glyco special hart
TRANSCRIPT
”Bittersweet Roles of O-GlcNAc in Physiology & Analytical Challenges”
Gerald W. Hart, Ph.D. DeLamar Professor & Director
Department of Biological Chemistry Johns Hopkins University
School of Medicine 725 N. Wolfe St., Baltimore, MD 21205-2185
Email: [email protected]
Disclosures: Supported by NIH R01CA42486, R01DK61671; N01-HV-00240; P01HL107153, R24DK084949 and Dr. Hart is ”The Beth W. and A. Ross Myers Scholar” of the Patrick C. Walsh Prostate Cancer Research Fund. Dr. Hart receives a share of royalty received by the university on sales of the CTD 1 1 0. 6 antibody, which are managed by JHU.
Warren Symp. CCFC August 2012
Common classes of animal glycans Essentials of Glycobiology Second Edition
Outside
Inside
O-GlcNAc is Different Than Other Glycans: Nucleocytoplasmic & Cycles
O-GlcNAc Discovered Early 1980s
Plasma Membrane
Detailed Glycomic Analysis of O-GlcNAc Structural Diversity:
--Ser (Thr)- ~3000 sites mapped to date
β
Highly Dynamic Enzymatic Modification of Ser and Thr residues by β-N-acetylglucosamine NOT elongated to more complex structures & Localized to the cytoplasm and nucleus. Present in all Metazoans studied, some bacteria & proteozoa, some fungi, plants & viruses. Highly abundant PTM (>~3000 identified proteins) & Often Reciprocal (Competitive) with
phosphorylation - Abundance = pancreas islets>>brain>>other tissues>liver. Dynamically cycling on Ser/Thr residues - Time scale & stoichiometry similar to phosphate. Cycling on proteins is Controlled by O-GlcNAc Transferase and by O-GlcNAcase
Properties of O-GlcNAc.
(Hart et al., Nature, April 2007)
O-GlcNAc is Abundant on Nuclear & Cytosolic Proteins
UDP-GlcNAc UDP
2-5% Glucose To Hexosamine Biosynthesis
Pan >O-GlcNAc Antibody Western Blot - HeLa
“O-GlcNAc or O-linked N-acetylglucosamine or hexosamine pathway” papers in PubMed = 1709 ~3000 proteins & 2500 Sites Mapped & Counting!
O-GlcNAc Has Extensive Crosstalk with Phosphorylation to Serve As A Nutrient Sensor that Regulates Many Cellular Processes
Ann. Rev. Biochem. (2011)
UDP-GlcNAc Is a Major Node of Metabolism.
“O-GlcNAc or O-linked N-acetylglucosamine or hexosamine pathway” papers in PubMed = 1737> 1,~3000 Proteins & 2500 Sites Mapped & Counting!
O-GlcNAc Regulates Many Cellular Processes:
Essential for Life - OGT is required at the single cell, tissue and organ level in plants and animals (Marth et al.)
Blocks Phosphorylation - eg.eNOS;-diabetic erectile dysfunction; glycogen synthase; RNA Pol II; ER, SV-40 Large T, c-myc,p53; Cancer
Regulates Protein Interactions - Prevents YY1 binding Rb; Reg. ß-catenin & E-cadherin trafficking (Andrews et al.)
Regulates Transcription - RNA Polymerase II, SP1, CREB, NeuroD1, PGC-1α, PDX-1, c-myc, p53, and mSin3A, most transcription factors; Histones are O-GlcNAcylated. OGT = Polycomb gene.
Regulates Translation - eg. p67 binding to EIF2 kinase (Gupta) Ribosome Proteins; mTOR pathway
Regulates the Proteasome & Ubiquitination of p53 - Reduced ATPase activity 19S cap;5/19 & 9/14 of catalytic and core subunits, respectively modified.
Neurodegeneration - OGase Maps to AD and OGT Parkinsons Loci; Most proteins linked are O-GlcNAcylated; O-GlcNAc reduced on tau etc. in AD; O-GlcNAc reduced on key synaptic proteins.
Short-Term, Protects Cells From Stress - Protects against multiple forms of cellular stress; Protects cardiac function after trauma and ischemia.
Regulates Cell Cycle & Cytokinesis - Affects Rate of Transition through cell-cycle; Elevated OGT causes Polyploidy by blocking cytokinesis - transient cytokinesis complex.
Regulates Growth Hormone (Gibberillic Acid) in Plants) - Spy and Secret Agent are both OGTs.
Diabetes - Elevated O-GlcNAc blocks insulin signaling; Hyper-O-GlcNAc of transcription factors important to glucose toxicity; O-GlcNAc on NeuroD1 and PDX-1 regulates insulin transcription.
