. B. Kolesnik, A. Low, S. L. Masters, T. A. Willson, L. Kedzierski, S. Yao, E. Handman, R. S. Norton and S. E. Nicholson (2010). "The SPRY domain-containing SOCS box protein SPSB2 targets iNOS for proteasomal degradation." J Cell Biol 190(1): 129-141.
Kubo, N., R. Okoshi, K. Nakashima, O. Shimozato, A. Nakagawara and T. Ozaki (2010). "MDM2 promotes the proteasomal degradation of p73 through the interaction with Itch in HeLa cells." Biochem Biophys Res Commun 403(3-4): 405-411.
Kubbutat, M. H., S. N. Jones and K. H. Vousden (1997). "Regulation of p53 stability by Mdm2." Nature 387(6630): 299-303.
Kunderfranco, P., M. Mello-Grand, R. Cangemi, S. Pellini, A. Mensah, V. Albertini, A. Malek, G. Chiorino, C. V. Catapano and G. M. Carbone (2010). "ETS transcription factors control transcription of EZH2 and epigenetic silencing of the tumor suppressor gene Nkx3.1 in prostate cancer." PLoS One 5(5): e10547.
Kunert, S., I. Meyer, S. Fleischhauer, M. Wannack, J. Fiedler, R. A. Shivdasani and H. Schulze (2009). "The microtubule modulator RanBP10 plays a critical role in regulation of platelet discoid shape and degranulation." Blood 114(27): 5532-5540.
Kurasawa, Y. and K. Todokoro (1999). "Identification of human APC10/Doc1 as a subunit of anaphase promoting complex." Oncogene 18(37): 5131-5137.
Lake, M. W., M. M. Wuebbens, K. V. Rajagopalan and H. Schindelin (2001). "Mechanism of ubiquitin activation revealed by the structure of a bacterial MoeB-MoaD complex." Nature 414(6861): 325-329.
Lamothe, B., A. Besse, A. D. Campos, W. K. Webster, H. Wu and B. G. Darnay (2007). "Site-specific Lys-63-linked tumor necrosis factor receptor-associated factor 6 auto-ubiquitination is a critical determinant of I kappa B kinase activation." J Biol Chem 282(6): 4102-4112.
Langdon, W. Y., J. W. Hartley, S. P. Klinken, S. K. Ruscetti and H. C. Morse, 3rd (1989). "v-cbl, an oncogene from a dual-recombinant murine retrovirus that induces early B-lineage lymphomas." Proc Natl Acad Sci U S A 86(4): 1168-1172.
Latres, E., D. S. Chiaur and M. Pagano (1999). "The human F box protein beta-Trcp associates with the Cul1/Skp1 complex and regulates the stability of beta-catenin." Oncogene 18(4): 849-854.
Latres, E., R. Chiarle, B. A. Schulman, N. P. Pavletich, A. Pellicer, G. Inghirami and M. Pagano (2001). "Role of the F-box protein Skp2 in lymphomagenesis." Proc Natl Acad Sci U S A 98(5): 2515-2520.
Chapter 8 References
324
Lau, I. C. E. (2008). Characterisation of FLJ22318 in Prostate Cancer Cells. Honours Thesis. School of Biological Sciences, Murdoch University, Perth, Western Australia.
Layfield, R., K. Franklin, M. Landon, G. Walker, P. Wang, R. Ramage, A. Brown, S. Love, K. Urquhart, T. Muir, R. Baker and R. J. Mayer (1999). "Chemically synthesized ubiquitin extension proteins detect distinct catalytic capacities of deubiquitinating enzymes." Anal Biochem 274(1): 40-49.
Leach, F. S., T. Tokino, P. Meltzer, M. Burrell, J. D. Oliner, S. Smith, D. E. Hill, D. Sidransky, K. W. Kinzler and B. Vogelstein (1993). "p53 Mutation and MDM2 amplification in human soft tissue sarcomas." Cancer Res 53(10 Suppl): 2231-2234.
Ledee, D. R., C. Y. Gao, R. Seth, R. N. Fariss, B. K. Tripathi and P. S. Zelenka (2005). "A specific interaction between muskelin and the cyclin-dependent kinase 5 activator p39 promotes peripheral localization of muskelin." J Biol Chem 280(22): 21376-21383.
Lee, D. H. and A. L. Goldberg (1998). "Proteasome inhibitors: valuable new tools for cell biologists." Trends Cell Biol 8(10): 397-403.
Lee, I. and H. Schindelin (2008). "Structural insights into E1-catalyzed ubiquitin activation and transfer to conjugating enzymes." Cell 134(2): 268-278.
Lee, Y. R., W. C. Yuan, H. C. Ho, C. H. Chen, H. M. Shih and R. H. Chen (2010). "The Cullin 3 substrate adaptor KLHL20 mediates DAPK ubiquitination to control interferon responses." EMBO J 29(10): 1748-1761.
Lehmann, A. R., A. Niimi, T. Ogi, S. Brown, S. Sabbioneda, J. F. Wing, P. L. Kannouche and C. M. Green (2007). "Translesion synthesis: Y-family polymerases and the polymerase switch." DNA Repair (Amst) 6(7): 891-899.
Lei, Q., J. Jiao, L. Xin, C. J. Chang, S. Wang, J. Gao, M. E. Gleave, O. N. Witte, X. Liu and H. Wu (2006). "NKX3.1 stabilizes p53, inhibits AKT activation, and blocks prostate cancer initiation caused by PTEN loss." Cancer Cell 9(5): 367-378.
Leng, R. P., Y. Lin, W. Ma, H. Wu, B. Lemmers, S. Chung, J. M. Parant, G. Lozano, R. Hakem and S. Benchimol (2003). "Pirh2, a p53-induced ubiquitin-protein ligase, promotes p53 degradation." Cell 112(6): 779-791.
Levkowitz, G., H. Waterman, E. Zamir, Z. Kam, S. Oved, W. Y. Langdon, L. Beguinot, B. Geiger and Y. Yarden (1998). "c-Cbl/Sli-1 regulates endocytic sorting and ubiquitination of the epidermal growth factor receptor." Genes Dev 12(23): 3663-3674.
Li, W., S. Nagaraja, G. P. Delcuve, M. J. Hendzel and J. R. Davie (1993). "Effects of histone acetylation, ubiquitination and variants on nucleosome stability." Biochem J 296 ( Pt 3): 737-744.
Li, J., J. Wang, Z. Nawaz, J. M. Liu, J. Qin and J. Wong (2000). "Both corepressor proteins SMRT and N-CoR exist in large protein complexes containing HDAC3." EMBO J 19(16): 4342-4350.
Li, M., D. Chen, A. Shiloh, J. Luo, A. Y. Nikolaev, J. Qin and W. Gu (2002). "Deubiquitination of p53 by HAUSP is an important pathway for p53 stabilization." Nature 416(6881): 648-653.
Li, M., C. L. Brooks, F. Wu-Baer, D. Chen, R. Baer and W. Gu (2003). "Mono- versus polyubiquitination: differential control of p53 fate by Mdm2." Science 302(5652): 1972-1975.
Li, S., M. P. Czubryt, J. McAnally, R. Bassel-Duby, J. A. Richardson, F. F. Wiebel, A. Nordheim and E. N. Olson (2005). "Requirement for serum response factor for skeletal muscle growth and maturation revealed by tissue-specific gene deletion in mice." Proc Natl Acad Sci U S A 102(4): 1082-1087.
Chapter 8 References
325
Li, X., B. Guan, S. Maghami and C. J. Bieberich (2006). "NKX3.1 is regulated by protein kinase CK2 in prostate tumor cells." Mol Cell Biol 26(8): 3008-3017.
Li, M., J. P. York and P. Zhang (2007a). "Loss of Cdc20 causes a securin-dependent metaphase arrest in two-cell mouse embryos." Mol Cell Biol 27(9): 3481-3488.
Li, W., D. Tu, A. T. Brunger and Y. Ye (2007b). "A ubiquitin ligase transfers preformed polyubiquitin chains from a conjugating enzyme to a substrate." Nature 446(7133): 333-337.
Li, J., L. M. Olson, Z. Zhang, L. Li, M. Bidder, L. Nguyen, J. Pfeifer and J. S. Rader (2008). "Differential display identifies overexpression of the USP36 gene, encoding a deubiquitinating enzyme, in ovarian cancer." Int J Med Sci 5(3): 133-142.
Li, W., D. Tu, L. Li, T. Wollert, R. Ghirlando, A. T. Brunger and Y. Ye (2009). "Mechanistic insights into active site-associated polyubiquitination by the ubiquitin-conjugating enzyme Ube2g2." Proc Natl Acad Sci U S A 106(10): 3722-3727.
Liang, J., M. Wan, Y. Zhang, P. Gu, H. Xin, S. Y. Jung, J. Qin, J. Wong, A. J. Cooney, D. Liu and Z. Songyang (2008). "Nanog and Oct4 associate with unique transcriptional repression complexes in embryonic stem cells." Nat Cell Biol 10(6): 731-739.
Liani, E., A. Eyal, E. Avraham, R. Shemer, R. Szargel, D. Berg, A. Bornemann, O. Riess, C. A. Ross, R. Rott and S. Engelender (2004). "Ubiquitylation of synphilin-1 and alpha-synuclein by SIAH and its presence in cellular inclusions and Lewy bodies imply a role in Parkinson's disease." Proc Natl Acad Sci U S A 101(15): 5500-5505.
Liew, C. W., H. Sun, T. Hunter and C. L. Day (2010). "RING domain dimerization is essential for RNF4 function." Biochem J 431(1): 23-29.
Lin, L., G. N. DeMartino and W. C. Greene (1998). "Cotranslational biogenesis of NF-kappaB p50 by the 26S proteasome." Cell 92(6): 819-828.
Lin, Y., W. C. Hwang and R. Basavappa (2002). "Structural and functional analysis of the human mitotic-specific ubiquitin-conjugating enzyme, UbcH10." J Biol Chem 277(24): 21913-21921.
Lin, D. I., O. Barbash, K. G. Kumar, J. D. Weber, J. W. Harper, A. J. Klein-Szanto, A. Rustgi, S. Y. Fuchs and J. A. Diehl (2006). "Phosphorylation-dependent ubiquitination of cyclin D1 by the SCF(FBX4-alphaB crystallin) complex." Mol Cell 24(3): 355-366.
Lin, H. K., Z. Chen, G. Wang, C. Nardella, S. W. Lee, C. H. Chan, W. L. Yang, J. Wang, A. Egia, K. I. Nakayama, C. Cordon-Cardo, J. Teruya-Feldstein and P. P. Pandolfi (2010). "Skp2 targeting suppresses tumorigenesis by Arf-p53-independent cellular senescence." Nature 464(7287): 374-379.
Linares, L. K., A. Hengstermann, A. Ciechanover, S. Muller and M. Scheffner (2003). "HdmX stimulates Hdm2-mediated ubiquitination and degradation of p53." Proc Natl Acad Sci U S A 100(21): 12009-12014.
Lind, G. E., R. I. Skotheim, M. F. Fraga, V. M. Abeler, R. Henrique, F. Saatcioglu, M. Esteller, M. R. Teixeira and R. A. Lothe (2005). "The loss of NKX3.1 expression in testicular--and prostate--cancers is not caused by promoter hypermethylation." Mol Cancer 4(1): 8.
Lindsten, K., F. M. de Vrij, L. G. Verhoef, D. F. Fischer, F. W. van Leeuwen, E. M. Hol, M. G. Masucci and N. P. Dantuma (2002). "Mutant ubiquitin found in neurodegenerative disorders is a ubiquitin fusion degradation substrate that blocks proteasomal degradation." J Cell Biol 157(3): 417-427.
Chapter 8 References
326
Linja, M. J., K. J. Savinainen, O. R. Saramaki, T. L. Tammela, R. L. Vessella and T. Visakorpi (2001). "Amplification and overexpression of androgen receptor gene in hormone-refractory prostate cancer." Cancer Res 61(9): 3550-3555.
Linke, K., P. D. Mace, C. A. Smith, D. L. Vaux, J. Silke and C. L. Day (2008). "Structure of the MDM2/MDMX RING domain heterodimer reveals dimerization is required for their ubiquitylation in trans." Cell Death Differ 15(5): 841-848.
Linossi, E. M. and S. E. Nicholson (2012). "The SOCS box-Adapting proteins for ubiquitination and proteasomal degradation." IUBMB Life 64(4): 316-323.
Lipkowitz, S. and A. M. Weissman (2011). "RINGs of good and evil: RING finger ubiquitin ligases at the crossroads of tumour suppression and oncogenesis." Nat Rev Cancer 11(9): 629-643.
Liu, Y., L. Fallon, H. A. Lashuel, Z. Liu and P. T. Lansbury, Jr. (2002). "The UCH-L1 gene encodes two opposing enzymatic activities that affect alpha-synuclein degradation and Parkinson's disease susceptibility." Cell 111(2): 209-218.
Liu, W., P. Zhang, W. Chen, C. Yu, F. Cui, F. Kong, J. Zhang and A. Jiang (2008).
"Characterization of two functional NKX3.1 binding sites upstream of the PCAN1 gene that are involved in the positive regulation of PCAN1 gene transcription." BMC Mol Biol 9: 45.
Liu, M., Z. Sun, A. Zhou, H. Li, L. Yang, C. Zhou, R. Liu, X. Hu, J. Zhou, S. Xiang and J. Zhang (2010). "Functional characterization of the promoter region of human TNFAIP1 gene." Mol Biol Rep 37(4): 1699-1705.
Livingstone, L. R., A. White, J. Sprouse, E. Livanos, T. Jacks and T. D. Tlsty (1992). "Altered cell cycle arrest and gene amplification potential accompany loss of wild-type p53." Cell 70(6): 923-935.
Locke, J. A., E. S. Guns, A. A. Lubik, H. H. Adomat, S. C. Hendy, C. A. Wood, S. L. Ettinger, M. E. Gleave and C. C. Nelson (2008). "Androgen levels increase by intratumoral de novo steroidogenesis during progression of castration-resistant prostate cancer." Cancer Res 68(15): 6407-6415.
Lorick, K. L., J. P. Jensen, S. Fang, A. M. Ong, S. Hatakeyama and A. M. Weissman (1999). "RING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination." Proc Natl Acad Sci U S A 96(20): 11364-11369.
Lorick, K. L., Y. Yang, J. P. Jensen, K. Iwai and A. M. Weissman (2006). "Studies of the ubiquitin proteasome system." Curr Protoc Cell Biol Chapter 15: Unit 15 19.
Lovering, R., I. M. Hanson, K. L. Borden, S. Martin, N. J. O'Reilly, G. I. Evan, D. Rahman, D. J. Pappin, J. Trowsdale and P. S. Freemont (1993). "Identification and preliminary characterization of a protein motif related to the zinc finger." Proc Natl Acad Sci U S A 90(6): 2112-2116.
Luo, Y., A. Batalao, H. Zhou and L. Zhu (1997). "Mammalian two-hybrid system: a complementary approach to the yeast two-hybrid system." Biotechniques 22(2): 350-352.
Luo, J., M. Li, Y. Tang, M. Laszkowska, R. G. Roeder and W. Gu (2004). "Acetylation of p53 augments its site-specific DNA binding both in vitro and in vivo." Proc Natl Acad Sci U S A 101(8): 2259-2264.
Mace, P. D., K. Linke, R. Feltham, F. R. Schumacher, C. A. Smith, D. L. Vaux, J. Silke and C. L. Day (2008). "Structures of the cIAP2 RING domain reveal conformational changes associated with ubiquitin-conjugating enzyme (E2) recruitment." J Biol Chem 283(46): 31633-31640.
Chapter 8 References
327
MacLachlan, T. K., R. Takimoto and W. S. El-Deiry (2002). "BRCA1 directs a selective p53-dependent transcriptional response towards growth arrest and DNA repair targets." Mol Cell Biol 22(12): 4280-4292.
Maiden, S. L. and J. Hardin (2011). "The secret life of alpha-catenin: moonlighting in morphogenesis." J Cell Biol 195(4): 543-552.
Malhotra, A. (2009). "Tagging for protein expression." Methods Enzymol 463: 239-258.
Mallery, D. L., C. J. Vandenberg and K. Hiom (2002). "Activation of the E3 ligase function of the BRCA1/BARD1 complex by polyubiquitin chains." EMBO J 21(24): 6755-6762.
Mancini, F., G. Di Conza, M. Pellegrino, C. Rinaldo, A. Prodosmo, S. Giglio, I. D'Agnano, F. Florenzano, L. Felicioni, F. Buttitta, A. Marchetti, A. Sacchi, A. Pontecorvi, S. Soddu and F. Moretti (2009). "MDM4 (MDMX) localizes at the mitochondria and facilitates the p53-mediated intrinsic-apoptotic pathway." EMBO J 28(13): 1926-1939.
Mann, M., R. C. Hendrickson and A. Pandey (2001). "Analysis of proteins and proteomes by mass spectrometry." Annu Rev Biochem 70: 437-473.
Marine, J. C. and G. Lozano (2010). "Mdm2-mediated ubiquitylation: p53 and beyond." Cell Death Differ 17(1): 93-102.
Markowski, M. C., C. Bowen and E. P. Gelmann (2008). "Inflammatory cytokines induce phosphorylation and ubiquitination of prostate suppressor protein NKX3.1." Cancer Res 68(17): 6896-6901.
Marques, R. B., S. Erkens-Schulze, C. M. de Ridder, K. G. Hermans, K. Waltering, T. Visakorpi, J. Trapman, J. C. Romijn, W. M. van Weerden and G. Jenster (2005). "Androgen receptor modifications in prostate cancer cells upon long-termandrogen ablation and antiandrogen treatment." Int J Cancer 117(2): 221-229.
Marszalek, B., P. Wojcicki, K. Kobus and W. H. Trzeciak (2002). "Clinical features, treatment and genetic background of Treacher Collins syndrome." J Appl Genet 43(2): 223-233.
Marti, A., C. Wirbelauer, M. Scheffner and W. Krek (1999). "Interaction between ubiquitin-protein ligase SCFSKP2 and E2F-1 underlies the regulation of E2F-1 degradation." Nat Cell Biol 1(1): 14-19.
Martinez-Noel, G., U. Muller and K. Harbers (2001). "Identification of molecular determinants required for interaction of ubiquitin-conjugating enzymes and RING finger proteins." Eur J Biochem 268(22): 5912-5919.
Matsumoto, M. L., K. E. Wickliffe, K. C. Dong, C. Yu, I. Bosanac, D. Bustos, L. Phu, D. S. Kirkpatrick, S. G. Hymowitz, M. Rape, R. F. Kelley and V. M. Dixit (2010). "K11-linked polyubiquitination in cell cycle control revealed by a K11 linkage-specific antibody." Mol Cell 39(3): 477-484.
Matusik, R. J., R. J. Jin, Q. Sun, Y. Wang, X. Yu, A. Gupta, S. Nandana, T. C. Case, M. Paul, J. Mirosevich, S. Oottamasathien and J. Thomas (2008). "Prostate epithelial cell fate." Differentiation 76(6): 682-698.
Mastracci, T. L., A. Shadeo, S. M. Colby, A. B. Tuck, F. P. O'Malley, S. B. Bull, W. L. Lam and I. L. Andrulis (2006). "Genomic alterations in lobular neoplasia: a microarray comparative genomic hybridization signature for early neoplastic proliferationin the breast." Genes Chromosomes Cancer 45(11): 1007-1017.
Mazet, F. and S. M. Shimeld (2002). "Gene duplication and divergence in the early evolution of vertebrates." Curr Opin Genet Dev 12(4): 393-396.
McCormack, E., I. Haaland, G. Venas, R. B. Forthun, S. Huseby, G. Gausdal, S. Knappskog, D. R. Micklem, J. B. Lorens, O. Bruserud and B. T. Gjertsen (2012). "Synergistic
Chapter 8 References
328
induction of p53 mediated apoptosis by valproic acid and nutlin-3 in acute myeloid leukemia." Leukemia 26(5): 910-917.
McCrea, P. D., C. W. Turck and B. Gumbiner (1991). "A homolog of the armadillo protein in Drosophila (plakoglobin) associated with E-cadherin." Science 254(5036): 1359-1361.
McDonnell, T. J., P. Troncoso, S. M. Brisbay, C. Logothetis, L. W. Chung, J. T. Hsieh, S. M. Tu and M. L. Campbell (1992). "Expression of the protooncogene bcl-2 in the prostate and its association with emergence of androgen-independent prostate cancer." Cancer Res 52(24): 6940-6944.
McGrath, J. P., S. Jentsch and A. Varshavsky (1991). "UBA 1: an essential yeast gene encoding ubiquitin-activating enzyme." EMBO J 10(1): 227-236.
McLean, J. R., D. Chaix, M. D. Ohi and K. L. Gould (2011). "State of the APC/C: organization, function, and structure." Crit Rev Biochem Mol Biol 46(2): 118-136.
Meeks, J. J. and E. M. Schaeffer (2011). "Genetic regulation of prostate development." J Androl 32(3): 210-217.
Mendes-da-Silva, P., A. Moreira, J. Duro-da-Costa, D. Matias and C. Monteiro (2000). "Frequent loss of heterozygosity on chromosome 5 in non-small cell lung carcinoma." Mol Pathol 53(4): 184-187.
Mendrysa, S. M., K. A. O'Leary, M. K. McElwee, J. Michalowski, R. N. Eisenman, D. A. Powell and M. E. Perry (2006). "Tumor suppression and normal aging in mice with constitutively high p53 activity." Genes Dev 20(1): 16-21.
Menon, R. P., T. J. Gibson and A. Pastore (2004). "The C terminus of fragile X mental retardation protein interacts with the multi-domain Ran-binding protein in the microtubule-organising centre." J Mol Biol 343(1): 43-53.
Menssen, R., J. Schweiggert, J. Schreiner, D. Kusevic, J. Reuther, B. Braun and D. H. Wolf (2012). "Exploring the topology of the Gid complex, the E3 ubiquitin ligase involved in catabolite induced degradation of gluconeogenic enzymes." J Biol Chem.
Meyer, H. J. and M. Rape (2011). "Processive ubiquitin chain formation by the anaphase-promoting complex." Semin Cell Dev Biol 22(6): 544-550.
Michelle, C., P. Vourc'h, L. Mignon and C. R. Andres (2009). "What was the set of ubiquitin and ubiquitin-like conjugating enzymes in the eukaryote common ancestor?" J Mol Evol 68(6): 616-628.
Middelberg, A. P. (2002). "Preparative protein refolding." Trends Biotechnol 20(10): 437-443.
Mihara, M., S. Erster, A. Zaika, O. Petrenko, T. Chittenden, P. Pancoska and U. M. Moll (2003). "p53 has a direct apoptogenic role at the mitochondria." Mol Cell 11(3): 577-590.
Mikami, S., T. Kobayashi, M. Masutani, S. Yokoyama and H. Imataka (2008). "A human cell-derived in vitro coupled transcription/translation system optimized for production of recombinant proteins." Protein Expr Purif 62(2): 190-198.
Mikolajka, A., X. Yan, G. M. Popowicz, P. Smialowski, E. A. Nigg and T. A. Holak (2006). "Structure of the N-terminal domain of the FOP (FGFR1OP) protein and implications for its dimerization and centrosomal localization." J Mol Biol 359(4): 863-875.
Mitsui, A. and P. A. Sharp (1999). "Ubiquitination of RNA polymerase II large subunit signaled by phosphorylation of carboxyl-terminal domain." Proc Natl Acad Sci U S A 96(11): 6054-6059.
Chapter 8 References
329
Millar, D. S., K. K. Ow, C. L. Paul, P. J. Russell, P. L. Molloy and S. J. Clark (1999). "Detailed methylation analysis of the glutathione S-transferase pi (GSTP1) gene in prostate cancer." Oncogene 18(6): 1313-1324.