Essential Role in Lymphocyte Activation - Both B- & T-lymphocytes; NFkB & NFAT Nuclear Localization; EMBO J. (2007) 26, 4368.
Role in Neuronal Plasticity & Synaptic Vesicle Trafficking & Axonal Branching- Vosseller et al.; Cole et al., Song et al.
O-GlcNAc on SP1 Regulates HIV-1 latency and activation, and links viral replication to the glucose metabolism of the host cell. J. Virol. (March 2009)
Regulates Histone Methylation - MLL5 GlcNAcylation triggers cell lineage determination of HL60 through activation of its HKMT activity. Nature April 2009
(Hart et al., Nature, April 2007)
2-5% Glucose To Hexosamine Biosynthesis
Crosstalk Between GlcNAcylation & Phosphorylation
is Surprisingly Extensive!
Recent Mass Spectrometric Methods:
Chemico- enzymatic Enrichment combined With Electron Transfer Dissociation (ETD)
a. Competitive occupancy at same site
b. Reciprocal occupancy at different sites
c. Simultaneous occupancy at different sites
d. Site-dependent reciprocal or simultaneous occupancy
1 439
TAD HLH
58
P
OR 1 439
TAD HLH
58
G
1 345
DBD OR
TAD RD
179 180 181 184 189
P P P
1 345
DBD TAD RD
179 180 181 184 189
G G
1 1242
PHD PTBD SRD PRD
1036 307 632 635
P P P G
C/EBPβ
c-myc
IRS-1
CaMKIV 1 473
CaMBD PKD AD
200
P
57/58
G
189 1 473
CaMBD PKD AD
200 57/58
G
189 OR
1009 914
G G
1041
G
In the absence of other stimuli, how Does a Short Term (~2.5 hours)
Elevation (~3 fold) in Global O-GlcNAcylation (caused by inhibiting O-GlcNAcase)
Affect Site Specific Phosphorylation?
Simple Question:
ROR (A/B) = [P-peptide] (A)
[Protein] (A)
[P-peptide] (B)
[Protein] (B)
= [P-peptide] (A)
[P-peptide] (B)
[Protein] (B)
[Protein] (A) X
= Ratio [1] X 1
Ratio [2]
A.
B. C.
Quantitative proteomic approach to delineate global interplay between phosphorylation and O-GlcNAcylation. A. Flow chart for phosphorylation detection and site-specific quantitation. B. Protein level quantitation using iTRAQ. C. Formula used to
calculate ROR
ROR = Relative Occupancy Ratio
IMAC at Polypeptide level
Titanium Dioxide
Exp. Design: Affect on Phosphorylation if O-GlcNAc is Globally Increased?:
Proceedings Natl. Acad. Sci. (USA) (2008) 105 ,13793–13798.
iTRAQ
Okadaic Acid OGase Inhibitors Both Inhibitors
ATP-citrate synthase
R.TASFSESR.A
Insulin receptor substrate 2 (IRS-2)
R.VASPTSGLK.R
Site-Specific Phosphorylation Dynamics: Selected Examples C OA P/N Both
C OA P/N Both
*All Data is Normalized to Spiked Internal STDs - 32 phosphorylation sites
P/N increases global O-GlcNAc about 2-3 fold in 3hours
Cycling Site – OA increases P/N decreases
Site is Not Cycling.
Effects of Inhibiting O-GlcNAcase on P Site Occupancy: Summary of ~700 Phosphorylation Sites Identified and Quantified: ~148 increased by P/N ~280 Sites Not Affected by 3h inhibitor ~280 decreased by P/N
Zihao Wang
Nearly Every Phosphorylation Site That is Actively Cycling is Affected by GlcNAcylation!
Proceedings Natl. Acad. Sci. (USA) (2008) 105 ,13793–13798.
Effects of a ~2-3-fold increase in Global O-GlcNAc for 2.5h:
Kinases Are Regulated by O-GlcNAcylation.
No OGT + OGT
CTD110.6 (1:5000) – O-GlcNAcylation of Yeast Kinase (human) Chip
Print Array OGT + UDP-GlcNAc Immunoblot + Fluor 2ndary Ab
Printed in Identical Pair Spots
Guanghui Han (collaboration Heng Zhu’s Lab.)