Mimeault, M. and S. K. Batra (2011). "Animal models relevant to human prostate carcinogenesis underlining the critical implication of prostatic stem/progenitor cells." Biochim Biophys Acta 1816(1): 25-37.
Molinari, E., M. Gilman and S. Natesan (1999). "Proteasome-mediated degradation of transcriptional activators correlates with activation domain potency in vivo." EMBO J 18(22): 6439-6447.
Momand, J., D. Jung, S. Wilczynski and J. Niland (1998). "The MDM2 gene amplification database." Nucleic Acids Res 26(15): 3453-3459.
Monia, B. P., D. J. Ecker, S. Jonnalagadda, J. Marsh, L. Gotlib, T. R. Butt and S. T. Crooke (1989). "Gene synthesis, expression, and processing of human ubiquitin carboxyl extension proteins." J Biol Chem 264(7): 4093-4103.
Montes de Oca Luna, R., D. S. Wagner and G. Lozano (1995). "Rescue of early embryonic lethality in mdm2-deficient mice by deletion of p53." Nature 378(6553): 203-206.
Morris, J. R. and E. Solomon (2004). "BRCA1 : BARD1 induces the formation of conjugated ubiquitin structures, dependent on K6 of ubiquitin, in cells during DNA replication and repair." Hum Mol Genet 13(8): 807-817.
Mosquera, J. M., R. Mehra, M. M. Regan, S. Perner, E. M. Genega, G. Bueti, R. B. Shah, S. Gaston, S. A. Tomlins, J. T. Wei, M. C. Kearney, L. A. Johnson, J. M. Tang, A. M. Chinnaiyan, M. A. Rubin and M. G. Sanda (2009). "Prevalence of TMPRSS2-ERG fusion prostate cancer among men undergoing prostate biopsy in the United States." Clin Cancer Res 15(14): 4706-4711.
Mosse, Y. P., J. Greshock, A. Margolin, T. Naylor, K. Cole, D. Khazi, G. Hii, C. Winter, S. Shahzad, M. U. Asziz, J. A. Biegel, B. L. Weber and J. M. Maris (2005). "High-resolution detection and mapping of genomic DNA alterations in neuroblastoma." Genes Chromosomes Cancer 43(4): 390-403.
Muhlbradt, E., E. Asatiani, E. Ortner, A. Wang and E. P. Gelmann (2009). "NKX3.1 activates expression of insulin-like growth factor binding protein-3 to mediate insulin-like growth factor-I signaling and cell proliferation." Cancer Res 69(6): 2615-2622.
Murrin, L. C. and J. N. Talbot (2007). "RanBPM, a scaffolding protein in the immune and nervous systems." J Neuroimmune Pharmacol 2(3): 290-295.
Nacusi, L. P. and D. J. Tindall (2011). "Targeting 5alpha-reductase for prostate cancer prevention and treatment." Nat Rev Urol 8(7): 378-384.
Naito, A. T., S. Okada, T. Minamino, K. Iwanaga, M. L. Liu, T. Sumida, S. Nomura, N. Sahara, T. Mizoroki, A. Takashima, H. Akazawa, T. Nagai, I. Shiojima and I. Komuro (2010). "Promotion of CHIP-mediated p53 degradation protects the heart from ischemic injury." Circ Res 106(11): 1692-1702.
Nakamura, M., H. Masuda, J. Horii, K. Kuma, N. Yokoyama, T. Ohba, H. Nishitani, T. Miyata, M. Tanaka and T. Nishimoto (1998). "When overexpressed, a novel centrosomal protein, RanBPM, causes ectopic microtubule nucleation similar to gamma-tubulin." J Cell Biol 143(4): 1041-1052.
Nakayama, K., H. Nagahama, Y. A. Minamishima, S. Miyake, N. Ishida, S. Hatakeyama, M. Kitagawa, S. Iemura, T. Natsume and K. I. Nakayama (2004). "Skp2-mediated degradation of p27 regulates progression into mitosis." Dev Cell 6(5): 661-672.
Chapter 8 References
330
Naramura, M., N. Nandwani, H. Gu, V. Band and H. Band (2010). "Rapidly fatal myeloproliferative disorders in mice with deletion of Casitas B-cell lymphoma (Cbl) and Cbl-b in hematopoietic stem cells." Proc Natl Acad Sci U S A 107(37): 16274-16279.
Nawaz, Z., D. M. Lonard, C. L. Smith, E. Lev-Lehman, S. Y. Tsai, M. J. Tsai and B. W. O'Malley (1999). "The Angelman syndrome-associated protein, E6-AP, is a coactivator for the nuclear hormone receptor superfamily." Mol Cell Biol 19(2): 1182-1189.
Nelson, D. L., M. M. Cox and A. L. Lehninger (2005). "Lehninger principles of biochemistry". New York, Freeman.
Nesvizhskii, A. I. and R. Aebersold (2005). "Interpretation of shotgun proteomic data: the protein inference problem." Mol Cell Proteomics 4(10): 1419-1440.
Newton, K., M. L. Matsumoto, I. E. Wertz, D. S. Kirkpatrick, J. R. Lill, J. Tan, D. Dugger, N. Gordon, S. S. Sidhu, F. A. Fellouse, L. Komuves, D. M. French, R. E. Ferrando, C. Lam, D. Compaan, C. Yu, I. Bosanac, S. G. Hymowitz, R. F. Kelley and V. M. Dixit (2008). "Ubiquitin chain editing revealed by polyubiquitin linkage-specific antibodies." Cell 134(4): 668-678.
Ngan, E. S., Y. Hashimoto, Z. Q. Ma, M. J. Tsai and S. Y. Tsai (2003). "Overexpression of Cdc25B, an androgen receptor coactivator, in prostate cancer." Oncogene 22(5): 734-739.
Nishitani, H., E. Hirose, Y. Uchimura, M. Nakamura, M. Umeda, K. Nishii, N. Mori and T. Nishimoto (2001). "Full-sized RanBPM cDNA encodes a protein possessing a long stretch of proline and glutamine within the N-terminal region, comprising a large protein complex." Gene 272(1-2): 25-33.
Nishitani, H., N. Sugimoto, V. Roukos, Y. Nakanishi, M. Saijo, C. Obuse, T. Tsurimoto, K. I. Nakayama, K. Nakayama, M. Fujita, Z. Lygerou and T. Nishimoto (2006). "Two E3 ubiquitin ligases, SCF-Skp2 and DDB1-Cul4, target human Cdt1 for proteolysis." EMBO J 25(5): 1126-1136.
Niu, J., Y. Shi, K. Iwai and Z. H. Wu (2011). "LUBAC regulates NF-kappaB activation upon genotoxic stress by promoting linear ubiquitination of NEMO." EMBO J 30(18): 3741-3753.
Nuber, U. and M. Scheffner (1999). "Identification of determinants in E2 ubiquitin-conjugating enzymes required for hect E3 ubiquitin-protein ligase interaction." J Biol Chem 274(11): 7576-7582.
Nupponen, N. N., E. R. Hyytinen, A. H. Kallioniemi and T. Visakorpi (1998). "Genetic alterations in prostate cancer cell lines detected by comparative genomic hybridization." Cancer Genet Cytogenet 101(1): 53-57.
Nymark, P., H. Wikman, S. Ruosaari, J. Hollmen, E. Vanhala, A. Karjalainen, S. Anttila and S. Knuutila (2006). "Identification of specific gene copy number changes in asbestos-related lung cancer." Cancer Res 66(11): 5737-5743.
O'Connor, P. M., J. Jackman, D. Jondle, K. Bhatia, I. Magrath and K. W. Kohn (1993). "Role of the p53 tumor suppressor gene in cell cycle arrest and radiosensitivity of Burkitt's lymphoma cell lines." Cancer Res 53(20): 4776-4780.
Oba, K., H. Matsuyama, S. Yoshihiro, F. Kishi, M. Takahashi, M. Tsukamoto, M. Kinjo, K. Sagiyama and K. Naito (2001). "Two putative tumor suppressor genes on chromosome arm 8p may play different roles in prostate cancer." Cancer Genet Cytogenet 124(1): 20-26.
Oberst, A., M. Malatesta, R. I. Aqeilan, M. Rossi, P. Salomoni, R. Murillas, P. Sharma, M. R. Kuehn, M. Oren, C. M. Croce, F. Bernassola and G. Melino (2007). "The Nedd4-
Chapter 8 References
331
binding partner 1 (N4BP1) protein is an inhibitor of the E3 ligase Itch." Proc Natl Acad Sci U S A 104(27): 11280-11285.
Oettgen, P., E. Finger, Z. Sun, Y. Akbarali, U. Thamrongsak, J. Boltax, F. Grall, A. Dube, A. Weiss, L. Brown, G. Quinn, K. Kas, G. Endress, C. Kunsch and T. A. Libermann (2000). "PDEF, a novel prostate epithelium-specific ets transcription factor, interacts with the androgen receptor and activates prostate-specific antigen gene expression." J Biol Chem 275(2): 1216-1225.
Ogunjimi, A. A., D. J. Briant, N. Pece-Barbara, C. Le Roy, G. M. Di Guglielmo, P. Kavsak, R. K. Rasmussen, B. T. Seet, F. Sicheri and J. L. Wrana (2005). "Regulation of Smurf2 ubiquitin ligase activity by anchoring the E2 to the HECT domain." Mol Cell 19(3): 297-308.
Oliner, J. D., K. W. Kinzler, P. S. Meltzer, D. L. George and B. Vogelstein (1992). "Amplification of a gene encoding a p53-associated protein in human sarcomas." Nature 358(6381): 80-83.
Olsson, P., T. K. Bera, M. Essand, V. Kumar, P. Duray, J. Vincent, B. Lee and I. Pastan (2001). "GDEP, a new gene differentially expressed in normal prostate and prostate cancer." Prostate 48(4): 231-241.
O'Neil, J., J. Grim, P. Strack, S. Rao, D. Tibbitts, C. Winter, J. Hardwick, M. Welcker, J. P. Meijerink, R. Pieters, G. Draetta, R. Sears, B. E. Clurman and A. T. Look (2007). "FBW7 mutations in leukemic cells mediate NOTCH pathway activation and resistance to gamma-secretase inhibitors." J Exp Med 204(8): 1813-1824.
Ong, S. E., B. Blagoev, I. Kratchmarova, D. B. Kristensen, H. Steen, A. Pandey and M. Mann (2002). "Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics." Mol Cell Proteomics 1(5): 376-386.
Orian, A., A. L. Schwartz, A. Israel, S. Whiteside, C. Kahana and A. Ciechanover (1999). "Structural motifs involved in ubiquitin-mediated processing of the NF-kappaB precursor p105: roles of the glycine-rich region and a downstream ubiquitination domain." Mol Cell Biol 19(5): 3664-3673.
Orian, A., H. Gonen, B. Bercovich, I. Fajerman, E. Eytan, A. Israel, F. Mercurio, K. Iwai, A. L. Schwartz and A. Ciechanover (2000). "SCF(beta)(-TrCP) ubiquitin ligase-mediated processing of NF-kappaB p105 requires phosphorylation of its C-terminus by IkappaB kinase." EMBO J 19(11): 2580-2591.
Orlicky, S., X. Tang, A. Willems, M. Tyers and F. Sicheri (2003). "Structural basis for phosphodependent substrate selection and orientation by the SCFCdc4 ubiquitin ligase." Cell 112(2): 243-256.
Ornstein, D. K., M. Cinquanta, S. Weiler, P. H. Duray, M. R. Emmert-Buck, C. D. Vocke, W. M. Linehan and J. A. Ferretti (2001). "Expression studies and mutational analysis of the androgen regulated homeobox gene NKX3.1 in benign and malignant prostate epithelium." J Urol 165(4): 1329-1334.
Ouchi, T., S. W. Lee, M. Ouchi, S. A. Aaronson and C. M. Horvath (2000). "Collaboration of signal transducer and activator of transcription 1 (STAT1) and BRCA1 in differential regulation of IFN-gamma target genes." Proc Natl Acad Sci U S A 97(10): 5208-5213.
Ozkan, E., H. Yu and J. Deisenhofer (2005). "Mechanistic insight into the allosteric activation of a ubiquitin-conjugating enzyme by RING-type ubiquitin ligases." Proc Natl Acad Sci U S A 102(52): 18890-18895.
Pandey, A., A. V. Podtelejnikov, B. Blagoev, X. R. Bustelo, M. Mann and H. F. Lodish (2000). "Analysis of receptor signaling pathways by mass spectrometry: identification of vav-2
Chapter 8 References
332
as a substrate of the epidermal and platelet-derived growth factor receptors." Proc Natl Acad Sci U S A 97(1): 179-184.
Pandey, U. B., Z. Nie, Y. Batlevi, B. A. McCray, G. P. Ritson, N. B. Nedelsky, S. L. Schwartz, N. A. DiProspero, M. A. Knight, O. Schuldiner, R. Padmanabhan, M. Hild, D. L. Berry, D. Garza, C. C. Hubbert, T. P. Yao, E. H. Baehrecke and J. P. Taylor (2007). "HDAC6 rescues neurodegeneration and provides an essential link between autophagy and the UPS." Nature 447(7146): 859-863.
Pant, V., S. Xiong, T. Iwakuma, A. Quintas-Cardama and G. Lozano (2011). "Heterodimerization of Mdm2 and Mdm4 is critical for regulating p53 activity during embryogenesis but dispensable for p53 and Mdm2 stability." Proc Natl Acad Sci U S A 108(29): 11995-12000.
Paul, F. E., F. Hosp and M. Selbach (2011). "Analyzing protein-protein interactions by quantitative mass spectrometry." Methods 54(4): 387-395.
Paul, I., S. F. Ahmed, A. Bhowmik, S. Deb and M. K. Ghosh (2012). "The ubiquitin ligase CHIP regulates c-Myc stability and transcriptional activity." Oncogene.
Pelzer, C., I. Kassner, K. Matentzoglu, R. K. Singh, H. P. Wollscheid, M. Scheffner, G. Schmidtke and M. Groettrup (2007). "UBE1L2, a novel E1 enzyme specific for ubiquitin." J Biol Chem 282(32): 23010-23014.
Perinchery, G., N. Bukurov, K. Nakajima, J. Chang, L. C. Li and R. Dahiya (1999). "High frequency of deletion on chromosome 9p21 may harbor several tumor-suppressor genes in human prostate cancer." Int J Cancer 83(5): 610-614.
Perissi, V., A. Aggarwal, C. K. Glass, D. W. Rose and M. G. Rosenfeld (2004). "A corepressor/coactivator exchange complex required for transcriptional activation by nuclear receptors and other regulated transcription factors." Cell 116(4): 511-526.
Perkins, D. N., D. J. C. Pappin, D. M. Creasy and J. S. Cottrell (1999). "Probability-based protein identification by searching sequence databases using mass spectrometry data." Electrophoresis 20: 3551-3567.
Perlmutter, M. A. and H. Lepor (2007). "Androgen deprivation therapy in the treatment of advanced prostate cancer." Rev Urol 9 Suppl 1: S3-8.
Persaud, A., P. Alberts, E. M. Amsen, X. Xiong, J. Wasmuth, Z. Saadon, C. Fladd, J. Parkinson and D. Rotin (2009). "Comparison of substrate specificity of the ubiquitin ligases Nedd4 and Nedd4-2 using proteome arrays." Mol Syst Biol 5: 333.
Petroski, M. D. and R. J. Deshaies (2005). "Mechanism of lysine 48-linked ubiquitin-chain synthesis by the cullin-RING ubiquitin-ligase complex SCF-Cdc34." Cell 123(6): 1107-1120.
Petroski, M. D., X. Zhou, G. Dong, S. Daniel-Issakani, D. G. Payan and J. Huang (2007). "Substrate modification with lysine 63-linked ubiquitin chains through the UBC13-UEV1A ubiquitin-conjugating enzyme." J Biol Chem 282(41): 29936-29945.
Pfleger, K. D. and K. A. Eidne (2006). "Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET)." Nat Methods 3(3): 165-174.
Pickart, C. M. and I. A. Rose (1985). "Ubiquitin carboxyl-terminal hydrolase acts on ubiquitin
carboxyl-terminal amides." J Biol Chem 260(13): 7903-7910. Pickart, C. M. (2001). "Mechanisms underlying ubiquitination." Annu Rev Biochem 70: 503-
533.
Pienta, K. J. and D. Bradley (2006). "Mechanisms underlying the development of androgen-independent prostate cancer." Clin Cancer Res 12(6): 1665-1671.
Chapter 8 References
333
Pise-Masison, C. A., M. Radonovich, K. Sakaguchi, E. Appella and J. N. Brady (1998). "Phosphorylation of p53: a novel pathway for p53 inactivation in human T-cell lymphotropic virus type 1-transformed cells." J Virol 72(8): 6348-6355.
Pitre, S., F. Dehne, A. Chan, J. Cheetham, A. Duong, A. Emili, M. Gebbia, J. Greenblatt, M. Jessulat, N. Krogan, X. Luo and A. Golshani (2006). "PIPE: a protein-protein interaction prediction engine based on the re-occurring short polypeptide sequences between known interacting protein pairs." BMC Bioinformatics 7: 365.
Plant, P. J., F. Lafont, S. Lecat, P. Verkade, K. Simons and D. Rotin (2000). "Apical membrane targeting of Nedd4 is mediated by an association of its C2 domain with annexin XIIIb." J Cell Biol 149(7): 1473-1484.
Poirier, M. B., L. Laflamme and M. F. Langlois (2006). "Identification and characterization of RanBPM, a novel coactivator of thyroid hormone receptors." J Mol Endocrinol 36(2): 313-325.
Polanowska, J., J. S. Martin, T. Garcia-Muse, M. I. Petalcorin and S. J. Boulton (2006). "A conserved pathway to activate BRCA1-dependent ubiquitylation at DNA damage sites." EMBO J 25(10): 2178-2188.
Polekhina, G., C. M. House, N. Traficante, J. P. Mackay, F. Relaix, D. A. Sassoon, M. W. Parker and D. D. Bowtell (2002). "Siah ubiquitin ligase is structurally related to TRAF and modulates TNF-alpha signaling." Nat Struct Biol 9(1): 68-75.
Pomerantz, J., N. Schreiber-Agus, N. J. Liegeois, A. Silverman, L. Alland, L. Chin, J. Potes, K. Chen, I. Orlow, H. W. Lee, C. Cordon-Cardo and R. A. DePinho (1998). "The Ink4a tumor suppressor gene product, p19Arf, interacts with MDM2 and neutralizes MDM2's inhibition of p53." Cell 92(6): 713-723.
Ponting, C., J. Schultz and P. Bork (1997). "SPRY domains in ryanodine receptors (Ca(2+)-release channels)." Trends Biochem Sci 22(6): 193-194.
Pootrakul, L., R. H. Datar, S. R. Shi, J. Cai, D. Hawes, S. G. Groshen, A. S. Lee and R. J. Cote (2006). "Expression of stress response protein Grp78 is associated with the development of castration-resistant prostate cancer." Clin Cancer Res 12(20 Pt 1): 5987-5993.
Porter, J. R. and M. K. Brawer (1993). "Prostatic intraepithelial neoplasia and prostate-specific antigen." World J Urol 11(4): 196-200.
Powell, I. J. (2011). "The precise role of ethnicity and family history on aggressive prostate cancer: a review analysis." Arch Esp Urol 64(8): 711-719.
Poyurovsky, M. V., C. Priest, A. Kentsis, K. L. Borden, Z. Q. Pan, N. Pavletich and C. Prives (2007). "The Mdm2 RING domain C-terminus is required for supramolecular assembly and ubiquitin ligase activity." EMBO J 26(1): 90-101.
Prag, S., G. D. Collett and J. C. Adams (2004). "Molecular analysis of muskelin identifies a conserved discoidin-like domain that contributes to protein self-association." Biochem J 381(Pt 2): 547-559.
Prag, G., S. Lee, R. Mattera, C. N. Arighi, B. M. Beach, J. S. Bonifacino and J. H. Hurley (2005). "Structural mechanism for ubiquitinated-cargo recognition by the Golgi-localized, gamma-ear-containing, ADP-ribosylation-factor-binding proteins." Proc Natl Acad Sci U S A 102(7): 2334-2339.
Prag, S., A. De Arcangelis, E. Georges-Labouesse and J. C. Adams (2007). "Regulation of post-translational modifications of muskelin by protein kinase C." Int J Biochem Cell Biol 39(2): 366-378.
Chapter 8 References
334
Preece, D. M., J. M. Harvey, J. M. Bentel and M. A. Thomas (2011). "ETS1 regulates NKX3.1 5' promoter activity and expression in prostate cancer cells." Prostate 71(4): 403-414.
Prescott, J. L., L. Blok and D. J. Tindall (1998). "Isolation and androgen regulation of the human homeobox cDNA, NKX3.1." Prostate 35(1): 71-80.
Punga, T., M. T. Bengoechea-Alonso and J. Ericsson (2006). "Phosphorylation and ubiquitination of the transcription factor sterol regulatory element-binding protein-1 in response to DNA binding." J Biol Chem 281(35): 25278-25286.
Pungaliya, P., D. Kulkarni, H. J. Park, H. Marshall, H. Zheng, H. Lackland, A. Saleem and E. H. Rubin (2007). "TOPORS functions as a SUMO-1 E3 ligase for chromatin-modifying proteins." J Proteome Res 6(10): 3918-3923.
Qiao, L. and J. Zhang (2009). "Inhibition of lysosomal functions reduces proteasomal activity." Neurosci Lett 456(1): 15-19.
Qin, H., Q. Shao, S. A. Igdoura, M. A. Alaoui-Jamali and D. W. Laird (2003). "Lysosomal and proteasomal degradation play distinct roles in the life cycle of Cx43 in gap junctional intercellular communication-deficient and -competent breast tumor cells." J Biol Chem 278(32): 30005-30014.
Qiu, L., C. Joazeiro, N. Fang, H. Y. Wang, C. Elly, Y. Altman, D. Fang, T. Hunter and Y. C. Liu (2000). "Recognition and ubiquitination of Notch by Itch, a hect-type E3 ubiquitin ligase." J Biol Chem 275(46): 35734-35737.
Quesnel, B., C. Preudhomme, J. Fournier, P. Fenaux and J. P. Peyrat (1994). "MDM2 gene
amplification in human breast cancer." Eur J Cancer 30A(7): 982-984. Rabhi-Essafi, I., A. Sadok, N. Khalaf and D. M. Fathallah (2007). "A strategy for high-level
expression of soluble and functional human interferon alpha as a GST-fusion protein in E. coli." Protein Eng Des Sel 20(5): 201-209.
Raiborg, C., T. E. Rusten and H. Stenmark (2003). "Protein sorting into multivesicular endosomes." Curr Opin Cell Biol 15(4): 446-455.
Raish, M., M. Khurshid, M. A. Ansari, P. K. Chaturvedi, S. M. Bae, J. H. Kim, E. K. Park, D. C. Park and W. S. Ahn (2012). "Analysis of molecular cytogenetic alterations in uterine leiomyosarcoma by array-based comparative genomic hybridization." J Cancer Res Clin Oncol.
Rajendra, R., D. Malegaonkar, P. Pungaliya, H. Marshall, Z. Rasheed, J. Brownell, L. F. Liu, S. Lutzker, A. Saleem and E. H. Rubin (2004). "Topors functions as an E3 ubiquitin ligase with specific E2 enzymes and ubiquitinates p53." J Biol Chem 279(35): 36440-36444.
Rao, M. A., H. Cheng, A. N. Quayle, H. Nishitani, C. C. Nelson and P. S. Rennie (2002). "RanBPM, a nuclear protein that interacts with and regulates transcriptional activity of androgen receptor and glucocorticoid receptor." J Biol Chem 277(50): 48020-48027.
Rape, M. and M. W. Kirschner (2004). "Autonomous regulation of the anaphase-promoting complex couples mitosis to S-phase entry." Nature 432(7017): 588-595.
Ravid, T. and M. Hochstrasser (2007). "Autoregulation of an E2 enzyme by ubiquitin-chain assembly on its catalytic residue." Nat Cell Biol 9(4): 422-427.
Regelmann, J., T. Schule, F. S. Josupeit, J. Horak, M. Rose, K. D. Entian, M. Thumm and D. H. Wolf (2003). "Catabolite degradation of fructose-1,6-bisphosphatase in the yeast Saccharomyces cerevisiae: a genome-wide screen identifies eight novel GID genes and indicates the existence of two degradation pathways." Mol Biol Cell 14(4): 1652-1663.
Chapter 8 References
335
Rehman, Y. and J. E. Rosenberg (2012). "Abiraterone acetate: oral androgen biosynthesis inhibitor for treatment of castration-resistant prostate cancer." Drug Des Devel Ther 6: 13-18.
Reuveny, S., Y. J. Kim, C. W. Kemp and J. Shiloach (1993). "Production of recombinant proteins in high-density insect cell cultures." Biotechnol Bioeng 42(2): 235-239.
Reyes-Turcu, F. E., K. H. Ventii and K. D. Wilkinson (2009). "Regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes." Annu Rev Biochem 78: 363-397.
Rideout, H. J., I. Lang-Rollin and L. Stefanis (2004). "Involvement of macroautophagy in the dissolution of neuronal inclusions." Int J Biochem Cell Biol 36(12): 2551-2562.
Ringshausen, I., C. C. O'Shea, A. J. Finch, L. B. Swigart and G. I. Evan (2006). "Mdm2 is critically and continuously required to suppress lethal p53 activity in vivo." Cancer Cell 10(6): 501-514.
Robinson, D. N. and L. Cooley (1997). "Drosophila kelch is an oligomeric ring canal actin organizer." J Cell Biol 138(4): 799-810.
Rock, K. L., C. Gramm, L. Rothstein, K. Clark, R. Stein, L. Dick, D. Hwang and A. L. Goldberg (1994). "Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules." Cell 78(5): 761-771.
Rodriguez Ortner, E., R. B. Hayes, J. Weissfeld and E. P. Gelmann (2006). "Effect of homeodomain protein NKX3.1 R52C polymorphism on prostate gland size." Urology 67(2): 311-315.
Rotin, D. and S. Kumar (2009). "Physiological functions of the HECT family of ubiquitin ligases." Nat Rev Mol Cell Biol 10(6): 398-409.
Rubin, M. A. and A. M. De Marzo (2004). "Molecular genetics of human prostate cancer." Mod Pathol 17(3): 380-388.
Ryu, Y. S., Y. Lee, K. W. Lee, C. Y. Hwang, J. S. Maeng, J. H. Kim, Y. S. Seo, K. H. You, B. Song and K. S. Kwon (2011). "TRIM32 protein sensitizes cells to tumor necrosis factor (TNFalpha)-induced apoptosis via its RING domain-dependent E3 ligase activity against X-linked inhibitor of apoptosis (XIAP)." J Biol Chem 286(29): 25729-25738.
Sahdev, S., S. K. Khattar and K. S. Saini (2008). "Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies." Mol Cell Biochem 307(1-2): 249-264.
Salghetti, S. E., A. A. Caudy, J. G. Chenoweth and W. P. Tansey (2001). "Regulation of transcriptional activation domain function by ubiquitin." Science 293(5535): 1651-1653.
Samaj, J., F. Baluska, B. Voigt, M. Schlicht, D. Volkmann and D. Menzel (2004). "Endocytosis, actin cytoskeleton, and signaling." Plant Physiol 135(3): 1150-1161.
Sanada, M., T. Suzuki, L. Y. Shih, M. Otsu, M. Kato, S. Yamazaki, A. Tamura, H. Honda, M. Sakata-Yanagimoto, K. Kumano, H. Oda, T. Yamagata, J. Takita, N. Gotoh, K. Nakazaki, N. Kawamata, M. Onodera, M. Nobuyoshi, Y. Hayashi, H. Harada, M. Kurokawa, S. Chiba, H. Mori, K. Ozawa, M. Omine, H. Hirai, H. Nakauchi, H. P. Koeffler and S. Ogawa (2009). "Gain-of-function of mutated C-CBL tumour suppressor in myeloid neoplasms." Nature 460(7257): 904-908.
Sanger, F., S. Nicklen and A. R. Coulson (1977). "DNA sequencing with chain-terminating inhibitors." Proc Natl Acad Sci U S A 74(12): 5463-5467.
Chapter 8 References
336
Santt, O., T. Pfirrmann, B. Braun, J. Juretschke, P. Kimmig, H. Scheel, K. Hofmann, M. Thumm and D. H. Wolf (2008). "The yeast GID complex, a novel ubiquitin ligase (E3) involved in the regulation of carbohydrate metabolism." Mol Biol Cell 19(8): 3323-3333.
Sargin, B., C. Choudhary, N. Crosetto, M. H. Schmidt, R. Grundler, M. Rensinghoff, C. Thiessen, L. Tickenbrock, J. Schwable, C. Brandts, B. August, S. Koschmieder, S. R. Bandi, J. Duyster, W. E. Berdel, C. Muller-Tidow, I. Dikic and H. Serve (2007). "Flt3-dependent transformation by inactivating c-Cbl mutations in AML." Blood 110(3): 1004-1012.
Sarker, D., A. H. Reid, T. A. Yap and J. S. de Bono (2009). "Targeting the PI3K/AKT pathway
for the treatment of prostate cancer." Clin Cancer Res 15(15): 4799-4805.
Sasaki, S., A. Shionoya, M. Ishida, M. J. Gambello, J. Yingling, A. Wynshaw-Boris and S. Hirotsune (2000). "A LIS1/NUDEL/cytoplasmic dynein heavy chain complex in the developing and adult nervous system." Neuron 28(3): 681-696.
Saville, M. K., A. Sparks, D. P. Xirodimas, J. Wardrop, L. F. Stevenson, J. C. Bourdon, Y. L. Woods and D. P. Lane (2004). "Regulation of p53 by the ubiquitin-conjugating enzymes UbcH5B/C in vivo." J Biol Chem 279(40): 42169-42181.
Scaglioni, P. P., T. M. Yung, S. Choi, C. Baldini, G. Konstantinidou and P. P. Pandolfi (2008). "CK2 mediates phosphorylation and ubiquitin-mediated degradation of the PML tumor suppressor." Mol Cell Biochem 316(1-2): 149-154.
Schauber, C., L. Chen, P. Tongaonkar, I. Vega, D. Lambertson, W. Potts and K. Madura (1998). "Rad23 links DNA repair to the ubiquitin/proteasome pathway." Nature 391(6668): 715-718.
Scheffner, M., J. M. Huibregtse, R. D. Vierstra and P. M. Howley (1993). "The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53." Cell 75(3): 495-505.
Scheffner, M., J. M. Huibregtse and P. M. Howley (1994). "Identification of a human ubiquitin-conjugating enzyme that mediates the E6-AP-dependent ubiquitination of p53." Proc Natl Acad Sci U S A 91(19): 8797-8801.
Scheffner, M., U. Nuber and J. M. Huibregtse (1995). "Protein ubiquitination involving an E1-E2-E3 enzyme ubiquitin thioester cascade." Nature 373(6509): 81-83.
Schottenfeld, D. and J. F. Fraumeni (2006). "Cancer epidemiology and prevention". Oxford, Oxford University Press.
Schork, S. M., G. Bee, M. Thumm and D. H. Wolf (1994). "Catabolite inactivation of fructose-1,6-bisphosphatase in yeast is mediated by the proteasome." FEBS Lett 349(2): 270-274.
Schork, S. M., M. Thumm and D. H. Wolf (1995). "Catabolite inactivation of fructose-1,6-
bisphosphatase of Saccharomyces cerevisiae. Degradation occurs via the ubiquitin pathway." J Biol Chem 270(44): 26446-26450.
Schuchner, S., V. Tembe, J. A. Rodriguez and B. R. Henderson (2005). "Nuclear targeting and cell cycle regulatory function of human BARD1." J Biol Chem 280(10): 8855-8861.
Schulman, B. A. and J. W. Harper (2009). "Ubiquitin-like protein activation by E1 enzymes: the apex for downstream signalling pathways." Nat Rev Mol Cell Biol 10(5): 319-331.
Schultz, J., F. Milpetz, P. Bork and C. P. Ponting (1998). "SMART, a simple modular architecture research tool: identification of signaling domains." Proc Natl Acad Sci U S A 95(11): 5857-5864.
Chapter 8 References
337
Schulze, H., M. Dose, M. Korpal, I. Meyer, J. E. Italiano, Jr. and R. A. Shivdasani (2008). "RanBP10 is a cytoplasmic guanine nucleotide exchange factor that modulates noncentrosomal microtubules." J Biol Chem 283(20): 14109-14119.
Sciavolino, P. J., E. W. Abrams, L. Yang, L. P. Austenberg, M. M. Shen and C. Abate-Shen (1997). "Tissue-specific expression of murine Nkx3.1 in the male urogenital system." Dev Dyn 209(1): 127-138.
Scott, P. M., P. S. Bilodeau, O. Zhdankina, S. C. Winistorfer, M. J. Hauglund, M. M. Allaman, W. R. Kearney, A. D. Robertson, A. L. Boman and R. C. Piper (2004). "GGA proteins bind ubiquitin to facilitate sorting at the trans-Golgi network." Nat Cell Biol 6(3): 252-259.
Scully, R., S. F. Anderson, D. M. Chao, W. Wei, L. Ye, R. A. Young, D. M. Livingston and J. D. Parvin (1997). "BRCA1 is a component of the RNA polymerase II holoenzyme." Proc Natl Acad Sci U S A 94(11): 5605-5610.
Sekine, K., Y. Hao, Y. Suzuki, R. Takahashi, T. Tsuruo and M. Naito (2005). "HtrA2 cleaves Apollon and induces cell death by IAP-binding motif in Apollon-deficient cells." Biochem Biophys Res Commun 330(1): 279-285.
Sellers, J. R. (2000). "Myosins: a diverse superfamily." Biochim Biophys Acta 1496(1): 3-22.
Seol, J. H., R. M. Feldman, W. Zachariae, A. Shevchenko, C. C. Correll, S. Lyapina, Y. Chi, M. Galova, J. Claypool, S. Sandmeyer, K. Nasmyth and R. J. Deshaies (1999). "Cdc53/cullin and the essential Hrt1 RING-H2 subunit of SCF define a ubiquitin ligase module that activates the E2 enzyme Cdc34." Genes Dev 13(12): 1614-1626.
Shang, F., G. Deng, M. Obin, C. C. Wu, X. Gong, D. Smith, R. A. Laursen, U. P. Andley, J. R. Reddan and A. Taylor (2001). "Ubiquitin-activating enzyme (E1) isoforms in lens epithelial cells: origin of translation, E2 specificity and cellular localization determined with novel site-specific antibodies." Exp Eye Res 73(6): 827-836.
Shang, Y., M. Myers and M. Brown (2002). "Formation of the androgen receptor transcription complex." Mol Cell 9(3): 601-610.
Shang, F., G. Deng, Q. Liu, W. Guo, A. L. Haas, B. Crosas, D. Finley and A. Taylor (2005). "Lys6-modified ubiquitin inhibits ubiquitin-dependent protein degradation." J Biol Chem 280(21): 20365-20374.
Sharifi, N., J. L. Gulley and W. L. Dahut (2010). "An update on androgen deprivation therapy for prostate cancer." Endocr Relat Cancer 17(4): R305-315.
Shaw, P., R. Bovey, S. Tardy, R. Sahli, B. Sordat and J. Costa (1992). "Induction of apoptosis by wild-type p53 in a human colon tumor-derived cell line." Proc Natl Acad Sci U S A 89(10): 4495-4499.
Shen, R., M. Chen, Y. J. Wang, H. Kaneki, L. Xing, J. O'Keefe R and D. Chen (2006). "Smad6 interacts with Runx2 and mediates Smad ubiquitin regulatory factor 1-induced Runx2 degradation." J Biol Chem 281(6): 3569-3576.
Shirley, R. B., I. Kaddour-Djebbar, D. M. Patel, V. Lakshmikanthan, R. W. Lewis and M. V. Kumar (2005). "Combination of proteasomal inhibitors lactacystin and MG132 induced synergistic apoptosis in prostate cancer cells." Neoplasia 7(12): 1104-1111.
Silver, E. T., T. J. Gwozd, C. Ptak, M. Goebl and M. J. Ellison (1992). "A chimeric ubiquitin conjugating enzyme that combines the cell cycle properties of CDC34 (UBC3) and the DNA repair properties of RAD6 (UBC2): implications for the structure, function and evolution of the E2s." EMBO J 11(8): 3091-3098.
Chapter 8 References
338
Simmons, S. O. and J. M. Horowitz (2006). "Nkx3.1 binds and negatively regulates the transcriptional activity of Sp-family members in prostate-derived cells." Biochem J 393(Pt 1): 397-409.
Singer, E. A., A. Kaushal, B. Turkbey, A. Couvillon, P. A. Pinto and H. L. Parnes (2012). "Active surveillance for prostate cancer: past, present and future." Curr Opin Oncol 24(3): 243-250.
Singh, L. N. and S. Hannenhalli (2008). "Functional diversification of paralogous transcription factors via divergence in DNA binding site motif and in expression." PLoS One 3(6): e2345.
Shore, N., M. Mason, et al. (2012). "New developments in castrate-resistant prostate cancer." BJU Int 109 Suppl 6: 22-32.
Suizu, F., Y. Hiramuki, F. Okumura, M. Matsuda, A. J. Okumura, N. Hirata, M. Narita, T. Kohno, J. Yokota, M. Bohgaki, C. Obuse, S. Hatakeyama, T. Obata and M. Noguchi (2009). "The E3 ligase TTC3 facilitates ubiquitination and degradation of phosphorylated Akt." Dev Cell 17(6): 800-810.
Skaar, J. R. and M. Pagano (2009). "Control of cell growth by the SCF and APC/C ubiquitin ligases." Curr Opin Cell Biol 21(6): 816-824.
Skowyra, D., K. L. Craig, M. Tyers, S. J. Elledge and J. W. Harper (1997). "F-box proteins are receptors that recruit phosphorylated substrates to the SCF ubiquitin-ligase complex." Cell 91(2): 209-219.
Skowyra, D., D. M. Koepp, T. Kamura, M. N. Conrad, R. C. Conaway, J. W. Conaway, S. J. Elledge and J. W. Harper (1999). "Reconstitution of G1 cyclin ubiquitination with complexes containing SCFGrr1 and Rbx1." Science 284(5414): 662-665.
Smith, J. R., D. Freije, J. D. Carpten, H. Gronberg, J. Xu, S. D. Isaacs, M. J. Brownstein, G. S. Bova, H. Guo, P. Bujnovszky, D. R. Nusskern, J. E. Damber, A. Bergh, M. Emanuelsson, O. P. Kallioniemi, J. Walker-Daniels, J. E. Bailey-Wilson, T. H. Beaty, D. A. Meyers, P. C. Walsh, F. S. Collins, J. M. Trent and W. B. Isaacs (1996). "Major susceptibility locus for prostate cancer on chromosome 1 suggested by a genome-wide search." Science 274(5291): 1371-1374.
Smith, D. S., M. Niethammer, R. Ayala, Y. Zhou, M. J. Gambello, A. Wynshaw-Boris and L. H. Tsai (2000). "Regulation of cytoplasmic dynein behaviour and microtubule organization by mammalian Lis1." Nat Cell Biol 2(11): 767-775.
Snowdon, C., C. Hlynialuk and G. van der Merwe (2008). "Components of the Vid30c are
needed for the rapamycin-induced degradation of the high-affinity hexose transporter Hxt7p in Saccharomyces cerevisiae." FEMS Yeast Res 8(2): 204-216.
Sobhian, B., G. Shao, D. R. Lilli, A. C. Culhane, L. A. Moreau, B. Xia, D. M. Livingston and R.
A. Greenberg (2007). "RAP80 targets BRCA1 to specific ubiquitin structures at DNA damage sites." Science 316(5828): 1198-1202.
Soltysik-Espanola, M., R. A. Rogers, S. Jiang, T. A. Kim, R. Gaedigk, R. A. White, H. Avraham and S. Avraham (1999). "Characterization of Mayven, a novel actin-binding protein predominantly expressed in brain." Mol Biol Cell 10(7): 2361-2375.
Song, S. U., S. H. Shin, S. K. Kim, G. S. Choi, W. C. Kim, M. H. Lee, S. J. Kim, I. H. Kim, M. S. Choi, Y. J. Hong and K. H. Lee (2003). "Effective transduction of osteogenic sarcoma cells by a baculovirus vector." J Gen Virol 84(Pt 3): 697-703.
Soni, S., S. Bala, B. Gwynn, K. E. Sahr, L. L. Peters and M. Hanspal (2006). "Absence of erythroblast macrophage protein (Emp) leads to failure of erythroblast nuclear extrusion." J Biol Chem 281(29): 20181-20189.
Chapter 8 References
339
Soni, S., S. Bala, A. Kumar and M. Hanspal (2007). "Changing pattern of the subcellular distribution of erythroblast macrophage protein (Emp) during macrophage differentiation." Blood Cells Mol Dis 38(1): 25-31.
Soni, S., S. Bala and M. Hanspal (2008). "Requirement for erythroblast-macrophage protein (Emp) in definitive erythropoiesis." Blood Cells Mol Dis 41(2): 141-147.
Song, H., B. Zhang, M. A. Watson, P. A. Humphrey, H. Lim and J. Milbrandt (2009). "Loss of Nkx3.1 leads to the activation of discrete downstream target genes during prostate tumorigenesis." Oncogene 28(37): 3307-3319.
Sood, A. K., H. Kim and J. Geradts (2012). "PDEF in prostate cancer." Prostate 72(6): 592-596.
Sorrentino, A., N. Thakur, S. Grimsby, A. Marcusson, V. von Bulow, N. Schuster, S. Zhang, C. H. Heldin and M. Landstrom (2008). "The type I TGF-beta receptor engages TRAF6 to activate TAK1 in a receptor kinase-independent manner." Nat Cell Biol 10(10): 1199-1207.
Spence, J., S. Sadis, A. L. Haas and D. Finley (1995). "A ubiquitin mutant with specific defects in DNA repair and multiubiquitination." Mol Cell Biol 15(3): 1265-1273.
Stacey, K. B., E. Breen and C. A. Jefferies (2012). "Tyrosine phosphorylation of the E3 ubiquitin ligase TRIM21 positively regulates interaction with IRF3 and hence TRIM21 activity." PLoS One 7(3): e34041.
Stad, R., N. A. Little, D. P. Xirodimas, R. Frenk, A. J. van der Eb, D. P. Lane, M. K. Saville and A. G. Jochemsen (2001). "Mdmx stabilizes p53 and Mdm2 via two distinct mechanisms." EMBO Rep 2(11): 1029-1034.
Stamenova, S. D., M. E. French, Y. He, S. A. Francis, Z. B. Kramer and L. Hicke (2007). "Ubiquitin binds to and regulates a subset of SH3 domains." Mol Cell 25(2): 273-284.
Starita, L. M., A. A. Horwitz, M. C. Keogh, C. Ishioka, J. D. Parvin and N. Chiba (2005). "BRCA1/BARD1 ubiquitinate phosphorylated RNA polymerase II." J Biol Chem 280(26): 24498-24505.
Staub, O. and D. Rotin (1996). "WW domains." Structure 4(5): 495-499.
Steffan, J. J. and H. K. Koul (2011). "Prostate derived ETS factor (PDEF): a putative tumor metastasis suppressor." Cancer Lett 310(1): 109-117.
Stephen, A. G., J. S. Trausch-Azar, A. Ciechanover and A. L. Schwartz (1996). "The ubiquitin-activating enzyme E1 is phosphorylated and localized to the nucleus in a cell cycle-dependent manner." J Biol Chem 271(26): 15608-15614.
Stommel, J. M. and G. M. Wahl (2004). "Accelerated MDM2 auto-degradation induced by DNA-damage kinases is required for p53 activation." EMBO J 23(7): 1547-1556.
Stone, S. L., H. Hauksdottir, A. Troy, J. Herschleb, E. Kraft and J. Callis (2005). "Functional analysis of the RING-type ubiquitin ligase family of Arabidopsis." Plant Physiol 137(1): 13-30.
Stueber, D., I. Ibrahimi, D. Cutler, B. Dobberstein and H. Bujard (1984). "A novel in vitro transcription-translation system: accurate and efficient synthesis of single proteins from cloned DNA sequences." EMBO J 3(13): 3143-3148.
Sudol, M., H. I. Chen, C. Bougeret, A. Einbond and P. Bork (1995). "Characterization of a novel protein-binding module--the WW domain." FEBS Lett 369(1): 67-71.
Summers, M. K., B. Pan, K. Mukhyala and P. K. Jackson (2008). "The unique N terminus of the UbcH10 E2 enzyme controls the threshold for APC activation and enhances checkpoint regulation of the APC." Mol Cell 31(4): 544-556.
Chapter 8 References
340
Sun, Y., X. Zhou and H. Ma (2007). "Genome-Wide Analysis of Kelch repeat-containing F-box family." J Integrative Plant Bio 49(6): 940-952.
Sun, L., L. Shi, W. Li, W. Yu, J. Liang, H. Zhang, X. Yang, Y. Wang, R. Li, X. Yao, X. Yi and Y. Shang (2009). "JFK, a Kelch domain-containing F-box protein, links the SCF complex to p53 regulation." Proc Natl Acad Sci U S A 106(25): 10195-10200.
Suresh, B., S. Ramakrishna, Y. S. Kim, S. M. Kim, M. S. Kim and K. H. Baek (2010). "Stability and function of mammalian lethal giant larvae-1 oncoprotein are regulated by the scaffolding protein RanBPM." J Biol Chem 285(46): 35340-35349.
Suresh, B., S. Ramakrishna and K. H. Baek (2012). "Diverse roles of the scaffolding protein RanBPM." Drug Discov Today 17(7-8): 379-387.
Sutterluty, H., E. Chatelain, A. Marti, C. Wirbelauer, M. Senften, U. Muller and W. Krek (1999). "p45SKP2 promotes p27Kip1 degradation and induces S phase in quiescent cells." Nat Cell Biol 1(4): 207-214.
Suzuki, T., A. Ueda, N. Kobayashi, J. Yang, K. Tomaru, M. Yamamoto, M. Takeno and Y. Ishigatsubo (2008). "Proteasome-dependent degradation of alpha-catenin is regulated by interaction with ARMc8alpha." Biochem J 411(3): 581-591.
Swanson, K. A., R. S. Kang, S. D. Stamenova, L. Hicke and I. Radhakrishnan (2003). "Solution structure of Vps27 UIM-ubiquitin complex important for endosomal sorting and receptor downregulation." EMBO J 22(18): 4597-4606.
Tai, C. Y., D. L. Dujardin, N. E. Faulkner and R. B. Vallee (2002). "Role of dynein, dynactin, and CLIP-170 interactions in LIS1 kinetochore function." J Cell Biol 156(6): 959-968.
Talbot, J. N., D. A. Skifter, E. Bianchi, D. T. Monaghan, M. L. Toews and L. C. Murrin (2009). "Regulation of mu opioid receptor internalization by the scaffold protein RanBPM." Neurosci Lett 466(3): 154-158.
Tan, P. Y., C. W. Chang, K. R. Chng, K. D. Wansa, W. K. Sung and E. Cheung (2012). "Integration of regulatory networks by NKX3-1 promotes androgen-dependent prostate cancer survival." Mol Cell Biol 32(2): 399-414.
Tanaka, M., I. Komuro, H. Inagaki, N. A. Jenkins, N. G. Copeland and S. Izumo (2000). "Nkx3.1, a murine homolog of Ddrosophila bagpipe, regulates epithelial ductal branching and proliferation of the prostate and palatine glands." Dev Dyn 219(2): 248-260.
Tang, Z., B. Li, R. Bharadwaj, H. Zhu, E. Ozkan, K. Hakala, J. Deisenhofer and H. Yu (2001). "APC2 Cullin protein and APC11 RING protein comprise the minimal ubiquitin ligase module of the anaphase-promoting complex." Mol Biol Cell 12(12): 3839-3851.
Taplin, M. E., B. Rajeshkumar, S. Halabi, C. P. Werner, B. A. Woda, J. Picus, W. Stadler, D. F. Hayes, P. W. Kantoff, N. J. Vogelzang and E. J. Small (2003). "Androgen receptor mutations in androgen-independent prostate cancer: Cancer and Leukemia Group B Study 9663." J Clin Oncol 21(14): 2673-2678.
Tasdemir, E., M. C. Maiuri, L. Galluzzi, I. Vitale, M. Djavaheri-Mergny, M. D'Amelio, A. Criollo, E. Morselli, C. Zhu, F. Harper, U. Nannmark, C. Samara, P. Pinton, J. M. Vicencio, R. Carnuccio, U. M. Moll, F. Madeo, P. Paterlini-Brechot, R. Rizzuto, G. Szabadkai, G. Pierron, K. Blomgren, N. Tavernarakis, P. Codogno, F. Cecconi and G. Kroemer (2008). "Regulation of autophagy by cytoplasmic p53." Nat Cell Biol 10(6): 676-687.
Tatham, M. H., M. C. Geoffroy, L. Shen, A. Plechanovova, N. Hattersley, E. G. Jaffray, J. J. Palvimo and R. T. Hay (2008). "RNF4 is a poly-SUMO-specific E3 ubiquitin ligase required for arsenic-induced PML degradation." Nat Cell Biol 10(5): 538-546.
Chapter 8 References
341
Tavtigian, S. V., J. Simard, D. H. Teng, V. Abtin, M. Baumgard, A. Beck, N. J. Camp, A. R. Carillo, Y. Chen, P. Dayananth, M. Desrochers, M. Dumont, J. M. Farnham, D. Frank, C. Frye, S. Ghaffari, J. S. Gupte, R. Hu, D. Iliev, T. Janecki, E. N. Kort, K. E. Laity, A. Leavitt, G. Leblanc, J. McArthur-Morrison, A. Pederson, B. Penn, K. T. Peterson, J. E. Reid, S. Richards, M. Schroeder, R. Smith, S. C. Snyder, B. Swedlund, J. Swensen, A. Thomas, M. Tranchant, A. M. Woodland, F. Labrie, M. H. Skolnick, S. Neuhausen, J. Rommens and L. A. Cannon-Albright (2001). "A candidate prostate cancer susceptibility gene at chromosome 17p." Nat Genet 27(2): 172-180.
Taylor, J., M. Lymboura, P. E. Pace, P. A'Hern R, A. J. Desai, S. Shousha, R. C. Coombes and S. Ali (1998). "An important role for BRCA1 in breast cancer progression is indicated by its loss in a large proportion of non-familial breast cancers." Int J Cancer 79(4): 334-342.
Tembe, V. and B. R. Henderson (2007). "BARD1 translocation to mitochondria correlates with Bax oligomerization, loss of mitochondrial membrane potential, and apoptosis." J Biol Chem 282(28): 20513-20522.
Tewari, R., E. Bailes, K. A. Bunting and J. C. Coates (2010). "Armadillo-repeat protein functions: questions for little creatures." Trends Cell Biol 20(8): 470-481.
Thien, C. B. and W. Y. Langdon (1997). "Tyrosine kinase activity of the EGF receptor is enhanced by the expression of oncogenic 70Z-Cbl." Oncogene 15(24): 2909-2919.
Thien, C. B., F. Walker and W. Y. Langdon (2001). "RING finger mutations that abolish c-Cbl-directed polyubiquitination and downregulation of the EGF receptor are insufficient for cell transformation." Mol Cell 7(2): 355-365.
Thomas, M. A., M. C. Hodgson, S. D. Loermans, J. Hooper, R. Endersby and J. M. Bentel (2006). "Transcriptional regulation of the homeobox gene NKX3.1 by all-trans retinoic acid in prostate cancer cells." J Cell Biochem 99(5): 1409-1419.
Thomas, M. A., D. M. Preece and J. M. Bentel (2010). "Androgen regulation of the prostatic tumour suppressor NKX3.1 is mediated by its 3' untranslated region." Biochem J 425(3): 575-583.
Thornton, B. R., T. M. Ng, M. E. Matyskiela, C. W. Carroll, D. O. Morgan and D. P. Toczyski (2006). "An architectural map of the anaphase-promoting complex." Genes Dev 20(4): 449-460.
Tofaris, G. K., R. Layfield and M. G. Spillantini (2001). "alpha-synuclein metabolism and aggregation is linked to ubiquitin-independent degradation by the proteasome." FEBS Lett 509(1): 22-26.
Tokunaga, F., S. Sakata, Y. Saeki, Y. Satomi, T. Kirisako, K. Kamei, T. Nakagawa, M. Kato, S. Murata, S. Yamaoka, M. Yamamoto, S. Akira, T. Takao, K. Tanaka and K. Iwai (2009). "Involvement of linear polyubiquitylation of NEMO in NF-kappaB activation." Nat Cell Biol 11(2): 123-132.
Togashi, H., E. F. Schmidt and S. M. Strittmatter (2006). "RanBPM contributes to Semaphorin3A signaling through plexin-A receptors." J Neurosci 26(18): 4961-4969.
Tomaru, K., A. Ueda, T. Suzuki, N. Kobayashi, J. Yang, M. Yamamoto, M. Takeno, T. Kaneko
and Y. Ishigatsubo (2010). "Armadillo Repeat Containing 8alpha Binds to HRS and Promotes HRS Interaction with Ubiquitinated Proteins." Open Biochem J 4: 1-8.
Tomlins, S. A., A. Bjartell, A. M. Chinnaiyan, G. Jenster, R. K. Nam, M. A. Rubin and J. A. Schalken (2009). "ETS gene fusions in prostate cancer: from discovery to daily clinical practice." Eur Urol 56(2): 275-286.
Tovar, C., J. Rosinski, Z. Filipovic, B. Higgins, K. Kolinsky, H. Hilton, X. Zhao, B. T. Vu, W. Qing, K. Packman, O. Myklebost, D. C. Heimbrook and L. T. Vassilev (2006). "Small-
Chapter 8 References
342
molecule MDM2 antagonists reveal aberrant p53 signaling in cancer: implications for therapy." Proc Natl Acad Sci U S A 103(6): 1888-1893.
Towfighi, J., C. M. Berlin, Jr., R. L. Ladda, E. E. Frauenhoffer and R. A. Lehman (1985). "Neuropathology of oral-facial-digital syndromes." Arch Pathol Lab Med 109(7): 642-646.
Traub, F., M. Mengel, H. J. Luck, H. H. Kreipe and R. von Wasielewski (2006). "Prognostic impact of Skp2 and p27 in human breast cancer." Breast Cancer Res Treat 99(2): 185-191.
Trempe, J. F. (2011). "Reading the ubiquitin postal code." Curr Opin Struct Biol 21(6): 792-801.
Trinkle-Mulcahy, L., S. Boulon, Y. W. Lam, R. Urcia, F. M. Boisvert, F. Vandermoere, N. A. Morrice, S. Swift, U. Rothbauer, H. Leonhardt and A. Lamond (2008). "Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes." J Cell Biol 183(2): 223-239.
Udeshi, N. D., D. R. Mani, T. Eisenhaure, P. Mertins, J. D. Jaffe, K. R. Clauser, N. Hacohen and S. A. Carr (2012). "Methods for quantification of in vivo changes in protein ubiquitination following proteasome and deubiquitinase inhibition." Mol Cell Proteomics 11(5): 148-159.
Ueda, T., N. Bruchovsky and M. D. Sadar (2002). "Activation of the androgen receptor N-terminal domain by interleukin-6 via MAPK and STAT3 signal transduction pathways." J Biol Chem 277(9): 7076-7085.
Umeda, M., H. Nishitani and T. Nishimoto (2003). "A novel nuclear protein, Twa1, and Muskelin comprise a complex with RanBPM." Gene 303: 47-54.
Valiyaveettil, M., A. A. Bentley, P. Gursahaney, R. Hussien, R. Chakravarti, N. Kureishy, S. Prag and J. C. Adams (2008). "Novel role of the muskelin-RanBP9 complex as a nucleocytoplasmic mediator of cell morphology regulation." J Cell Biol 182(4): 727-739.
VanDemark, A. P., R. M. Hofmann, C. Tsui, C. M. Pickart and C. Wolberger (2001). "Molecular insights into polyubiquitin chain assembly: crystal structure of the Mms2/Ubc13 heterodimer." Cell 105(6): 711-720.
van der Merwe, G. K., T. G. Cooper and H. J. van Vuuren (2001). "Ammonia regulates VID30 expression and Vid30p function shifts nitrogen metabolism toward glutamate formation especially when Saccharomyces cerevisiae is grown in low concentrations of ammonia." J Biol Chem 276(31): 28659-28666.
VanGuilder, H. D., K. E. Vrana and W. M. Freeman (2008). "Twenty-five years of quantitative PCR for gene expression analysis." Biotechniques 44(5): 619-626.
van Wijk, S. J., S. J. de Vries, P. Kemmeren, A. Huang, R. Boelens, A. M. Bonvin and H. T. Timmers (2009). "A comprehensive framework of E2-RING E3 interactions of the human ubiquitin-proteasome system." Mol Syst Biol 5: 295.
van Wijk, S. J. and H. T. Timmers (2010). "The family of ubiquitin-conjugating enzymes (E2s): deciding between life and death of proteins." FASEB J 24(4): 981-993.
Varshavsky, A. (1997). "The N-end rule pathway of protein degradation." Genes Cells 2(1): 13-28.
Vasilescu, J., X. Guo and J. Kast (2004). "Identification of protein-protein interactions using in vivo cross-linking and mass spectrometry." Proteomics 4(12): 3845-3854.
Chapter 8 References
343
Veldscholte, J., C. A. Berrevoets, C. Ris-Stalpers, G. G. Kuiper, G. Jenster, J. Trapman, A. O. Brinkmann and E. Mulder (1992). "The androgen receptor in LNCaP cells contains a mutation in the ligand binding domain which affects steroid binding characteristics and response to antiandrogens." J Steroid Biochem Mol Biol 41(3-8): 665-669.
Verdecia, M. A., C. A. Joazeiro, N. J. Wells, J. L. Ferrer, M. E. Bowman, T. Hunter and J. P. Noel (2003). "Conformational flexibility underlies ubiquitin ligation mediated by the WWP1 HECT domain E3 ligase." Mol Cell 11(1): 249-259.
Verma, R., R. M. Feldman and R. J. Deshaies (1997). "SIC1 is ubiquitinated in vitro by a pathway that requires CDC4, CDC34, and cyclin/CDK activities." Mol Biol Cell 8(8): 1427-1437.
Verma, R., R. Oania, J. Graumann and R. J. Deshaies (2004). "Multiubiquitin chain receptors define a layer of substrate selectivity in the ubiquitin-proteasome system." Cell 118(1): 99-110.
Vijay-Kumar, S., C. E. Bugg and W. J. Cook (1987a). "Structure of ubiquitin refined at 1.8 A resolution." J Mol Biol 194(3): 531-544.
Vijay-Kumar, S., C. E. Bugg, K. D. Wilkinson, R. D. Vierstra, P. M. Hatfield and W. J. Cook (1987b). "Comparison of the three-dimensional structures of human, yeast, and oat ubiquitin." J Biol Chem 262(13): 6396-6399.
Villaverde, A. and M. M. Carrio (2003). "Protein aggregation in recombinant bacteria: biological role of inclusion bodies." Biotechnol Lett 25(17): 1385-1395.
Visakorpi, T., E. Hyytinen, P. Koivisto, M. Tanner, R. Keinanen, C. Palmberg, A. Palotie, T. Tammela, J. Isola and O. P. Kallioniemi (1995a). "In vivo amplification of the androgen receptor gene and progression of human prostate cancer." Nat Genet 9(4): 401-406.
Visakorpi, T., A. H. Kallioniemi, A. C. Syvanen, E. R. Hyytinen, R. Karhu, T. Tammela, J. J. Isola and O. P. Kallioniemi (1995b). "Genetic changes in primary and recurrent prostate cancer by comparative genomic hybridization." Cancer Res 55(2): 342-347.
Visakorpi, T. (2003). "The molecular genetics of prostate cancer." Urology 62(5 Suppl 1): 3-10.
Vlahovicek, K., L. Kajan, V. Agoston and S. Pongor (2005). "The SBASE domain sequence resource, release 12: prediction of protein domain-architecture using support vector machines." Nucleic Acids Res 33(Database issue): D223-225.
Vocke, C. D., R. O. Pozzatti, D. G. Bostwick, C. D. Florence, S. B. Jennings, S. E. Strup, P. H. Duray, L. A. Liotta, M. R. Emmert-Buck and W. M. Linehan (1996). "Analysis of 99 microdissected prostate carcinomas reveals a high frequency of allelic loss on chromosome 8p12-21." Cancer Res 56(10): 2411-2416.
Vodermaier, H. C., C. Gieffers, S. Maurer-Stroh, F. Eisenhaber and J. M. Peters (2003). "TPR subunits of the anaphase-promoting complex mediate binding to the activator protein CDH1." Curr Biol 13(17): 1459-1468.
Voeller, H. J., M. Augustus, V. Madike, G. S. Bova, K. C. Carter and E. P. Gelmann (1997). "Coding region of NKX3.1, a prostate-specific homeobox gene on 8p21, is not mutated in human prostate cancers." Cancer Res 57(20): 4455-4459.
Vostal, J. G. and N. R. Shulman (1993). "Vinculin is a major platelet protein that undergoes Ca(2+)-dependent tyrosine phosphorylation." Biochem J 294 ( Pt 3): 675-680.
Wade, M., Y. V. Wang and G. M. Wahl (2010). "The p53 orchestra: Mdm2 and Mdmx set the tone." Trends Cell Biol 20(5): 299-309.
Walczak, H., K. Iwai and I. Dikic (2012). "Generation and physiological roles of linear ubiquitin chains." BMC Biol 10: 23.
Chapter 8 References
344
Walden, H., M. S. Podgorski and B. A. Schulman (2003). "Insights into the ubiquitin transfer cascade from the structure of the activating enzyme for NEDD8." Nature 422(6929): 330-334.
Walsh, C. T. (2005). "Posttranslational modification of proteins: Expanding nature's inventory". Greenwood Village, CO, Roberts and Company Publishers.
Wang, D., Z. Li, E. M. Messing and G. Wu (2002a). "Activation of Ras/Erk pathway by a novel MET-interacting protein RanBPM." J Biol Chem 277(39): 36216-36222.
Wang, L., S. K. McDonnell, D. A. Elkins, S. L. Slager, E. Christensen, A. F. Marks, J. M. Cunningham, B. J. Peterson, S. J. Jacobsen, J. R. Cerhan, M. L. Blute, D. J. Schaid and S. N. Thibodeau (2002b). "Analysis of the RNASEL gene in familial and sporadic prostate cancer." Am J Hum Genet 71(1): 116-123.
Wang, Q., C. Moyret-Lalle, F. Couzon, C. Surbiguet-Clippe, J. C. Saurin, T. Lorca, C. Navarro and A. Puisieux (2003a). "Alterations of anaphase-promoting complex genes in human colon cancer cells." Oncogene 22(10): 1486-1490.
Wang, S., J. Gao, Q. Lei, N. Rozengurt, C. Pritchard, J. Jiao, G. V. Thomas, G. Li, P. Roy-Burman, P. S. Nelson, X. Liu and H. Wu (2003b). "Prostate-specific deletion of the murine Pten tumor suppressor gene leads to metastatic prostate cancer." Cancer Cell 4(3): 209-221.
Wang, D., Z. Li, S. R. Schoen, E. M. Messing and G. Wu (2004). "A novel MET-interacting protein shares high sequence similarity with RanBPM, but fails to stimulate MET-induced Ras/Erk signaling." Biochem Biophys Res Commun 313(2): 320-326.
Wang, D., Z. Li, E. M. Messing and G. Wu (2005). "The SPRY domain-containing SOCS box protein 1 (SSB-1) interacts with MET and enhances the hepatocyte growth factor-induced Erk-Elk-1-serum response element pathway." J Biol Chem 280(16): 16393-16401.
Wang, M. and C. M. Pickart (2005). "Different HECT domain ubiquitin ligases employ distinct mechanisms of polyubiquitin chain synthesis." EMBO J 24(24): 4324-4333.
Wang, M., D. Cheng, J. Peng and C. M. Pickart (2006). "Molecular determinants of polyubiquitin linkage selection by an HECT ubiquitin ligase." EMBO J 25(8): 1710-1719.
Wang, B. and S. J. Elledge (2007). "Ubc13/Rnf8 ubiquitin ligases control foci formation of the Rap80/Abraxas/Brca1/Brcc36 complex in response to DNA damage." Proc Natl Acad Sci U S A 104(52): 20759-20763.
Wang, B., S. Matsuoka, B. A. Ballif, D. Zhang, A. Smogorzewska, S. P. Gygi and S. J. Elledge (2007). "Abraxas and RAP80 form a BRCA1 protein complex required for the DNA damage response." Science 316(5828): 1194-1198.
Wang, J., Q. Peng, Q. Lin, C. Childress, D. Carey and W. Yang (2010). "Calcium activates Nedd4 E3 ubiquitin ligases by releasing the C2 domain-mediated auto-inhibition." J Biol Chem 285(16): 12279-12288.
Wang, X., J. Wang and X. Jiang (2011). "MdmX protein is essential for Mdm2 protein-mediated p53 polyubiquitination." J Biol Chem 286(27): 23725-23734.
Wang, L., C. Fu, Y. Cui, Y. Xie, Y. Yuan, X. Wang, H. Chen and B. R. Huang (2012). "The
Ran-binding protein RanBPM can depress the NF-kappaB pathway by interacting with TRAF6." Mol Cell Biochem 359(1-2): 83-94.
Waterman, H., G. Levkowitz, I. Alroy and Y. Yarden (1999). "The RING finger of c-Cbl mediates desensitization of the epidermal growth factor receptor." J Biol Chem 274(32): 22151-22154.
Chapter 8 References
345
Webb, J. L., B. Ravikumar, J. Atkins, J. N. Skepper and D. C. Rubinsztein (2003). "Alpha-Synuclein is degraded by both autophagy and the proteasome." J Biol Chem 278(27): 25009-25013.
Weissman, A. M. (2001). "Themes and variations on ubiquitylation." Nat Rev Mol Cell Biol 2(3): 169-178.
Welcsh, P. L. and M. C. King (2001). "BRCA1 and BRCA2 and the genetics of breast and ovarian cancer." Hum Mol Genet 10(7): 705-713.
Wenzel, D. M., K. E. Stoll and R. E. Klevit (2011). "E2s: structurally economical and functionally replete." Biochem J 433(1): 31-42.
Westermark, U. K., M. Reyngold, A. B. Olshen, R. Baer, M. Jasin and M. E. Moynahan (2003). "BARD1 participates with BRCA1 in homology-directed repair of chromosome breaks." Mol Cell Biol 23(21): 7926-7936.
Wickliffe, K. E., A. Williamson, H. J. Meyer, A. Kelly and M. Rape (2011). "K11-linked ubiquitin chains as novel regulators of cell division." Trends Cell Biol 21(11): 656-663.
Wiesner, S., A. A. Ogunjimi, H. R. Wang, D. Rotin, F. Sicheri, J. L. Wrana and J. D. Forman-Kay (2007). "Autoinhibition of the HECT-type ubiquitin ligase Smurf2 through its C2 domain." Cell 130(4): 651-662.
Wiklund, F., B. A. Jonsson, A. J. Brookes, L. Stromqvist, J. Adolfsson, M. Emanuelsson, H. O. Adami, K. Augustsson-Balter and H. Gronberg (2004). "Genetic analysis of the RNASEL gene in hereditary, familial, and sporadic prostate cancer." Clin Cancer Res 10(21): 7150-7156.
Willems, A. R., M. Schwab and M. Tyers (2004). "A hitchhiker's guide to the cullin ubiquitin ligases: SCF and its kin." Biochim Biophys Acta 1695(1-3): 133-170.
Williamson, A., K. E. Wickliffe, B. G. Mellone, L. Song, G. H. Karpen and M. Rape (2009). "Identification of a physiological E2 module for the human anaphase-promoting complex." Proc Natl Acad Sci U S A 106(43): 18213-18218.
Williamson, A., S. Banerjee, X. Zhu, I. Philipp, A. T. Iavarone and M. Rape (2011). "Regulation of ubiquitin chain initiation to control the timing of substrate degradation." Mol Cell 42(6): 744-757.
Wilson, C. A., L. Ramos, M. R. Villasenor, K. H. Anders, M. F. Press, K. Clarke, B. Karlan, J. J. Chen, R. Scully, D. Livingston, R. H. Zuch, M. H. Kanter, S. Cohen, F. J. Calzone and D. J. Slamon (1999). "Localization of human BRCA1 and its loss in high-grade, non-inherited breast carcinomas." Nat Genet 21(2): 236-240.
Wiltz, A. L., S. Shikanov, S. E. Eggener, M. H. Katz, A. E. Thong, G. D. Steinberg, A. L.
Shalhav, G. P. Zagaja and K. C. Zorn (2009). "Robotic radical prostatectomy in overweight and obese patients: oncological and validated-functional outcomes." Urology 73(2): 316-322.
Windheim, M., M. Peggie and P. Cohen (2008). "Two different classes of E2 ubiquitin-conjugating enzymes are required for the mono-ubiquitination of proteins and elongation by polyubiquitin chains with a specific topology." Biochem J 409(3): 723-729.
Winn, P. J., T. L. Religa, J. N. Battey, A. Banerjee and R. C. Wade (2004). "Determinants of functionality in the ubiquitin conjugating enzyme family." Structure 12(9): 1563-1574.
Wong, J. M., K. Mafune, H. Yow, E. N. Rivers, T. S. Ravikumar, G. D. Steele, Jr. and L. B. Chen (1993). "Ubiquitin-ribosomal protein S27a gene overexpressed in human colorectal carcinoma is an early growth response gene." Cancer Res 53(8): 1916-1920.
Chapter 8 References
346
Wu-Baer, F., K. Lagrazon, W. Yuan and R. Baer (2003). "The BRCA1/BARD1 heterodimer assembles polyubiquitin chains through an unconventional linkage involving lysine residue K6 of ubiquitin." J Biol Chem 278(37): 34743-34746.
Wu, P. and L. Brand (1994). "Resonance energy transfer: methods and applications." Anal Biochem 218(1): 1-13.
Wu, L. C., Z. W. Wang, J. T. Tsan, M. A. Spillman, A. Phung, X. L. Xu, M. C. Yang, L. Y. Hwang, A. M. Bowcock and R. Baer (1996). "Identification of a RING protein that can interact in vivo with the BRCA1 gene product." Nat Genet 14(4): 430-440.
Wu, G., G. Xu, B. A. Schulman, P. D. Jeffrey, J. W. Harper and N. P. Pavletich (2003a). "Structure of a beta-TrCP1-Skp1-beta-catenin complex: destruction motif binding and lysine specificity of the SCF(beta-TrCP1) ubiquitin ligase." Mol Cell 11(6): 1445-1456.
Wu, P. Y., M. Hanlon, M. Eddins, C. Tsui, R. S. Rogers, J. P. Jensen, M. J. Matunis, A. M. Weissman, C. Wolberger and C. M. Pickart (2003b). "A conserved catalytic residue in the ubiquitin-conjugating enzyme family." EMBO J 22(19): 5241-5250.
Wu, Y., X. Sun, E. Kaczmarek, K. M. Dwyer, E. Bianchi, A. Usheva and S. C. Robson (2006). "RanBPM associates with CD39 and modulates ecto-nucleotidase activity." Biochem J 396(1): 23-30.
Wu, R. C., Q. Feng, D. M. Lonard and B. W. O'Malley (2007a). "SRC-3 coactivator functional lifetime is regulated by a phospho-dependent ubiquitin time clock." Cell 129(6): 1125-1140.
Wu, Y., Q. Li and X. Z. Chen (2007b). "Detecting protein-protein interactions by Far western
blotting." Nat Protoc 2(12): 3278-3284.
Xia, Y., G. M. Pao, H. W. Chen, I. M. Verma and T. Hunter (2003). "Enhancement of BRCA1 E3 ubiquitin ligase activity through direct interaction with the BARD1 protein." J Biol Chem 278(7): 5255-5263.
Xie, J., M. Sun, L. Guo, W. Liu, J. Jiang, X. Chen, L. Zhou and J. Gu (2006). "Human Dectin-1 isoform E is a cytoplasmic protein and interacts with RanBPM." Biochem Biophys Res Commun 347(4): 1067-1073.
Xirodimas, D. P., M. K. Saville, J. C. Bourdon, R. T. Hay and D. P. Lane (2004). "Mdm2-mediated NEDD8 conjugation of p53 inhibits its transcriptional activity." Cell 118(1): 83-97.
Xu, J., D. Meyers, D. Freije, S. Isaacs, K. Wiley, D. Nusskern, C. Ewing, E. Wilkens, P. Bujnovszky, G. S. Bova, P. Walsh, W. Isaacs, J. Schleutker, M. Matikainen, T. Tammela, T. Visakorpi, O. P. Kallioniemi, R. Berry, D. Schaid, A. French, S. McDonnell, J. Schroeder, M. Blute, S. Thibodeau, H. Gronberg, M. Emanuelsson, J. E. Damber, A. Bergh, B. A. Jonsson, J. Smith, J. Bailey-Wilson, J. Carpten, D. Stephan, E. Gillanders, I. Amundson, T. Kainu, D. Freas-Lutz, A. Baffoe-Bonnie, A. Van Aucken, R. Sood, F. Collins, M. Brownstein and J. Trent (1998). "Evidence for a prostate cancer susceptibility locus on the X chromosome." Nat Genet 20(2): 175-179.
Xu, Y., D. W. Piston and C. H. Johnson (1999). "A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins." Proc Natl Acad Sci U S A 96(1): 151-156.
Xu, L. L., V. Srikantan, I. A. Sesterhenn, M. Augustus, R. Dean, J. W. Moul, K. C. Carter and S. Srivastava (2000). "Expression profile of an androgen regulated prostate specific homeobox gene NKX3.1 in primary prostate cancer." J Urol 163(3): 972-979.
Xu, J., L. Dimitrov, B. L. Chang, T. S. Adams, A. R. Turner, D. A. Meyers, R. A. Eeles, D. F. Easton, W. D. Foulkes, J. Simard, G. G. Giles, J. L. Hopper, L. Mahle, P. Moller, T.
Chapter 8 References
347
Bishop, C. Evans, S. Edwards, J. Meitz, S. Bullock, Q. Hope, C. L. Hsieh, J. Halpern, R. N. Balise, I. Oakley-Girvan, A. S. Whittemore, C. M. Ewing, M. Gielzak, S. D. Isaacs, P. C. Walsh, K. E. Wiley, W. B. Isaacs, S. N. Thibodeau, S. K. McDonnell, J. M. Cunningham, K. E. Zarfas, S. Hebbring, D. J. Schaid, D. M. Friedrichsen, K. Deutsch, S. Kolb, M. Badzioch, G. P. Jarvik, M. Janer, L. Hood, E. A. Ostrander, J. L. Stanford, E. M. Lange, J. L. Beebe-Dimmer, C. E. Mohai, K. A. Cooney, T. Ikonen, A. Baffoe-Bonnie, H. Fredriksson, M. P. Matikainen, T. Tammela, J. Bailey-Wilson, J. Schleutker, C. Maier, K. Herkommer, J. J. Hoegel, W. Vogel, T. Paiss, F. Wiklund, M. Emanuelsson, E. Stenman, B. A. Jonsson, H. Gronberg, N. J. Camp, J. Farnham, L. A. Cannon-Albright and D. Seminara (2005). "A combined genomewide linkage scan of 1,233 families for prostate cancer-susceptibility genes conducted by the international consortium for prostate cancer genetics." Am J Hum Genet 77(2): 219-229.
Xu, P., D. M. Duong, N. T. Seyfried, D. Cheng, Y. Xie, J. Robert, J. Rush, M. Hochstrasser, D. Finley and J. Peng (2009). "Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation." Cell 137(1): 133-145.
Yamamoto, A., A. Friedlein, Y. Imai, R. Takahashi, P. J. Kahle and C. Haass (2005). "Parkin phosphorylation and modulation of its E3 ubiquitin ligase activity." J Biol Chem 280(5): 3390-3399.
Yan, J., Y. S. Kim, X. P. Yang, L. P. Li, G. Liao, F. Xia and A. M. Jetten (2007). "The ubiquitin-interacting motif containing protein RAP80 interacts with BRCA1 and functions in DNA damage repair response." Cancer Res 67(14): 6647-6656.
Yan, Q., S. Dutt, R. Xu, K. Graves, P. Juszczynski, J. P. Manis and M. A. Shipp (2009). "BBAP monoubiquitylates histone H4 at lysine 91 and selectively modulates the DNA damage response." Mol Cell 36(1): 110-120.
Yang, J., M. F. Siqueira, Y. Behl, M. Alikhani and D. T. Graves (2008). "The transcription factor ST18 regulates proapoptotic and proinflammatory gene expression in fibroblasts." FASEB J 22(11): 3956-3967.
Yang, W. L., J. Wang, C. H. Chan, S. W. Lee, A. D. Campos, B. Lamothe, L. Hur, B. C. Grabiner, X. Lin, B. G. Darnay and H. K. Lin (2009). "The E3 ligase TRAF6 regulates Akt ubiquitination and activation." Science 325(5944): 1134-1138.
Yang, Z., Y. Zhang and L. Wang (2012). "Mdm2 is a novel activator of ApoCIII promoter which is
antagonized by p53 and SHP inhibition." Biochem Biophys Res Commun 417(2): 744-746.
Yarden, R. I., S. Pardo-Reoyo, M. Sgagias, K. H. Cowan and L. C. Brody (2002). "BRCA1 regulates the G2/M checkpoint by activating Chk1 kinase upon DNA damage." Nat Genet 30(3): 285-289.
Yasukawa, T., C. Kanei-Ishii, T. Maekawa, J. Fujimoto, T. Yamamoto and S. Ishii (1995). "Increase of solubility of foreign proteins in Escherichia coli by coproduction of the bacterial thioredoxin." J Biol Chem 270(43): 25328-25331.
Ye, Y. and M. Rape (2009). "Building ubiquitin chains: E2 enzymes at work." Nat Rev Mol Cell Biol 10(11): 755-764.
Yin, Q., S. C. Lin, B. Lamothe, M. Lu, Y. C. Lo, G. Hura, L. Zheng, R. L. Rich, A. D. Campos, D. G. Myszka, M. J. Lenardo, B. G. Darnay and H. Wu (2009). "E2 interaction and dimerization in the crystal structure of TRAF6." Nat Struct Mol Biol 16(6): 658-666.
Yin, Y. X., Z. P. Sun, S. H. Huang, L. Zhao, Z. Geng and Z. Y. Chen (2010). "RanBPM contributes to TrkB signaling and regulates brain-derived neurotrophic factor-induced neuronal morphogenesis and survival." J Neurochem 114(1): 110-121.
Chapter 8 References
348
Yin, Y., A. Seifert, J. S. Chua, J. F. Maure, F. Golebiowski and R. T. Hay (2012). "SUMO-targeted ubiquitin E3 ligase RNF4 is required for the response of human cells to DNA damage." Genes Dev 26(11): 1196-1208.
Yoon, H. G., D. W. Chan, Z. Q. Huang, J. Li, J. D. Fondell, J. Qin and J. Wong (2003). "Purification and functional characterization of the human N-CoR complex: the roles of HDAC3, TBL1 and TBLR1." EMBO J 22(6): 1336-1346.
Yoon, H. G. and J. Wong (2006). "The corepressors silencing mediator of retinoid and thyroid hormone receptor and nuclear receptor corepressor are involved in agonist- and antagonist-regulated transcription by androgen receptor." Mol Endocrinol 20(5): 1048-1060.
Young, P., Q. Deveraux, R. E. Beal, C. M. Pickart and M. Rechsteiner (1998). "Characterization of two polyubiquitin binding sites in the 26 S protease subunit 5a." J Biol Chem 273(10): 5461-5467.
Yu, Z. K., J. L. Gervais and H. Zhang (1998). "Human CUL-1 associates with the SKP1/SKP2 complex and regulates p21(CIP1/WAF1) and cyclin D proteins." Proc Natl Acad Sci U S A 95(19): 11324-11329.
Yu, C. X., T. Jin, W. W. Chen, P. J. Zhang, W. W. Liu, H. Y. Guan, J. Zhang, Q. W. Liu and A. L. Jiang (2009). "Identification of Sp1-elements in the promoter region of human homeobox gene NKX3.1." Mol Biol Rep 36(8): 2353-2360.
Yuan, Y., C. Fu, H. Chen, X. Wang, W. Deng and B. R. Huang (2006). "The Ran binding protein RanBPM interacts with TrkA receptor." Neurosci Lett 407(1): 26-31.
Zeng, W., Y. Wang, Y. Yuan, Y. Deng and Y. Li (2006). "CTLH: A Novel Domain with a
Typical "U" Shape Architecture." Res J Biol Sci 1(4): 12-15.
Zheng, N., P. Wang, P. D. Jeffrey and N. P. Pavletich (2000). "Structure of a c-Cbl-UbcH7 complex: RING domain function in ubiquitin-protein ligases." Cell 102(4): 533-539.
Zhang, J., M. Kalkum, B. T. Chait and R. G. Roeder (2002). "The N-CoR-HDAC3 nuclear receptor corepressor complex inhibits the JNK pathway through the integral subunit GPS2." Mol Cell 9(3): 611-623.
Zhang, C., D. R. Dowd, A. Staal, C. Gu, J. B. Lian, A. J. van Wijnen, G. S. Stein and P. N. MacDonald (2003). "Nuclear coactivator-62 kDa/Ski-interacting protein is a nuclear matrix-associated coactivator that may couple vitamin D receptor-mediated transcription and RNA splicing." J Biol Chem 278(37): 35325-35336.
Zhang, M., M. Windheim, S. M. Roe, M. Peggie, P. Cohen, C. Prodromou and L. H. Pearl (2005). "Chaperoned ubiquitylation--crystal structures of the CHIP U box E3 ubiquitin ligase and a CHIP-Ubc13-Uev1a complex." Mol Cell 20(4): 525-538.
Zhang, H., M. H. Muders, J. Li, F. Rinaldo, D. J. Tindall and K. Datta (2008a). "Loss of NKX3.1 favors vascular endothelial growth factor-C expression in prostate cancer." Cancer Res 68(21): 8770-8778.
Zhang, Y., R. A. Fillmore and W. E. Zimmer (2008b). "Structural and functional analysis of domains mediating interaction between the bagpipe homologue, Nkx3.1 and serum response factor." Exp Biol Med (Maywood) 233(3): 297-309.
Zhang, Q., Y. Meng, L. Zhang, J. Chen and D. Zhu (2009). "RNF13: a novel RING-type ubiquitin ligase over-expressed in pancreatic cancer." Cell Res 19(3): 348-357.
Zhang, P. J., X. Y. Hu, C. Y. Liu, Z. B. Chen, N. N. Ni, Y. Yu, L. N. Yang, Z. Q. Huang, Q. W. Liu and A. L. Jiang (2012). "The inhibitory effects of NKX3.1 on IGF-1R expression and its signalling pathway in human prostatic carcinoma PC3 cells." Asian J Androl 14(3): 493-498.
Chapter 8 References
349
Zhao, X. Y., P. J. Malloy, A. V. Krishnan, S. Swami, N. M. Navone, D. M. Peehl and D. Feldman (2000). "Glucocorticoids can promote androgen-independent growth of prostate cancer cells through a mutated androgen receptor." Nat Med 6(6): 703-706.
Zhao, J., Y. Liu, X. Wei, C. Yuan, X. Yuan and X. Xiao (2009). "A novel WD-40 repeat protein WDR26 suppresses H2O2-induced cell death in neural cells." Neurosci Lett 460(1): 66-71.
Zhao, D., H. Q. Zheng, Z. Zhou and C. Chen (2010). "The Fbw7 tumor suppressor targets KLF5 for ubiquitin-mediated degradation and suppresses breast cell proliferation." Cancer Res 70(11): 4728-4738.
Zheng, N., B. A. Schulman, L. Song, J. J. Miller, P. D. Jeffrey, P. Wang, C. Chu, D. M. Koepp, S. J. Elledge, M. Pagano, R. C. Conaway, J. W. Conaway, J. W. Harper and N. P. Pavletich (2002). "Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex." Nature 416(6882): 703-709.
Zheng, S. L., J. H. Ju, B. L. Chang, E. Ortner, J. Sun, S. D. Isaacs, K. E. Wiley, W. Liu, M. Zemedkun, P. C. Walsh, J. Ferretti, J. Gruschus, W. B. Isaacs, E. P. Gelmann and J. Xu (2006). "Germ-line mutation of NKX3.1 cosegregates with hereditary prostate cancer and alters the homeodomain structure and function." Cancer Res 66(1): 69-77.
Zhong, Q., W. Gao, F. Du and X. Wang (2005). "Mule/ARF-BP1, a BH3-only E3 ubiquitin ligase, catalyzes the polyubiquitination of Mcl-1 and regulates apoptosis." Cell 121(7): 1085-1095.
Zhou, J., L. Qin, J. C. Tien, L. Gao, X. Chen, F. Wang, J. T. Hsieh and J. Xu (2012). "Nkx3.1 functions as para-transcription factor to regulate gene expression and cell proliferation in non-cell autonomous manner." J Biol Chem 287(21): 17248-17256.
Zhu, Y., Y. Wang, C. Xia, D. Li, Y. Li, W. Zeng, W. Yuan, H. Liu, C. Zhu, X. Wu and M. Liu (2004). "WDR26: a novel Gbeta-like protein, suppresses MAPK signaling pathway." J Cell Biochem 93(3): 579-587.
Appendix I Buffers and Solutions
Appendix I: Buffers and Solutions
Appendix I Buffers and Solutions
350
Buffers and Solutions 1. 2% Acetic Acid Acetic Acid 10mL ddH2O 490mL Acetic acid and ddH2O were combined and the solution was stored at room temperature. 2. 1% and 2 % Agarose Gel 1%
Agarose Gel
2% Agarose
Gel Agarose Powder
3g 6g
1X TAE100 300mL 300mL Ethidium Bromide26(10mg/mL)
8μL 8μL
Agarose powder was added to TAE100, the solution was microwaved until the agarose had dissolved and ethidium bromide26 was added. Agarose was stored at room temperature and liquefied by heating in a microwave prior to use. 3. 1M Ammonium Chloride (NH4Cl) Ammonium Chloride 5.36g ddH2O 100mL Ammonium chloride was dissolved in ddH2O and the solution was stored at room temperature.
4. 10% Ammonium Persulphate (APS) Ammonium Persulphate 0.1g ddH2O 1mL Ammonium persulphate was dissolved in ddH2O and the solution was stored at 4°C. 5. Ampicillin (100 mg/mL) Ampicillin Powder 100mg ddH2O 1mL Ampicillin powder was dissolved in ddH2O and the solution was stored at -20°C. 6. 50mM Calcium Chloride (CaCl2) 1M Calcium Chloride7 5mL ddH2O 100mL The reagents were mixed and the solution sterilised using a 0.2μM filter and stored at room temperature. 7. 1M Calcium Chloride (CaCl2) Calcium Chloride 27g ddH2O 100mL Calcium chloride was dissolved in ddH2O and the solution then sterilised using a 0.2μM filter and stored at room temperature. 8. 50mM Chloroquine Chloroquine 257mg ddH2O 10mL Chloroquine was dissolved in ddH2O and the solution stored shielded from light at room temperature.
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9. Confocal Blocking Buffer Horse Serum 5mL BSA Fraction V 500mg Sodium Azide 10mg PBS56 45mL Reagents were combined and the solution was stored at 4˚C shielded from light. 10. Coomassie Blue Destaining Solution Methanol 450mL Glacial Acetic Acid 100mL ddH2O 450mL Reagents were combined and the solution stored at room temperature. 11. Coomassie Blue Staining Solution Coomassie Brilliant Blue G 1g Methanol 450mL Glacial Acetic Acid 100mL ddH2O 450mL Coomassie Brilliant Blue G was dissolved in the methanol and ddH2O mixture. The glacial acetic acid was added in a fumehood and the solution was stored at room temperature.
12. Coomassie Blue Staining Solution for Mass Spectrometry Ammonium Sulphate 100g Coomassie Brilliant Blue G 1.2g Orthophosphoric Acid 100mL Methanol 200mL ddH2O 700mL Coomassie Brilliant Blue G was dissolved in 200mL methanol. In a separate bottle, ammonium sulphate was dissolved in 600mL ddH2O, orthophosphoric acid was added and the solution was made up to 800mL using ddH2O. The Coomassie Brilliant Blue G solution was then added and the solution was stored at room temperature.
13. 20mg/mL Cycloheximide Cycloheximide 20mg DMSO 1mL Cycloheximide was dissolved in DMSO and the solution stored shielded from light at -20°C in 100µL aliquots.
14. 10mM dATP 100mM dATP 10µL ddH2O 90µL Reagents were combined and the solution stored at -20°C.
15. DEPC Treated ddH2O Diethylpyrocarbonate (DEPC) 1mL ddH2O 1L DEPC was added to ddH2O in a fumehood, the solution was shaken, left overnight to allow the DEPC to evaporate, autoclaved and stored at room temperature.
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16. 10-2M Dihydrotestosterone 5α-Dihydrotestosterone 2.9024mg Absolute Ethanol 1mL Reagents were combined and the solution stored at -20°C shielded from light. 17. 10-4M Dihydrotestosterone 10-2M DHT16 10µL Absolute Ethanol 990µL Reagents were combined and the solution stored at -20°C shielded from light. 18. 10-5M Dihydrotestosterone 10-4M DHT17 10µL Absolute Ethanol 90µL Reagents were combined and the solution stored at -20°C, shielded from light. The solution was diluted 1:1000 in culture medium to obtain a concentration of 10-8M. 19. 1M Disodium Hydrogen Orthophosphate (Na2HPO4) Disodium Hydrogen Orthophosphate 35.48g ddH2O 250mL Disodium Hydrogen Orthophosphate was dissolved in ddH2O by heating in a waterbath at 30°C. Once dissolved the solution was stored at room temperature.
20. 1M Dithiothreitol (DTT) Dithiothreitol 1.25g ddH2O 8mL DTT was dissolved in ddH2O, the solution was divided into 1mL aliquots and stored at -20°C. 21. 50mM Dithiothreitol (DTT) in 20mM Tris pH7.5 1M DTT20 0.5mL 1M Tris108 pH7.5 0.2mL ddH2O 9.3mL Reagents were combined and the solution used immediately. 22. 6X DNA Loading Dye Bromophenol Blue 10mg Sucrose 7g 0.1M EDTA27 pH8.0 2mL ddH2O 8mL Reagents were combined and the solution was made up to 10mL with ddH2O then stored at room temperature. 23. 10mM and 25mM dNTP (PCR) 10mM
dNTP 25mM dNTP
100mM dATP
10µL 25µL
100mM dTTP
10µL 25µL
100mM dGTP
10µL 25µL
100mM dCTP
10µL 25µL
ddH2O 60µL - Reagents were combined and the solution was stored at -20ºC.
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24. ECL PlusTM Detection Reagent Equal volumes of Solution A and Solution B (GE Healthcare) were combined at room temperature immediately prior to use. 25. 70% , 75% and 95% (v/v) Ethanol 70%
Ethanol 75%
Ethanol 95%
Ethanol Absolute Ethanol
70mL 75mL 95mL
ddH2O 30mL 25mL 5mL Reagents were combined and the solution was stored at room temperature. 26. Ethidium Bromide (10mg/mL) Ethidium Bromide 10mg ddH2O 1mL Ethidium bromide was dissolved in ddH2O and the solution was stored shielded from light at room temperature. 27. 0.1M Ethylenediaminetetra-acetic acid (EDTA) pH8.0 0.5M EDTA26 pH8.0 100mL ddH2O 400mL 0.5M EDTA29 was diluted in ~400mL ddH2O, the pH adjusted with sodium hydroxide, and the solution made up to 500mL, autoclaved and stored at room temperature.
28. 0.5M Ethylenediaminetetra-acetic acid (EDTA) pH 8.0 EDTA 186.12g Sodium Hydroxide ~20g EDTA was dissolved in 500mL ddH2O, the pH adjusted to 8.0 with sodium hydroxide pellets and the volume adjusted to 1L with ddH2O. The solution was mixed and left overnight, autoclaved and stored at room temperature. 29. 4% Formaldehyde 40% Formaldehyde 5 mL PBS56 45mL Reagents were combined and the solution was stored at room temperature shielded from light. 30. 50% Glutathione Sepharose Stock Glutathione Sepharose 4B 1 bottle PBS56 pH7.3 500mL PBS pH7.3 /0.02% Sodium Azide58
~20mL Glutathione Sepharose 4B beads were transferred into a 50mL tube and allowed to settle at 4°C. The supernatant was discarded and the beads resuspended in 50mL cold PBS56 pH7.3 then stored at 4°C to allow to settle again. This procedure was carried out five times. After the last wash, the beads were allowed to settle at 4°C, the supernatant removed and the beads resuspended in an equal volume PBS pH7.3/0.02% sodium azide58 then stored at 4°C.
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31. 50% Glutathione Sepharose 50% Glutathione Sepharose Stock30
300μL PBS56 pH 7.3 ~30mL For each experimental sample, 300μL 50% Glutathione sepharose stock30 bead slurry was centrifuged for 1 minute at 3000rpm 4°C, the supernatant removed and the beads resuspended in 5mL cold PBS56. This wash was repeated four times then the 150μL beads were resuspended in 150μL PBS56 to produce a 50% Glutathione sepharose slurry which was used immediately. 32. GST Elution Buffer Reduced Glutathione 0.062g 1M Tris108 pH8 1mL 50mM ZnCl2
115 25µL ddH2O 9mL Reagents were combined and the solution used immediately. 33. Glycerol/PIPES Buffer 1M CaCl2
7 12mL 0.5M PIPES pH7.059 4mL Glycerol 30mL ddH2O 138mL Reagents were combined and the solution sterilised using a 0.2μM filter then stored at 4°C. 34. 10mg/mL Hœchst 33258 Hœchst 33258 25mg DMSO 2.5mL Hœchst 33258 was dissolved in DMSO, the solution divided into 50μL aliquots and stored at -20°C.
35. 100mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) IPTG 1.2g ddH2O 50mL IPTG was dissolved in ~40mL ddH2O and the final volume adjusted to 50mL, the solution was sterilised using a 0.2μM filter, divided into 1mL aliquots and stored at -20°C. 36. Kanamycin (100 mg/mL) Kanamycin Powder 100mg ddH2O 1mL Kanamycin powder was dissolved in ddH2O and the solution was stored at -20°C. 37. 10mM Lactacystin Lactacystin 2mg ddH2O 531μL Lactacystin was dissolved in ddH2O, the solution was divided into 50μL aliquots and stored at -20°C. The solution was diluted 1:1000 to obtain a working concentration of 10µM in culture medium. 38. LB Agar LB Broth43 500mL Agar Bacteriologica 7.5g Agar was dissolved in the LB broth43, the solution was autoclaved then stored at room temperature. Prior to use, agar was liquefied in a microwave.
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39. LB Agar/Ampicillin LB agar38 was liquefied in a microwave, 15mL per petri dish was poured into a sterile 50mL tube, the solution was cooled to ~60°C, 1µL per mL of 100mg/mL ampicillin5 was added (final concentration 100µg/mL ampicillin), the solution was inverted to mix, poured into petri dishes and allowed to set at room temperature. 40. LB Agar/Ampicillin/IPTG/X-gal LB Agar/Ampicillin plates39 were spread with 100μL 100mM IPTG35 and 20μL 50mg/mL X-Gal113 then allowed to dry for 30 minutes at 37°C before use. 41. LB Agar/Kanamycin LB agar38 was liquefied in a microwave, 15mL per petri dish was poured into a sterile 50mL tube, the solution was cooled to ~60°C, 1µL per mL of 100mg/mL kanamycin36 was added (final concentration 100µg/mL kanamycin), the solution was inverted to mix, poured into petri dishes and allowed to set at room temperature. 42. LB Agar/Kanamycin/IPTG/X-gal LB Agar/Kanamycin plates41 were spread with 100μL 100mM IPTG35 and 20μL 50mg/mL X-Gal113 then allowed to dry for 30 minutes at 37°C before use.
43. Luria-Bertani (LB) Broth Tryptone 10g Yeast Extract 5g Sodium Chloride 10g Reagents were dissolved in ~800mL ddH2O, the pH was adjusted to 7.0, the solution was made up to 1L with ddH2O, autoclaved and stored at room temperature. 44. LB Broth/Ampicillin For a final concentration of 100µg/mL ampicillin, 1µL 100mg/mL ampicillin5 was added per mL LB Broth43 immediately prior to use. 45. LB Broth/10% Glycerol LB Broth43 9mL Glycerol 1mL Glycerol was mixed with LB broth43 and the solution was stored at -20°C. 46. LB Broth/Kanamycin For a final concentration of 100µg/mL kanamycin, 1µL of 100mg/mL kanamycin36 was added per 1mL of LB Broth43. 47. Lysis Buffer (Miltenyi) for Immunoprecipitation Lysis Buffer 4850µL 200mM PMSF 60 25µL 40x Protease Inhibitor Cocktail63
125µL Reagents were combined and used immediately at 4°C.
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48. Lysozyme (10mg/mL) Lysozyme 100mg 1M Tris108 pH8.0 2mL ddH2O 8mL Reagents were combined, the solution was sterilised using a 0.2µM filter then stored at -20ºC in 1mL aliquots. 49. 1M Magnesium Chloride (MgCl2) MgCl2 203g ddH2O 1L MgCl2 was dissolved in ddH2O, the solution was autoclaved then stored at room temperature. 50. 10mM MG132 MG132 4.756mg Ethanol 1mL MG132 was dissolved in ethanol and the solution stored at -20°C in 100µL aliquots. The solution was diluted 1:1000 in culture medium to obtain a concentration of 10µM. 51. 20x MOPS Buffer (Gradient Polyacrylamide Gels) MOPS 104.6g Tris Base 60.6g SDS 10g EDTA 3g ddH2O 500mL Reagents were dissolved in 400mL ddH2O, the volume made up to 500mL and the solution stored at 4°C. 20X MOPS buffer was diluted to 1X with ddH2O and used as required.
52. Mounting Medium Tris-PO4 buffer110 5mL ddH2O 75mL Polyvinylalcohol 20g Glycerol 30mL Chlorobutanol 100mg 1% Phenol Red 2-3 drops Tris-PO4 buffer and ddH2O were combined in an Erhlemeyer flask, PVA and 2-3 drops Phenol red were added and the flask placed in a 60ºC waterbath and shaken intermittently to dissolve. Glycerol was slowly added, then chlorobutanol. The pH was adjusted to 8.2 with Tris-PO4 buffer and the mounting medium stored at 4º. 53. NETN Buffer NP40 2.5mL 4M NaCl81 12.5mL 1M Tris108 pH8.0 10mL 0.5M EDTA27 pH8.0 1mL Reagents were combined and the solution stored at 4°C. For the production of GST-RING domains, 0.5M EDTA27 pH8.0 was omitted from the NETN buffer. 54. 5X PCR Buffer Taq 10X PCR Buffer 5mL (without MgCl2) 25mM dNTP23 200µL ddH2O 4.8mL Reagents were combined and the solution stored at -20°C in 1mL aliquots.
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55. 300μM Phalloidin Stock TRITC-Phalloidin 0.1mg DMSO 255μL Phalloidin was dissolved in DMSO and stored in 20μL aliquots at -20°C. 56. Phosphate Buffered Saline (PBS) pH7.3, pH7.4 Sodium Chloride 8g Potassium Chloride 0.2g Disodium Hydrogen Orthophosphate 1.44g Potassium Dihydrogen Orthophosphate 0.44g Reagents were dissolved in ~800mL ddH2O, the pH was adjusted using 10M HCl, the solution was made up to 1L using ddH2O, autoclaved, and stored at room temperature. 57. PBS/1% BSA BSA Fraction V 100mg PBS56 pH7.4 10mL Reagents were combined and the solution was stored at 4˚C. 58. Phosphate Buffered Saline (PBS) pH7.3/0.02% Sodium Azide PBS56 pH7.3 19.96mL 10% Sodium Azide80 40μL Reagents were combined and the solution stored at 4°C.
59. 0.5M PIPES PIPES 15.15g ddH2O 100mL PIPES was dissolved in ~80mL ddH2O, the pH adjusted to 7.0 using 1M NaOH and the volume made up to 100mL with ddH2O. The solution was sterilised by passing through a 0.2μM filter and stored at room temperature. 60. 200mM PMSF PMSF 348.4mg Isopropanol 10mL Reagents were combined in a fumehood and the solution was stored at room temperature. 61. 1Kb PlusTM Molecular Weight Marker (DNA Ladder) 1Kb Plus Molecular Weight Marker 250µL 6X DNA Loading Dye22 833.3µL The reagents were combined, the volume adjusted to 5mL using Storage Buffer98 and the solution was stored in 1mL aliquots at -20º. 62. 5M Potassium Acetate Potassium Acetate 24.5g ddH2O 50mL Potassium acetate was dissolved in ~40mL ddH2O, the solution was made up to 50mL with ddH2O, autoclaved then stored at room temperature.
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63. 40X Protease Inhibitor Cocktail Protease Inhibitor Cocktail Tablets 4 ddH2O 1mL Tablets were dissolved in ddH2O, the solution was divided into aliquots and stored at 4°C. 64. 50% Protein A Sepharose Stock Protein A Sepharose 1 bottle 50mM Tris107 pH 7.0 500mL 50mM Tris pH7.0 /0.02% Sodium Azide109 ~20mL
Protein A sepharose beads were transferred into a 50mL tube and allowed to settle at 4°C. The supernatant was discarded and the beads resuspended in 50mL cold 50mM Tris107 pH7.0 and stored at 4°C to allow the beads to settle again. This procedure was carried out five times. After the last wash, the beads were allowed to settle at 4°C, the supernatant removed, the beads resuspended in an equal volume of 50mM Tris pH7.0/0.02% sodium azide109 and stored at 4°C.
65. 50% Protein A Sepharose 50% Protein A Sepharose Stock64
300μL 50mM Tris107 pH7.0 ~10mL For each experimental sample, 300μL 50% Protein A sepharose stock64 bead slurry was centrifuged for 1 minute at 3000rpm/4°C, the supernatant removed and the beads resuspended in 1mL cold 50mM Tris107 pH7.0. The wash was repeated four times then the 150μL beads were resuspended in 150μL PBS56 pH7.4 to produce 300μL of a 50% Protein A sepharose slurry, which was divided into 100μL aliquots, stored at 4°C and used within 2 days. 66. 50% Protein G Sepharose Stock Protein G Sepharose 4 Fast Flow 1 bottle 20mM Sodium Phosphate90 pH 7.0 500mL 20mM Sodium Phosphate pH7.0 /0.02% Sodium Azide91 ~20mL
Protein G sepharose beads were transferred into a 50mL tube and allowed to settle at 4°C. The supernatant was discarded and the beads resuspended in 50mL cold 20mM sodium phosphate90 pH7.0 and stored at 4°C to allow the beads to settle again. This procedure was carried out five times. After the last wash the beads were allowed to settle at 4°C, the supernatant removed and the beads resuspended in an equal volume of 20mM sodium phosphate pH7.0/0.02% sodium azide91 and stored at 4°C.
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67. 50% Protein G Sepharose 50% Protein G Sepharose Stock66
300μL 50mM Tris107 pH7.0 ~10mL For each experimental sample, 300μL 50% Protein G sepharose stock66 bead slurry was centrifuged for 1 minute at 3000rpm/4°C, the supernatant removed and the beads resuspended in 1mL cold 50mM Tris107 pH7.0. The wash was repeated four times then the 150μL beads were resuspended in 150μL PBS56 pH7.4 to produce 300μL of a 50% Protein G sepharose slurry, which was divided into 100μL aliquots, stored at 4°C and used within 2 days. 68. 100U/mL Pyrophosphatase Pyrophosphatase 96.8 units 20mM Tris107 pH7.5 968μL Pyrophosphatase was dissolved in 20mM Tris107 pH7.5, the solution divided into 100μL aliquots and stored at -20°C.
69. RIPA Buffer 1% Sodium Deoxycholate87 625µL 1M Tris108 pH7.4 125µL NP40 25µL 200mM Sodium Orthovanadate88 12.5µL 0.5M EDTA28 pH8.0 5µL 0.5M Sodium Fluoride85 5µL 200mM PMSF60 12.5µL 40X Protease Inhibitor Cocktail63 50µL 4M NaCl81 93.8μL ddH2O 1.6 mL Reagents were combined, with PMSF60 and 40x protease inhibitor cocktail63 added last, and the solution was used immediately. 70. RNase A (10mg/mL) RNase A 100mg 1M Tris108 pH 7.4 100µL 4M Sodium Chloride81 37.5µl Reagents were combined, the volume was adjusted to 10mL using ddH2O, heated to 100ºC for 15 minutes and allowed to cool to room temperature. The solution was then stored in 1mL aliquots at -20ºC. 71. RPMI 1640 Media RPMI 1640 1 sachet Sodium Hydrogen Carbonate 2g ddH2O 1L RPMI was dissolved in ddH2O, sodium hydrogen carbonate was added and the solution was filtered using a 0.2µm filter into 1L sterile bottles, then stored at 4°C.
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72. RPMI 1640/PS RPMI 164071 990mL PS 10mL Reagents were combined and stored at 4°C. 73. RPMI 1640/PS/5%CSS Charcoal Stripped Serum 10mL RPMI1640/PS72 190mL Reagents were combined and the solution stored at 4°C. 74. RPMI 1640/PS/10% FCS RPMI 1640/PS72 450mL Foetal Calf Serum 50mL Reagents were combined and the solution was stored at 4°C. 75. RPMI 1640/PS/10% FCS/10% DMSO RPMI 1640/PS/10% FCS74 9mL DMSO 1mL
DMSO was dissolved in RPMI 1640/ PS/10%FCS74 and the solution was used immediately. 76. 10X Running Buffer Tris 30g Glycine 144g SDS 10g ddH2O 1L Reagents were dissolved in ddH2O, the volume adjusted to 1L and the solution was stored at room temperature. 10X Running Buffer was diluted to 1X with ddH2O and used as required.
77. 2X and 10X SDS-PAGE Loading Buffer 2X 10X Glycerol 1mL 12.5mL 1M Tris108 pH6.8
2.5mL 2.5mL
20% SDS84 2mL 2.5mL 2-Mercaptoethanol
200µL 2.5mL
Bromophenol Blue
0.01g 0.25g
ddH2O To 10mL
To 25mL
Reagents were combined, the volume adjusted with ddH2O and the solution was stored at -20ºC in 1mL aliquots. 78. 12% Separating Gel (Western Blotting) 1 M Tris108 pH8.8 1.775mL 40% Acrylamide 3mL (37:5:1) 20% SDS84 37.5µL 10% APS4 37.5µL TEMED 3.75µL ddH2O 2.515mL Reagents were combined, with 10% APS and TEMED added last, the solution was inverted to mix and used immediately. 79. 3M Sodium Acetate (pH4.6) Sodium Acetate 49.2g ddH2O 200mL Sodium acetate was dissolved in 160mL ddH2O, the pH adjusted to 4.6, the volume adjusted to 200mL and the solution autoclaved and stored at room temperature.
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80. 10% Sodium Azide (NaN3) Sodium Azide 0.1g ddH2O 1mL Sodium azide was dissolved in ddH2O and the solution stored at 4°C. 81. 4M Sodium Chloride (NaCl) Sodium Chloride 117.2g ddH2O 500mL Sodium chloride was dissolved in ddH2O, the solution was autoclaved and stored at room temperature. 82. 1% Sodium Deoxycholate Sodium Deoxycholate 1g ddH2O 100mL Sodium deoxycholate was dissolved in ddH2O, the solution was autoclaved and stored at -20°C shielded from light. 83. 1M Sodium Dihydrogen Orthophosphate (NaHPO42H2O) Sodium Dihydrogen Orthophosphate 39g ddH2O 250mL Reagents were combined and the solution was stored at room temperature. 84. 20% Sodium Dodecyl Sulphate (SDS) Sodium Dodecyl Sulphate 10g ddH20 50mL SDS was dissolved in ddH2O and the solution was stored at room temperature.
85. 0.5M Sodium Fluoride (NaF) Sodium Fluoride 2.1g ddH2O 100mL Sodium fluoride was dissolved in ddH2O and the solution was stored at room temperature. 86. Sodium Hydrogen Phosphate (Na2HPO4) Na2HPO4 156.2g ddH2O 1L Na2HPO4 was dissolved in ~800mL ddH2O, the solution was made up to 1L and stored at room temperature. 87. 10M Sodium Hydroxide (NaOH) Sodium Hydroxide 20g ddH2O 50mL Sodium hydroxide was dissolved in ddH2O and the solution was stored at room temperature. 88. 200mM Sodium Orthovanadate Sodium Orthovanadate 3.7g ddH2O 100mL Sodium orthovanadate was dissolved in ~80mL ddH2O, the pH adjusted to 10.0, the solution heated at ~95°C until colourless and the pH readjusted to 10.0. The procedure was repeated until the solution was colourless at pH 10.0, then the solution was made up to 100mL with ddH2O, divided into 1mL aliquots and stored at -20°C.
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89. 0.1M Sodium Phosphate pH7 1M Na2HPO4
86 57.7mL 1M NaHPO42H2O83 42.3mL ddH2O 900mL Reagents were dissolved in ~800mL ddH2O and the pH adjusted to 7. The solution was made up to 1L, autoclaved and stored at room temperature. 90. 20mM Sodium Phosphate pH7 Sodium Phosphate 100mL ddH2O 400mL The reagents were combined, the solution autoclaved then stored at room temperature. 91. 20mM Sodium Phosphate pH7/0.02% Sodium Azide 20mM Sodium Phosphate90 19.96mL 10% Sodium Azide80 40μL Reagents were combined and the solution stored at 4°C. 92. Solution I (Plasmid Preparation) Glucose 0.9g 1M Tris108 pH 8.8 2.5mL 0.5M EDTA28 pH8.0 2.0mL ddH2O 95.5mL Reagents were combined and the solution was autoclaved then stored at 4°C.
93. Solution II (Plasmid Preparation) 20% SDS84 250µL 10M Sodium Hydroxide87 100µL ddH2O 4.65mL Reagents were combined and the solution was stored at room temperature and used on the day of preparation. 94. Solution III (Plasmid Preparation) 5M Potassium Acetate62 60mL Glacial Acetic Acid 11.5mL ddH2O 28.5mL The reagents were combined, the pH adjusted to 8.0 and the solution was autoclaved and stored at 4°C. 95. Sonication Buffer 1M Tris108 pH8.0 25mL 4M NaCl81 6.25mL 0.5M EDTA28 pH 8 1mL ddH2O 467.5mL Reagents were combined and the solution stored at 4°C. 96. 4% Stacking Gel (Western Blotting) 1 M Tris108 pH6.8 1.25mL 40% Acrylamide (37:5:1) 1.3mL 20% SDS84 50µL 10% APS4 50µL TEMED 10µL ddH20 7.4mL Reagents were combined, with 10% APS and TEMED added last, the solution was inverted to mix and used immediately.
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97. Streptavidin-HRP Solution TBS101 5mL Solution A (Vectastain) 2 drops Solution B (Vectastain) 2 drops Solution A and Solution B were added to the TBS101, the solution mixed and allowed to stand for 30 minutes. A working solution was made by diluting 100µL Streptavidin-HRP solution per 3mL buffer in 1% TBST/BSA104 and used on the day of preparation. 98. Storage Buffer (for 1Kb Plus DNA Ladder) 1M Tris108 pH 7.4 50µL 0.5M EDTA28 pH 8.0 10µL 4M Sodium Chloride81 62.5µL Reagents were combined, the volume adjusted to 5mL with ddH2O and the solution was stored at room temperature. 99. 20% Sucrose Sucrose 10g ddH20 50mL Sucrose was dissolved in ~25mL ddH2O, the solution was made up to 50mL with ddH2O and stored at room temperature.
100. 50X Tris Acetate EDTA (TAE) Tris Base 242g Glacial Acetic Acid 57.1mL 0.5M EDTA28 pH8.0 100mL Tris base was dissolved in ddH2O, the glacial acetic acid and EDTA were added, the volume made up to 1L with ddH2O and the solution autoclaved then stored at room temperature. 50X TAE was diluted to 1X with ddH2O as required and stored at room temperature. 101. Tris Buffered Saline (TBS) 1M Tris108 pH 7.4 50mL 4M Sodium Chloride81 37.5mL Tris and sodium chloride were combined, the volume adjusted to 1L with ddH2O and the solution was stored at room temperature. 102. TBS Tween (TBST) TBS101 500mL Tween-20 1mL The reagents were combined, mixed by inversion and the solution was stored at room temperature. 103. TBST/1%, 3% and 5% Blotto
Skim milk powder was dissolved in TBST102. The solution was made on the day of use and stored at room temperature.
1% Blotto
2% Blotto
5% Blotto
Skim Milk Powder
0.3g 0.9g 1.5g
TBST102 30mL 30mL 30mL
Appendix I Buffers and Solutions
364
104. TBST/1% BSA Bovine Serum Albumin 0.1g TBST102 10mL BSA was dissolved in TBST102 and the solution used immediately. 105. Transfer Buffer (Western Blotting) Glycine 14.4g Tris 3.03g Methanol 200mL ddH2O 800mL Glycine and Tris were dissolved in ~500mL ddH2O, the volume made up to 800mL, 200mL methanol was added and the solution was stored at -20°C for >1 hour prior to use. Solution was used on the day of preparation. 106. 0.5M Tris pH7.0 Tris Base 60.55g ddH2O 1L Tris was dissolved in ~800mL ddH2O, the pH was adjusted using 10M HCl to 7.0 and the solution was made up to 1L with ddH2O. The solution was autoclaved and stored at room temperature. 107. 20mM and 50mM Tris pH7.0 20mM 50mM
0.5M Tris106 pH7.0
40mL 100mL
ddH2O 1L 1L 0.5M Tris was diluted to ~800mL with ddH2O, the pH adjusted to 7.0 using 10M HCl, the solution made up to 1L, autoclaved and stored at room temperature.
108. 1M Tris (pH6.8, 7.5, 7.5, 8.0, 8.8) Tris Base 121.1g ddH2O 1L Tris was dissolved in ~800mL ddH2O, the pH was adjusted using 10M HCl and the solution was made up to 1L with ddH2O. The solution was autoclaved and stored at room temperature. 109. 50mM Tris pH7/0.02% Sodium Azide 50mM Tris pH7.0107 19.96mL 10% Sodium Azide80 40μL Reagents were combined and the solution stored at 4°C. 110. Tris-PO4 pH9.0 Tris Base 121.9g ddH2O 1L Tris was dissolved in ~800mL ddH2O, the pH adjusted to 9 with 1M NaH2PO4
86 and the volume made up to 1L with ddH2O. The solution was autoclaved and stored at room temperature. 111. 0.1%/10% (v/v) Triton X-100 0.1% Triton
X-100 10% Triton
X-100 Triton X-100
50μL 5mL
PBS56 pH7.4
49.95mL 45mL
Reagents were combined and the solution was stored at room temperature shielded from light.
Appendix I Buffers and Solutions
365
112. Whole Cell Lysis Buffer 20% Sucrose99 2.5mL 20% SDS84 0.5mL 1M Tris108 pH6.8 0.25mL 2-Mercaptoethanol 0.25mL ddH2O 1.5mL Reagents were combined, the solution inverted to mix and stored in a fumehood protected from light for up to 4 weeks. 113. 50mg/mL X-gal X-gal 0.5g Dimethylformamide 10mL X-gal was dissolved in dimethylformamide, the solution aliquoted and stored at -20°C. 114. 1M Zinc Chloride (ZnCl2) ZnCl2 6.815g ddH2O 50mL Reagents were combined and the ZnCl2 dissolved by heating. The solution was stored at room temperature. 115. 50mM ZnCl2 1M ZnCl2
114 5mL ddH2O 95mL Reagents were combined and the solution stored at room temperature.
Appendix II Primer Sequences
Appendix II: Primer Sequences
Appendix II Primer Sequences
366
Sequencing Primers pEGFP1266-S 5’ CAT GGT CCT GCT GGA GTT CGT G 3’ M13-S 5’ GTT TTC CCA GTC ACG AC 3’ M13-AS 5’ CAG GAA ACA GCT ATG AC 3’ pGEX-S 5’ GGG CTGGCA AGC CAC GTT TGG TG 3’ pGEX-AS 5’ CCG GGA GCT GCA TGT GTC AGA GG 3’ RMND5BSalI1-S 5’ ATG GGA TCC ATG GAG CAG TGT 3’ RMND5BSalI1182-AS 5’ATG GGA TCC TCA GAA TAT GAT GCG 3’ RMND5B790-S 5’ ATC TGT GAG ACC TTT ACC CGG 3’ RMND5A603-S 5’ CTT GTT AAT GGG TGG AAC CA 3’ RMND5A490-AS 5’CTC CAC CAT CAC TTC ATT GA 3’ RanBPM1259-AS 5’ CCC ATT CTT CCT GCT AAT AC 3’ RanBPM687-S 5’ AAG CTT TAA TGG GAA TTG GTC TTT CTG 3’ Cloning Primers RMND5BBamHI1-S 5’ATG GGA TCC ATG GAG CAG TGT 3’ RMND5BBamHI1182-AS 5’ ATG GGA TCC TCA GAA TAT GAT GCG 3' RMND5A1-S 5’ GAA TTC ATG GAT CAG TGC GTG ACG 3’ RMND5ABamHI1-S 5’ GGA TCC ATG GAT CAG TGC GTG ACG 3’ RMND5A1176-AS 5’ GAA TTC TCA GAA AAA TAT CTG TTT GGC 3’ RanBPM1-S 5’ AAG CTT TAA TGT CCG GGC AGC CGC 3’ RanBPM1029-S 5’ GGA GTG GAG AAC CAA AAT CC 3’ RanBPM2190-AS 5’ GTC GAC CTA ATG TAG GTA GTC TTC CAC 3’
Appendix II Primer Sequences
367
RMND5ARING1006-S 5’ GGA TCC TGC CCC ATT CTT CGT CAG 3’ RMND5ARING1131-AS 5’ GAA TTC CAC AGT AGG GAC ATT TTA ATT 3’ RMND5BRING1012-S 5’ GGA TCC TGC CCC ATC CTC CGC CAG 3’ RMND5BRING1137-AS 5’ GAA TTC CAC AGT AGG GAC ACT TCA GC 3’ CBLRING1141-S 5’ GGA TCC TGT AAA ATA TGT GCT GAA AAT GA 3’ CBLRING1257-AS 5’ GAA TTC CGC AGA AAG GAC AGC CCT G 3’ Gene Expression Primers βActin146-S 5’ AGA AGG ATT CCT ATG TGG GCG ACG A 3’ βActin464-AS 5’ CGA GTC CAT CAC GAT GCC AGT GGT A 3’ Twa1335-S 5’ TGA TCC GCC AGC GGG AGA CA 3’ Twa1681-AS 5’ GGG CTC CTC AAT CAC ACC CTT GC 3’ EMP580-S 5’ AGC TGC CTG GAG TTC AGC CTC A 3’ EMP1083-AS 5’ GAC GTA GCC GTT GGG CAG CA 3’ ARMC81103-S 5’ AGG TGC GGT TAG CTG CCG TC 3’ ARMC81668-AS 5’ TGT CCC ATC CGC TAT GTT GGC T 3’ Muskelin1675-S 5’ TGT CCA AGG TTT GCC CAT CAG C 3’ Muskelin2120-AS 5’ ACC AGG TTG CCT TTA GGA GGA GT 3’ C17orf39298-S 5’ TCC GCG GCC TCA CTC ATC CC 3’ C17orf39814-AS 5’ AGC CCT CTA TGG AGG CTG CTG A 3’ RanBPM1550-S 5’ TAATATCAAATAAAGCACATCAAT CAT 3’ RanBPM2190-AS 5’ GTC GAC CTA ATG TAG GTA GTC TTC CAC 3’
Appendix II Primer Sequences
368
RMND5B790-S 5’ ATC TGT GAG ACC TTT ACC CGG 3’ RMND5BTOPO1182-AS 5’ GAA TAT GAT GCG TTT CCC ATC TGC 3’ RMND5A603-S 5’ CTT GTT AAT GGG TGG AAC CA 3’ RMND5A1176-AS 5’ GAA TTC TCA GAA AAA TAT CTG TTT GGC 3’ Site Directed Mutagenesis Primers RMND5A(C356S)1045-S 5’ CCC ATG AAA TTG GTC TCTG GTCA TAT TAT ATC AAG AGA TGC C 3’ RMND5A(C356S)1083-AS 5’ ATC TCT TGA TAT AAT ATG ACC AGA GAC CAA TTT CAT GGG TGG 3’ Primer Set 1 RMND5A(C356A)1045-S 5’ CCC ATG AAA TTG GTC GCT GGT CAT ATT ATA TCA AGA 3’ RMND5A(C356A)1083-AS 5’ ATC TCT TGA TAT AAT ATG ACC AGC GAC CAA TTT CAT GGG TGG 3’ Primer Set 2 RMND5A(C356A/H358A)1045-S 5’ CCC ATG AAA TTG GTC GCT GGT GCT ATT ATA TCA AGA GAT GCC 3’ RMND5A(C356A/H358A)1083-AS 5’ ATC TCT TGA TAT AAT AGC ACC AGC GAC CAA TTT CAT GGG TGG 3’ Primer Set 1 RMND5B(C358S)1051-S 5’ CCC ATC AAG CTC ATC TCT GGC CAT GTT ATC TCC CGA GAT GCA 3’ RMND5B(C358S)1089-AS 5’ ATC TCG GGA GAT AAC ATG GCC AGA GAT GAG CTT GAT GGG AGG 3’ Primer Set 2 RMND5B(C358S)1057-S 5’ AAG CTC ATC TCT GGC CAT GTT ATC TCC 3’ RMND5B(C358S)1080-AS 5’ GAT AAC ATG GCC AGA GAT GAG CTT GAT GGG 3’ RMND5B(C358A/H360S)1054-S 5’ATC AAG CTC ATC GCT GGC GCT GTT ATC TCC CGA 3’ RMND5B(C358A/H360A)1083-AS 5’ GGA GAT AAC AGC GCC AGC GAT GAG CTT GAT GGG AGG 3’
Appendix III Sequencing
Appendix III: Sequencing
Appendix III Sequences
369
pGEX-RMND5A – Representative Sequence >gi|15082505|gb|BC012165.1| Homo sapiens required for meiotic nuclear division 5 homolog A (S. cerevisiae), mRNA (cDNA clone MGC:20406 IMAGE:4636136), complete cds Length=3239 GENE ID: 64795 RMND5A | required for meiotic nuclear division 5 homolog A (S. cerevisiae) [Homo sapiens] (10 or fewer PubMed links) Score = 1581 bits (1752), Expect = 0.0 Identities = 975/1023 (96%), Gaps = 23/1023 (2%) Strand=Plus/Plus Query 99 CATGGATCAGTGCGTGACGGTGGAGCGCGAGCTGGAGAAGGTGCTGCACAAGTTCTCAGG 158 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 98 CATGGATCAGTGCGTGACGGTGGAGCGCGAGCTGGAGAAGGTGCTGCACAAGTTCTCAGG 157 Query 159 CTACGGGCAGCTGTGCGAGCGCGGCCTGGAGGAGCTCATCGACTACACCGGCGGCCTCAA 218 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 158 CTACGGGCAGCTGTGCGAGCGCGGCCTGGAGGAGCTCATCGACTACACCGGCGGCCTCAA 217 Query 219 GCACGAGATCCTGCAGAGCCACGGCCAAGATGCTGAATTATCAGGGACACTTTCACTTGT 278 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 218 GCACGAGATCCTGCAGAGCCACGGCCAAGATGCTGAATTATCAGGGACACTTTCACTTGT 277 Query 279 TTTGACACAGTGCTGTAAAAGAATAAAGGATACTGTTCAAAAATTGGCCTCCGACCACAA 338 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 278 TTTGACACAGTGCTGTAAAAGAATAAAGGATACTGTTCAAAAATTGGCCTCCGACCACAA 337 Query 339 AGACATCCACAGCAGTGTTTCTCGGGTTGGAAAAGCCATTGATAAGAATTTTGATTCTGA 398 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 338 AGACATCCACAGCAGTGTTTCTCGGGTTGGAAAAGCCATTGATAAGAATTTTGATTCTGA 397 Query 399 CATTAGCAGTGTGGGAATAGATGGCTGCTGGCAGGCAGACAGCCAAAGGCTTCTCAATGA 458 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 398 CATTAGCAGTGTGGGAATAGATGGCTGCTGGCAGGCAGACAGCCAAAGGCTTCTCAATGA 457 Query 459 AGTGATGGTGGAGCACTTCTTTCGACAAGGAATGCTGGATGTGGCTGAGGAGCTCTGTCA 518 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 458 AGTGATGGTGGAGCACTTCTTTCGACAAGGAATGCTGGATGTGGCTGAGGAGCTCTGTCA 517
Appendix III Sequences
370
Appendix III Sequences
371
pGEX-RMND5A wild type RING domain
RefSeq -----------------------------------------TGCCCCATTCTTCGTCAGC RING5A CTCCCGGCCGCCATGGCGGCCGCGGGAATTCGATTGGATCCTGCCCCATTCTTCGTCAGC ******************* RefSeq AAACAACAGATAACAATCCACCCATGAAATTGGTCTGTGGTCATATTATATCAAGAGATG RING5A AAACAACAGATAACAATCCACCCATGAAATTGGTCTGTGGTCATATTATATCAAGAGATG ************************************************************ RefSeq CCCTGAATAAAATGTTTAATGGTAGCAAATTAAAATGTCCCTACTGT------------- RING5A CCCTGAATAAAATGTTTAATGGTAGCAAATTAAAATGTCCCTACTGTGGAATTCAATCAC *********************************************** RefSeq = RMND5A RING domain reference sequence
RING5A = pGEX-RMND5A RING domain (Wild-type)
Appendix III Sequences
372
pGEX-RMND5B wild type RING domain
RefSeq ---------------------------------------TGCCCCATCCTCCGCCAGCAG RING5B CCCGGCCGCCATGGCGGCCGCGGGAATTCGATTGGATCCTGCCCCATCCTCCGCCAGCAG ********************* RefSeq ACGTCAGATTCCAACCCTCCCATCAAGCTCATCTGTGGCCATGTTATCTCCCGAGATGCA RING5B ACGTCAGATTCCAACCCTCCCATCAAGCTCATCTGTGGCCATGTTATCTCCCGAGATGCA ************************************************************ RefSeq CTCAATAAGCTCATTAATGGAGGAAAGCTGAAGTGTCCCTACTGT--------------- RING5B CTCAATAAGCTCATTAATGGAGGAAAGCTGAAGTGTCCCTACTGTGGAATTCAATCACTA *********************************************
RefSeq = RMND5B RING domain reference sequence
RING5B = pGEX-RMND5B RING domain (Wild-type)
Appendix III Sequences
373
pGEX-CBL RING domain
RefSeq --------------------------------------TGTAAAATATGTGCTGAAAATG CBL CCGGCCGCCATGGCGGCCGCGGGAATTCGATTGGATCCTGTAAAATATGTGCTGAAAATG ********************** RefSeq ATAAGGATGTAAAGATTGAGCCCTGTGGACACCTCATGTGCACATCCTGTCTTACATCCT CBL ATAAGGATGTAAAGATTGAGCCCTGTGGACACCTCATGTGCACATCCTGTCTTACATCCT ************************************************************ RefSeq GGCAGGAATCAGAAGGTCAGGGCTGTCCTTTCTGC------------------------- CBL GGCAGGAATCAGAAGGTCAGGGCTGTCCTTTCTGCGGAATTCAATCACTAGTGAATTCGC ***********************************
RefSeq = CBL RING domain reference sequence
CBL = pGEX-CBL RING domain
Appendix III Sequences
374
pEGFP-RMND5A (C356S) RING domain
RING5A TGCCCCATTCTTCGTCAGCAAACAACAGATAACAATCCACCCATGAAATTGGT ||||||||||||||||||||||||||||||||||||||||||||||||||||| RefSeq TGCCCCATTCTTCGTCAGCAAACAACAGATAACAATCCACCCATGAAATTGGT RING5A CTCTGGTCATATTATATCAAGAGATGCCCTGAATAAAATGTTTAATGGTAGCAAATTAAA || ||||||||||||||||||||||||||||||||||||||||||||||||||||||||| RefSeq CTGTGGTCATATTATATCAAGAGATGCCCTGAATAAAATGTTTAATGGTAGCAAATTAAA RING5A ATGTCCCTACTGT ||||||||||||| RefSeq ATGTCCCTACTGT
RefSeq = RMND5A RING domain reference sequence
RING5A = pEGFP-RMND5A RING (C356S) mutant
G1061C (C356S) (Base change marked in red)
Single base change at position 425 C (*) on chromatogram
*
Appendix III Sequences
375
pEGFP-RMND5B (C358S) RING domain
RING5B TGCCCCATCCTCCGCCAGCAGACGTCAGATTCCAACCCTCCCATCAAGCTCATCTCTGGCC ||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||| RefSeq TGCCCCATCCTCCGCCAGCAGACGTCAGATTCCAACCCTCCCATCAAGCTCATCTGTGGCC RING5B ATGTTATCTCCCGAGATGCACTCAATAAGCTCATTAATGGAGGAAAGCTGAAGTGTCCCT |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| RefSeq ATGTTATCTCCCGAGATGCACTCAATAAGCTCATTAATGGAGGAAAGCTGAAGTGTCCCT RING5B ACTGT ||||| RefSeq ACTGT
RefSeq = RMND5B RING domain reference sequence
RING5B = pEGFP-RMND5B RING (C358S) mutant
G1067C (C358S) (Base change marked in red)
Single base change at position 251 C (*) on chromatogram
*
Appendix III Sequences
376
pEGFP-RMND5A (C356A/H358A) RING domain
RING5A TGCCCCATTCTTCGTCAGCAAACAACAGATAACAATCCACCCATGAAATTGGTC |||||||||||||||||||||||||||||||||||||||||||||||||||||| RefSeq TGCCCCATTCTTCGTCAGCAAACAACAGATAACAATCCACCCATGAAATTGGTC RING5A GCTGGTGCTATTATATCAAGAGATGCCCTGAATAAAATGTTTAATGGTAGCAAATTAA |||| |||||||||||||||||||||||||||||||||||||||||||||||||| RefSeq TGTGGTCATATTATATCAAGAGATGCCCTGAATAAAATGTTTAATGGTAGCAAATTAA RING5A AATGTCCCTACTGT |||||||||||||| RefSeq AATGTCCCTACTGT
RefSeq = RMND5A RING domain reference sequence
RING5A = pEGFP-RMND5A RING (C356A/H358A) mutant
T1060G/G1061C, C1066G/A1067C (C356A/H358A) (Base changes marked in red)
Base changes at positions 434 G, 435 C (*) and 440 G, 441 C (#) on chromatogram
* #
Appendix III Sequences
377
pEGFP-RMND5B (C358A/H360A) RING domain
RING5B TGCCCCATCCTCCGCCAGCAGACGTCAGATTCCAACCCTCCCATCAAGCTCATCGCTGGC |||||||||||||||||||||||||||||||||||||||||||||||||||||| |||| RefSeq TGCCCCATCCTCCGCCAGCAGACGTCAGATTCCAACCCTCCCATCAAGCTCATCTGTGGC RING5B GCTGTTATCTCCCGAGATGCACTCAATAAGCTCATTAATGGAGGAAAGCTGAAGTGTCCC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||| RefSeq CATGTTATCTCCCGAGATGCACTCAATAAGCTCATTAATGGAGGAAAGCTGAAGTGTCCC RING5B TACTGT |||||| RefSeq TACTGT
RefSeq = RMND5B RING domain reference sequence
RING5B = pEGFP-RMND5B RING (C358A/H360A) mutant
T1066G/G1067C, C1072G/A1073C (C358A/H360A) (Base changes marked in red)
Base changes at positions 161 G, 162 C (*) and 167 G, 168 C (#) on chromatogram
* #
Appendix III Sequences
378
pmCherry-RanBPM (55kDa) - Representative Sequence >gi|39812377|ref|NM_005493.2| Homo sapiens RAN binding protein 9 (RANBP9), mRNA Length=3132 GENE ID: 10048 RANBP9 | RAN binding protein 9 [Homo sapiens] (Over 10 PubMed links) Score = 1842 bits (2042), Expect = 0.0 Identities = 1088/1126 (96%), Gaps = 10/1126 (0%) Strand=Plus/Plus Query 11 AATAGACTACCAGGTTGGGATAAGCATTCATATGGTTACCATGGGGATGATGGACATTCG 70 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 783 AATAGACTACCAGGTTGGGATAAGCATTCATATGGTTACCATGGGGATGATGGACATTCG 842 Query 71 TTTTGTTCTTCTGGAACTGGACAACCTTATGGACCAACTTTCACTACTGGTGATGTCATT 130 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 843 TTTTGTTCTTCTGGAACTGGACAACCTTATGGACCAACTTTCACTACTGGTGATGTCATT 902 Query 131 GGCTGTTGTGTTAATCTTATCAACAATACCTGCTTTTACACCAAGAATGGACATAGTTTA 190 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 903 GGCTGTTGTGTTAATCTTATCAACAATACCTGCTTTTACACCAAGAATGGACATAGTTTA 962 Query 191 GGTATTGCTTTCACTGACCTACCGCCAAATTTGTATCCTACTGTGGGGCTTCAAACACCA 250 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 963 GGTATTGCTTTCACTGACCTACCGCCAAATTTGTATCCTACTGTGGGGCTTCAAACACCA 1022 Query 251 GGAGAAGTGGTCGATGCCAATTTTGGGCAACATCCTTTCGTGTTTGATATAGAAGACTAT 310 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1023 GGAGAAGTGGTCGATGCCAATTTTGGGCAACATCCTTTCGTGTTTGATATAGAAGACTAT 1082 Query 311 ATGCGGGAGTGGAGAACCAAAATCCAGGCACAGATAGATCGATTTCCTATCGGAGATCGA 370 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1083 ATGCGGGAGTGGAGAACCAAAATCCAGGCACAGATAGATCGATTTCCTATCGGAGATCGA 1142
Appendix III Sequences
379
Appendix III Sequences
380
pmCherry-RMND5B - Representative Sequence >gi|95044658|gb|DQ494789.1| Homo sapiens RMND5B mRNA, complete cds Length=1825 GENE ID: 64777 RMND5B | required for meiotic nuclear division 5 homolog B (S. cerevisiae) [Homo sapiens] (10 or fewer PubMed links) Score = 609 bits (674), Expect = 2e-170 Identities = 337/337 (100%), Gaps = 0/337 (0%) Strand=Plus/Plus Query 22 CCCCCTTAGCGTCAGCTTTGCCTCTGGCTGTGTGGCGCTGCCTGTGTTGATGAACATCAA 81 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1026 CCCCCTTAGCGTCAGCTTTGCCTCTGGCTGTGTGGCGCTGCCTGTGTTGATGAACATCAA 1085 Query 82 GGCTGTGATTGAGCAGCGGCAGTGCACTGGGGTCTGGAATCACAAGGACGAGTTACCGAT 141 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1086 GGCTGTGATTGAGCAGCGGCAGTGCACTGGGGTCTGGAATCACAAGGACGAGTTACCGAT 1145 Query 142 TGAGATTGAACTAGGCATGAAGTGCTGGTACCACTCCGTGTTCGCTTGCCCCATCCTCCG 201 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1146 TGAGATTGAACTAGGCATGAAGTGCTGGTACCACTCCGTGTTCGCTTGCCCCATCCTCCG 1205 Query 202 CCAGCAGACGTCAGATTCCAACCCTCCCATCAAGCTCATCTGTGGCCATGTTATCTCCCG 261 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1206 CCAGCAGACGTCAGATTCCAACCCTCCCATCAAGCTCATCTGTGGCCATGTTATCTCCCG 1265 Query 262 AGATGCACTCAATAAGCTCATTAATGGAGGAAAGCTGAAGTGTCCCTACTGTCCCATGGA 321 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1266 AGATGCACTCAATAAGCTCATTAATGGAGGAAAGCTGAAGTGTCCCTACTGTCCCATGGA 1325 Query 322 GCAGAACCCGGCAGATGGGAAACGCATCATATTCTGA 358 ||||||||||||||||||||||||||||||||||||| Sbjct 1326 GCAGAACCCGGCAGATGGGAAACGCATCATATTCTGA 1362
Appendix III Sequences
381
Appendix IV Mass Spectrometry
Appendix IV: Mass Spectrometry
User : Matt F
Email :
Search title : Submitted from PROJ13881 with Decoy database by Mascot Daemon on APAF-WS-08
MS data file : \\Apaf-hpv-file\User_Shared\MFitzhenry\2012 Reports\PROJ13881_JackyBentel(RPerthH)\Band1.mgf
Database : SwissProt 2012x (536029 sequences; 190235160 residues)
Taxonomy : Homo sapiens (human) (20319 sequences)
Timestamp : 8 Jun 2012 at 04:52:21 GMT
Protein hits : HSP7C_HUMAN Heat shock cognate 71 kDa protein OS=Homo sapiens GN=HSPA8 PE=1 SV=1
HSP71_HUMAN Heat shock 70 kDa protein 1A/1B OS=Homo sapiens GN=HSPA1A PE=1 SV=5
HNRPM_HUMAN Heterogeneous nuclear ribonucleoprotein M OS=Homo sapiens GN=HNRNPM PE=1 SV=3
PUF60_HUMAN Poly(U)-binding-splicing factor PUF60 OS=Homo sapiens GN=PUF60 PE=1 SV=1
GRP75_HUMAN Stress-70 protein, mitochondrial OS=Homo sapiens GN=HSPA9 PE=1 SV=2
K2C1_HUMAN
Keratin, type II cytoskeletal 1 OS=Homo sapiens GN=KRT1 PE=1 SV=6
RMD5A_HUMAN Protein RMD5 homolog A OS=Homo sapiens GN=RMND5A PE=1 SV=1
K1C9_HUMAN
Keratin, type I cytoskeletal 9 OS=Homo sapiens GN=KRT9 PE=1 SV=3
K1C10_HUMAN Keratin, type I cytoskeletal 10 OS=Homo sapiens GN=KRT10 PE=1 SV=6
SNW1_HUMAN
SNW domain-containing protein 1 OS=Homo sapiens GN=SNW1 PE=1 SV=1
DDX41_HUMAN Probable ATP-dependent RNA helicase DDX41 OS=Homo sapiens GN=DDX41 PE=1 SV=2
XRCC6_HUMAN X-ray repair cross-complementing protein 6 OS=Homo sapiens GN=XRCC6 PE=1 SV=2
K22E_HUMAN
Keratin, type II cytoskeletal 2 epidermal OS=Homo sapiens GN=KRT2 PE=1 SV=2
K2C6B_HUMAN Keratin, type II cytoskeletal 6B OS=Homo sapiens GN=KRT6B PE=1 SV=5
DDX5_HUMAN
Probable ATP-dependent RNA helicase DDX5 OS=Homo sapiens GN=DDX5 PE=1 SV=1
K2C5_HUMAN
Keratin, type II cytoskeletal 5 OS=Homo sapiens GN=KRT5 PE=1 SV=3
MERL_HUMAN
Merlin OS=Homo sapiens GN=NF2 PE=1 SV=1
RS27A_HUMAN Ubiquitin-40S ribosomal protein S27a OS=Homo sapiens GN=RPS27A PE=1 SV=2
PABP1_HUMAN Polyadenylate-binding protein 1 OS=Homo sapiens GN=PABPC1 PE=1 SV=2
ALBU_HUMAN
Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=2
LMNB1_HUMAN Lamin-B1 OS=Homo sapiens GN=LMNB1 PE=1 SV=2
DHX35_HUMAN Probable ATP-dependent RNA helicase DHX35 OS=Homo sapiens GN=DHX35 PE=1 SV=2
KHDR1_HUMAN KH domain-containing, RNA-binding, signal transduction-associated protein 1 OS=Homo
sapiens GN=KHDRBS1 PE=1 SV=1
PCKGM_HUMAN Phosphoenolpyruvate carboxykinase [GTP], mitochondrial OS=Homo sapiens GN=PCK2 PE=1 SV=3
HNRPQ_HUMAN Heterogeneous nuclear ribonucleoprotein Q OS=Homo sapiens GN=SYNCRIP PE=1 SV=2
MYL10_HUMAN Myosin regulatory light chain 10 OS=Homo sapiens GN=MYL10 PE=2 SV=2
ODP2_HUMAN
Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, mitochondrial OS=Homo
CN16B_HUMAN Uncharacterized protein C14orf166B OS=Homo sapiens GN=C14orf166B PE=2 SV=2
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data
/201
2060
8/F0
7733
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8/06
/201
2 3:
00 P
M
User
Search title
MS data file
Database
Taxonomy
Timestamp
Protein hits
: Matt F
: : Submitted from PROJ13881 with Decoy database by Mascot Daemon on APAF-WS-08
: \\Apaf-hpv-file\User_Shared\MFitzhenry\2012 Reports\PROJ13881_JackyBentel(RPerthH)\Band-all.mgf
: SwissProt 2012x (536029 sequences; 190235160 residues)
: Homo sapiens (human) (20319 sequences)
: 8 Jun 2012 at 04:50:59 GMT
:PUF60_HUMAN Poly(U)-binding-splicing factor PUF60 OS=Homo sapiens GN=PUF60 PE=1 SV=1
K2C1_HUMAN
Keratin, type II cytoskeletal 1 OS=Homo sapiens GN=KRT1 PE=1 SV=6
K1C9_HUMAN
Keratin, type I cytoskeletal 9 OS=Homo sapiens GN=KRT9 PE=1 SV=3
ACTB_HUMAN
Actin, cytoplasmic 1 OS=Homo sapiens GN=ACTB PE=1 SV=1
HSP7C_HUMAN Heat shock cognate 71 kDa protein OS=Homo sapiens GN=HSPA8 PE=1 SV=1
TBB5_HUMAN
Tubulin beta chain OS=Homo sapiens GN=TUBB PE=1 SV=2
K1C10_HUMAN Keratin, type I cytoskeletal 10 OS=Homo sapiens GN=KRT10 PE=1 SV=6
TBB4B_HUMAN Tubulin beta-4B chain OS=Homo sapiens GN=TUBB4B PE=1 SV=1
GRP78_HUMAN 78 kDa glucose-regulated protein OS=Homo sapiens GN=HSPA5 PE=1 SV=2
HNRPC_HUMAN Heterogeneous nuclear ribonucleoproteins C1/C2 OS=Homo sapiens GN=HNRNPC PE=1 SV=4
PABP1_HUMAN Polyadenylate-binding protein 1 OS=Homo sapiens GN=PABPC1 PE=1 SV=2
K22E_HUMAN
Keratin, type II cytoskeletal 2 epidermal OS=Homo sapiens GN=KRT2 PE=1 SV=2
IF4A3_HUMAN Eukaryotic initiation factor 4A-III OS=Homo sapiens GN=EIF4A3 PE=1 SV=4
ATPB_HUMAN
ATP synthase subunit beta, mitochondrial OS=Homo sapiens GN=ATP5B PE=1 SV=3
HSP71_HUMAN Heat shock 70 kDa protein 1A/1B OS=Homo sapiens GN=HSPA1A PE=1 SV=5
K1C14_HUMAN Keratin, type I cytoskeletal 14 OS=Homo sapiens GN=KRT14 PE=1 SV=4
DDX41_HUMAN Probable ATP-dependent RNA helicase DDX41 OS=Homo sapiens GN=DDX41 PE=1 SV=2
K2C6B_HUMAN Keratin, type II cytoskeletal 6B OS=Homo sapiens GN=KRT6B PE=1 SV=5
HS71L_HUMAN Heat shock 70 kDa protein 1-like OS=Homo sapiens GN=HSPA1L PE=1 SV=2
POTEE_HUMAN POTE ankyrin domain family member E OS=Homo sapiens GN=POTEE PE=1 SV=3
K1C16_HUMAN Keratin, type I cytoskeletal 16 OS=Homo sapiens GN=KRT16 PE=1 SV=4
K2C6C_HUMAN Keratin, type II cytoskeletal 6C OS=Homo sapiens GN=KRT6C PE=1 SV=3
HNRH1_HUMAN Heterogeneous nuclear ribonucleoprotein H OS=Homo sapiens GN=HNRNPH1 PE=1 SV=4
K2C5_HUMAN
Keratin, type II cytoskeletal 5 OS=Homo sapiens GN=KRT5 PE=1 SV=3
TBA1A_HUMAN Tubulin alpha-1A chain OS=Homo sapiens GN=TUBA1A PE=1 SV=1
K1C17_HUMAN Keratin, type I cytoskeletal 17 OS=Homo sapiens GN=KRT17 PE=1 SV=2
K2C8_HUMAN
Keratin, type II cytoskeletal 8 OS=Homo sapiens GN=KRT8 PE=1 SV=7
SF3B3_HUMAN Splicing factor 3B subunit 3 OS=Homo sapiens GN=SF3B3 PE=1 SV=4
HNRPM_HUMAN Heterogeneous nuclear ribonucleoprotein M OS=Homo sapiens GN=HNRNPM PE=1 SV=3
IF4A1_HUMAN Eukaryotic initiation factor 4A-I OS=Homo sapiens GN=EIF4A1 PE=1 SV=1
MYO6_HUMAN
Unconventional myosin-VI OS=Homo sapiens GN=MYO6 PE=1 SV=4
RMD5A_HUMAN Protein RMD5 homolog A OS=Homo sapiens GN=RMND5A PE=1 SV=1
RS27A_HUMAN Ubiquitin-40S ribosomal protein S27a OS=Homo sapiens GN=RPS27A PE=1 SV=2
TBA4A_HUMAN Tubulin alpha-4A chain OS=Homo sapiens GN=TUBA4A PE=1 SV=1
PABP5_HUMAN Polyadenylate-binding protein 5 OS=Homo sapiens GN=PABPC5 PE=1 SV=1
ATPA_HUMAN
ATP synthase subunit alpha, mitochondrial OS=Homo sapiens GN=ATP5A1 PE=1 SV=1
FLOT2_HUMAN Flotillin-2 OS=Homo sapiens GN=FLOT2 PE=1 SV=2
HNRPF_HUMAN Heterogeneous nuclear ribonucleoprotein F OS=Homo sapiens GN=HNRNPF PE=1 SV=3
ILF2_HUMAN
Interleukin enhancer-binding factor 2 OS=Homo sapiens GN=ILF2 PE=1 SV=2
1 of
37
Pept
ide
Sum
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port
(Sub
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3881
with
Dec
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ht
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2/m
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/mas
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data
/201
2060
8/F0
7733
1.da
t&RE
PTY
PE=p
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DX39A_HUMAN ATP-dependent RNA helicase DDX39A OS=Homo sapiens GN=DDX39A PE=1 SV=2
IDHP_HUMAN
Isocitrate dehydrogenase [NADP], mitochondrial OS=Homo sapiens GN=IDH2 PE=1 SV=2
EF1A1_HUMAN Elongation factor 1-alpha 1 OS=Homo sapiens GN=EEF1A1 PE=1 SV=1
K1C24_HUMAN Keratin, type I cytoskeletal 24 OS=Homo sapiens GN=KRT24 PE=1 SV=1
GRP75_HUMAN Stress-70 protein, mitochondrial OS=Homo sapiens GN=HSPA9 PE=1 SV=2
H3C_HUMAN
Histone H3.3C OS=Homo sapiens GN=H3F3C PE=1 SV=3
RALYL_HUMAN RNA-binding Raly-like protein OS=Homo sapiens GN=RALYL PE=2 SV=2
SPF45_HUMAN Splicing factor 45 OS=Homo sapiens GN=RBM17 PE=1 SV=1
CRNL1_HUMAN Crooked neck-like protein 1 OS=Homo sapiens GN=CRNKL1 PE=1 SV=4
XPO2_HUMAN
Exportin-2 OS=Homo sapiens GN=CSE1L PE=1 SV=3
MACF1_HUMAN Microtubule-actin cross-linking factor 1, isoforms 1/2/3/5 OS=Homo sapiens GN=MACF1 PE=1 SV=4
HORN_HUMAN
Hornerin OS=Homo sapiens GN=HRNR PE=1 SV=2
VIP2_HUMAN
Inositol hexakisphosphate and diphosphoinositol-pentakisphosphate kinase 2 OS=Homo sapiens GN=PPIP5K2 PE=1 SV=3
THOC3_HUMAN THO complex subunit 3 OS=Homo sapiens GN=THOC3 PE=1 SV=1
ANXA5_HUMAN Annexin A5 OS=Homo sapiens GN=ANXA5 PE=1 SV=2
PCBP1_HUMAN Poly(rC)-binding protein 1 OS=Homo sapiens GN=PCBP1 PE=1 SV=2
Sw
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Dec
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alse
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247
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%
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ide
mat
ches
abo
ve h
omol
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entit
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262
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4.58
%
Mas
cot S
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togr
am
Ions
scor
e is
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Log(
P), w
here
P is
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abili
ty th
at th
e ob
serv
ed m
atch
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.In
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scor
es >
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cate
iden
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xten
sive
hom
olog
y (p
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es a
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ed fr
om io
ns sc
ores
as a
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is fo
r ran
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tein
hits
.
8/06
/201
2 3:
00 P
M
Pept
ide
Sum
mar
y R
epor
t
Hel
p
2 of
37
User : Matt F
Email :
Search title : Submitted from JackyBentel by Mascot Daemon on APAF-WS-08
MS data file : \\Apaf-hpv-file\User_Shared\MFitzhenry\2012 Reports\PROJ14099_Bentel\IP_ALL.mgf
Database : SwissProt 2012x (536789 sequences; 190518892 residues)
Taxonomy : Homo sapiens (human) (20232 sequences)
Timestamp : 25 Jul 2012 at 22:29:54 GMT
Protein hits : PUF60_HUMAN Poly(U)-binding-splicing factor PUF60 OS=Homo sapiens GN=PUF60 PE=1 SV=1
K1C10_HUMAN Keratin, type I cytoskeletal 10 OS=Homo sapiens GN=KRT10 PE=1 SV=6
K22E_HUMAN
Keratin, type II cytoskeletal 2 epidermal OS=Homo sapiens GN=KRT2 PE=1 SV=2
K2C1_HUMAN
Keratin, type II cytoskeletal 1 OS=Homo sapiens GN=KRT1 PE=1 SV=6
K1C9_HUMAN
Keratin, type I cytoskeletal 9 OS=Homo sapiens GN=KRT9 PE=1 SV=3
HSP7C_HUMAN Heat shock cognate 71 kDa protein OS=Homo sapiens GN=HSPA8 PE=1 SV=1
ACTB_HUMAN
Actin, cytoplasmic 1 OS=Homo sapiens GN=ACTB PE=1 SV=1
HNRPC_HUMAN Heterogeneous nuclear ribonucleoproteins C1/C2 OS=Homo sapiens GN=HNRNPC PE=1 SV=4
K2C6B_HUMAN Keratin, type II cytoskeletal 6B OS=Homo sapiens GN=KRT6B PE=1 SV=5
POTEE_HUMAN POTE ankyrin domain family member E OS=Homo sapiens GN=POTEE PE=1 SV=3
HSP71_HUMAN Heat shock 70 kDa protein 1A/1B OS=Homo sapiens GN=HSPA1A PE=1 SV=5
PABP1_HUMAN Polyadenylate-binding protein 1 OS=Homo sapiens GN=PABPC1 PE=1 SV=2
GRP78_HUMAN 78 kDa glucose-regulated protein OS=Homo sapiens GN=HSPA5 PE=1 SV=2
ALBU_HUMAN
Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=2
K2C8_HUMAN
Keratin, type II cytoskeletal 8 OS=Homo sapiens GN=KRT8 PE=1 SV=7
PGAM5_HUMAN Serine/threonine-protein phosphatase PGAM5, mitochondrial OS=Homo sapiens GN=PGAM5 PE=1 SV=2
HNRH1_HUMAN Heterogeneous nuclear ribonucleoprotein H OS=Homo sapiens GN=HNRNPH1 PE=1 SV=4
K1C19_HUMAN Keratin, type I cytoskeletal 19 OS=Homo sapiens GN=KRT19 PE=1 SV=4
RS27A_HUMAN Ubiquitin-40S ribosomal protein S27a OS=Homo sapiens GN=RPS27A PE=1 SV=2
SF3A3_HUMAN Splicing factor 3A subunit 3 OS=Homo sapiens GN=SF3A3 PE=1 SV=1
SF3B3_HUMAN Splicing factor 3B subunit 3 OS=Homo sapiens GN=SF3B3 PE=1 SV=4
HNRPK_HUMAN Heterogeneous nuclear ribonucleoprotein K OS=Homo sapiens GN=HNRNPK PE=1 SV=1
RMD5B_HUMAN Protein RMD5 homolog B OS=Homo sapiens GN=RMND5B PE=2 SV=1
PTBP1_HUMAN Polypyrimidine tract-binding protein 1 OS=Homo sapiens GN=PTBP1 PE=1 SV=1
U5S1_HUMAN
116 kDa U5 small nuclear ribonucleoprotein component OS=Homo sapiens GN=EFTUD2 PE=1 SV=1
TBB4A_HUMAN Tubulin beta-4A chain OS=Homo sapiens GN=TUBB4A PE=1 SV=2
EF1A1_HUMAN Elongation factor 1-alpha 1 OS=Homo sapiens GN=EEF1A1 PE=1 SV=1
TAF6_HUMAN
Transcription initiation factor TFIID subunit 6 OS=Homo sapiens GN=TAF6 PE=1 SV=1
DDX41_HUMAN Probable ATP-dependent RNA helicase DDX41 OS=Homo sapiens GN=DDX41 PE=1 SV=2
PRP19_HUMAN Pre-mRNA-processing factor 19 OS=Homo sapiens GN=PRPF19 PE=1 SV=1
K1H1_HUMAN
Keratin, type I cuticular Ha1 OS=Homo sapiens GN=KRT31 PE=2 SV=3
HSPB1_HUMAN Heat shock protein beta-1 OS=Homo sapiens GN=HSPB1 PE=1 SV=2
NEP_HUMAN
Neprilysin OS=Homo sapiens GN=MME PE=1 SV=2
ATPB_HUMAN
ATP synthase subunit beta, mitochondrial OS=Homo sapiens GN=ATP5B PE=1 SV=3
MYO6_HUMAN
Unconventional myosin-VI OS=Homo sapiens GN=MYO6 PE=1 SV=4
RN128_HUMAN E3 ubiquitin-protein ligase RNF128 OS=Homo sapiens GN=RNF128 PE=1 SV=1
PLCA_HUMAN
1-acyl-sn-glycerol-3-phosphate acyltransferase alpha OS=Homo sapiens GN=AGPAT1 PE=2 SV=2
Sw
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Dec
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Pept
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Sum
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http
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af-b
l-04/
mas
cot/c
gi/m
aste
r_re
sults
.pl?
file=
../da
ta/2
0120
726/
F078
415.
dat&
REPT
YPE
=...
1 of
24
26/0
7/20
12 8
:44
AM
User : Matt F
Email :
Search title : Submitted from JackyBentel by Mascot Daemon on APAF-WS-08
MS data file : \\Apaf-hpv-file\User_Shared\MFitzhenry\2012 Reports\PROJ14099_Bentel\M_ALL.mgf
Database : SwissProt 2012x (536789 sequences; 190518892 residues)
Taxonomy : Homo sapiens (human) (20232 sequences)
Timestamp : 25 Jul 2012 at 22:27:48 GMT
Protein hits : PUF60_HUMAN Poly(U)-binding-splicing factor PUF60 OS=Homo sapiens GN=PUF60 PE=1 SV=1
HSP7C_HUMAN Heat shock cognate 71 kDa protein OS=Homo sapiens GN=HSPA8 PE=1 SV=1
K2C1_HUMAN
Keratin, type II cytoskeletal 1 OS=Homo sapiens GN=KRT1 PE=1 SV=6
K2C6B_HUMAN Keratin, type II cytoskeletal 6B OS=Homo sapiens GN=KRT6B PE=1 SV=5
K2C6A_HUMAN Keratin, type II cytoskeletal 6A OS=Homo sapiens GN=KRT6A PE=1 SV=3
DDX41_HUMAN Probable ATP-dependent RNA helicase DDX41 OS=Homo sapiens GN=DDX41 PE=1 SV=2
K1C10_HUMAN Keratin, type I cytoskeletal 10 OS=Homo sapiens GN=KRT10 PE=1 SV=6
HNRPC_HUMAN Heterogeneous nuclear ribonucleoproteins C1/C2 OS=Homo sapiens GN=HNRNPC PE=1 SV=4
K22E_HUMAN
Keratin, type II cytoskeletal 2 epidermal OS=Homo sapiens GN=KRT2 PE=1 SV=2
K1C16_HUMAN Keratin, type I cytoskeletal 16 OS=Homo sapiens GN=KRT16 PE=1 SV=4
HNRH1_HUMAN Heterogeneous nuclear ribonucleoprotein H OS=Homo sapiens GN=HNRNPH1 PE=1 SV=4
PABP1_HUMAN Polyadenylate-binding protein 1 OS=Homo sapiens GN=PABPC1 PE=1 SV=2
IF4A3_HUMAN Eukaryotic initiation factor 4A-III OS=Homo sapiens GN=EIF4A3 PE=1 SV=4
K1C9_HUMAN
Keratin, type I cytoskeletal 9 OS=Homo sapiens GN=KRT9 PE=1 SV=3
K1C14_HUMAN Keratin, type I cytoskeletal 14 OS=Homo sapiens GN=KRT14 PE=1 SV=4
HSP71_HUMAN Heat shock 70 kDa protein 1A/1B OS=Homo sapiens GN=HSPA1A PE=1 SV=5
HNRPF_HUMAN Heterogeneous nuclear ribonucleoprotein F OS=Homo sapiens GN=HNRNPF PE=1 SV=3
PGAM5_HUMAN Serine/threonine-protein phosphatase PGAM5, mitochondrial OS=Homo sapiens GN=PGAM5 PE=1 SV=2
GRP78_HUMAN 78 kDa glucose-regulated protein OS=Homo sapiens GN=HSPA5 PE=1 SV=2
U5S1_HUMAN
116 kDa U5 small nuclear ribonucleoprotein component OS=Homo sapiens GN=EFTUD2 PE=1 SV=1
ACTB_HUMAN
Actin, cytoplasmic 1 OS=Homo sapiens GN=ACTB PE=1 SV=1
ILF2_HUMAN
Interleukin enhancer-binding factor 2 OS=Homo sapiens GN=ILF2 PE=1 SV=2
DHX35_HUMAN Probable ATP-dependent RNA helicase DHX35 OS=Homo sapiens GN=DHX35 PE=1 SV=2
RALY_HUMAN
RNA-binding protein Raly OS=Homo sapiens GN=RALY PE=1 SV=1
KRT84_HUMAN Keratin, type II cuticular Hb4 OS=Homo sapiens GN=KRT84 PE=1 SV=2
PRP19_HUMAN Pre-mRNA-processing factor 19 OS=Homo sapiens GN=PRPF19 PE=1 SV=1
SF3B3_HUMAN Splicing factor 3B subunit 3 OS=Homo sapiens GN=SF3B3 PE=1 SV=4
TAF6_HUMAN
Transcription initiation factor TFIID subunit 6 OS=Homo sapiens GN=TAF6 PE=1 SV=1
RBMX_HUMAN
RNA-binding motif protein, X chromosome OS=Homo sapiens GN=RBMX PE=1 SV=3
SF3A3_HUMAN Splicing factor 3A subunit 3 OS=Homo sapiens GN=SF3A3 PE=1 SV=1
CDC5L_HUMAN Cell division cycle 5-like protein OS=Homo sapiens GN=CDC5L PE=1 SV=2
PRP8_HUMAN
Pre-mRNA-processing-splicing factor 8 OS=Homo sapiens GN=PRPF8 PE=1 SV=2
FL2D_HUMAN
Pre-mRNA-splicing regulator WTAP OS=Homo sapiens GN=WTAP PE=1 SV=2
HNRPR_HUMAN Heterogeneous nuclear ribonucleoprotein R OS=Homo sapiens GN=HNRNPR PE=1 SV=1
HNRPM_HUMAN Heterogeneous nuclear ribonucleoprotein M OS=Homo sapiens GN=HNRNPM PE=1 SV=3
PCBP2_HUMAN Poly(rC)-binding protein 2 OS=Homo sapiens GN=PCBP2 PE=1 SV=1
RSMB_HUMAN
Small nuclear ribonucleoprotein-associated proteins B and B' OS=Homo sapiens GN=SNRPB PE=1 SV=2
THOC1_HUMAN THO complex subunit 1 OS=Homo sapiens GN=THOC1 PE=1 SV=1
PCBP1_HUMAN Poly(rC)-binding protein 1 OS=Homo sapiens GN=PCBP1 PE=1 SV=2
SNW1_HUMAN
SNW domain-containing protein 1 OS=Homo sapiens GN=SNW1 PE=1 SV=1
PTBP2_HUMAN Polypyrimidine tract-binding protein 2 OS=Homo sapiens GN=PTBP2 PE=1 SV=1
U520_HUMAN
U5 small nuclear ribonucleoprotein 200 kDa helicase OS=Homo sapiens GN=SNRNP200 PE=1 SV=2
HNRPK_HUMAN Heterogeneous nuclear ribonucleoprotein K OS=Homo sapiens GN=HNRNPK PE=1 SV=1
SF3B1_HUMAN Splicing factor 3B subunit 1 OS=Homo sapiens GN=SF3B1 PE=1 SV=3
Pept
ide
Sum
mar
y Re
port
(Sub
mitt
ed fr
om Ja
ckyB
ente
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Mas
cot D
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http
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af-b
l-04/
mas
cot/c
gi/m
aste
r_re
sults
.pl?
file=
../da
ta/2
0120
726/
F078
414.
dat&
REPT
YPE
=...
1 of
26
26/0
7/20
12 8
:43
AM
SF3B2_HUMAN Splicing factor 3B subunit 2 OS=Homo sapiens GN=SF3B2 PE=1 SV=2
CCD25_HUMAN Coiled-coil domain-containing protein 25 OS=Homo sapiens GN=CCDC25 PE=1 SV=2
VAPA_HUMAN
Vesicle-associated membrane protein-associated protein A OS=Homo sapiens GN=VAPA PE=1 SV=3
DX39B_HUMAN Spliceosome RNA helicase DDX39B OS=Homo sapiens GN=DDX39B PE=1 SV=1
CC150_HUMAN Coiled-coil domain-containing protein 150 OS=Homo sapiens GN=CCDC150 PE=2 SV=2
RU2A_HUMAN
U2 small nuclear ribonucleoprotein A' OS=Homo sapiens GN=SNRPA1 PE=1 SV=2
Sw
issP
rot
Dec
oyFa
lse d
iscov
ery
rate
Pept
ide
mat
ches
abo
ve id
entit
y th
resh
old
172
95.
23 %
Pept
ide
mat
ches
abo
ve h
omol
ogy
or id
entit
y th
resh
old
179
126.
70 %
Mas
cot S
core
His
togr
am
Ions
scor
e is
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Log(
P), w
here
P is
the
prob
abili
ty th
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e ob
serv
ed m
atch
is a
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om e
vent
.In
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dual
ions
scor
es >
30
indi
cate
iden
tity
or e
xten
sive
hom
olog
y (p
<0.0
5).
Prot
ein
scor
es a
re d
eriv
ed fr
om io
ns sc
ores
as a
non
-pro
babi
listic
bas
is fo
r ran
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pro
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hits
.
Pept
ide
Sum
mar
y R
epor
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Si
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Max
. num
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f hits
Sh
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1. PUF60_HUMAN Mass: 59838 Score: 1105 Matches: 47(30) Sequences: 19(17) emPAI: 2.34
Poly(U)-binding-splicing factor PUF60 OS=Homo sapiens GN=PUF60 PE=1 SV=1
Check to include this hit in error tolerant search or archive report
Query
Observed
Mr(expt)
Mr(calc)
ppm Miss Score
Expect Rank Unique Peptide
Pept
ide
Sum
mar
y Re
port
(Sub
mitt
ed fr
om Ja
ckyB
ente
l by
Mas
cot D
aem
on o
n A
PAF-
WS-
08)
http
://ap
af-b
l-04/
mas
cot/c
gi/m
aste
r_re
sults
.pl?
file=
../da
ta/2
0120
726/
F078
414.
dat&
REPT
YPE
=...
2 of
26
26/0
7/20
12 8
:43
AM