Some O-GlcNAcylated Kinases I.D. so far: ERK-5 MAP2K3 p38 MAP3K14 MAPK1 MAP2K6 MAP3K6 CDK2 CDK5 CCRK Polo-like K1 AURKB PCTAIRE K1 pim-1 oncogene src
PKCζ PKCalpha AKT PIK3C3 TANK-binding K1 casein kinase 2* PRKD2 C20orf97 CAMKK2 beta CAMK IV CAMK1G CAMKII
Ser/thr K16 Ser/thr K24 Ser/thr K25 Ser/thr K17B Ser/thr K33
S6K1 PKLR PDK3 PTK9 PAK4 BRD3 APEG1 SNARK STE20-like kinase AP2 associated K1 Heat Shock 27 p8 PACE-1 SFRS K1 RIO K2 RIO K3
46 O-GlcNAcylated Kinases in Synapse -”Disproportionately O-GlcNAcylated” MCP Papers in Press. Published on May 29, 2012 (Burlingame) – i.d. 1750 O-GlcNAc sites in synapse.
LATS1 NEK8 FLJ23356 TESK2 CAMKK2 KSR2 ARK5 CAMKK2 TNK2 MAPK14 NUAK1
MATK FGR ACUR2B PNCK CAMK2A BMPRIB CSNK1G2 CDC2 CSNK2A1 CSNK1E ITK URK1
TRIB2 CDK9 RIPK2 ALS2CR2 STK16 PRKC1 PRKX STK4 CAMKK1 HIPK1 PRK2 MAPK8 MYO3A PKN3
MAPK10 STK17A RP6-213H19.1
Dias et al. Biochemical and Biophysical Research Communications (2012)
O-GlcNAc at the Active Site Inhibits CAMKIV:
*Song et al. Cellular Signaling (2007) O-GlcNAc transferase is activated by CaMKIV-dependent phosphorylation under potassium chloride-induced depolarization in NG-108-15 cells
Relative Sizes of pT200 and og189 on CAMKIV
Surface models of N-acetylglucosamine (left foreground) and inorganic phosphate (right foreground), along with a cartoon model of the kinase domain from human wild-type CaMKIV (center background) modeled from an X-ray crystal structure of human CaMKIγ. The amino acid residues colored in green and red are those that are modified by GlcNAcylation and phosphorylation, respectively.
CaMKK
CaMKIV
GS GS
GS
137
189
356
OGA
137
CaMKIV GS
GS 356
200 PT
CaM Ca2+ CaMKIV
GS GS
GS
137
189
356
*
OGT
Stimulation
Inactive (Basal State)
Active (Stimulated State)
Activation loop
Ca2+ increase
A
B
C
GS GS T57/S58 T57/S58
GS T57/S58
O-GlcNAc is a Negatively Modulates - Preventing Activation of CAMKIV
Activation is a 2 Step Process: 1 . Removal of O- GlcNAc 2. Activation by Phosphorylation &
Ca/CM *Song et al. Cellular Signaling (2007) O-GlcNAc transferase is
activated by CaMKIV-dependent phosphorylation under potassium chloride-induced depolarization = O-GlcNAc-Phosphate Reg. Cycle.
ATP Pocket PT
CaMKIV Activates OGT
J. Cell Sci. 1 23, 13-22.
Dias et al. (2009) J. Biol. Chem. 284, 21327–21337
DYNAMIC CROSSTALK BETWEEN TWO ESSENTIAL NUTRIENT-SENSITIVE ENZYMES:
O-GlcNAc Transferase (OGT) and AMPK-activated protein kinase (AMPK)
HIGHLIGHTS
1) AMPK regulates nutrient-sensitive nuclear localization of OGT in myotubes, affecting global O-GlcNAcylation of nuclear protein and K9 acetylation on histone 3.
2) AMPK phosphorylates Thr-444 on OGT, a residue that regulates nuclear localization of OGT in response to AMPK activation in myotubes
3) AMPK is extensively and dynamically O-GlcNAcylated (alpha 1 and 2 subunits & gamma subunits) 4) Acute global inhibition of O-GlcNAc cycling blunts
activation of AMPK
John Bullen et al. submitted
3000
Intein Chemical Ligation to Produce P344 & G347 CK2 Isoforms
Tarrant et al. Nature Chemical Biology 2012
Tarrant et al. Nature Chemical Biology 2012
Using a Large Protein Array As Substrates, The Different Modified Forms of CK2 Have Different Substrate Specificities.
Good Substrate For O-GlcNAc Isoform.
Good Substrate For Phospho Isoform.
Pin1 Assoc. Also Regulates
S-Adenyosyl homocystein lyase
Nucleosomal Assembly Protein 1
Tarrant et al. Nature Chemical Biology 2012
CK2 Specificity Is, In Part, Controlled by Its Modifications:
Extensive Crosstalk Between GlcNAcylation And Phosphorylation Regulates Cytokinesis
A.
B.
OGT DNA
OGT DNA OGT
DNA
Tubulin O-GlcNAc
Tubulin O-GlcNAc
O-GlcNAc transferase is associated with the spindle and midbody
Over-expression of OGT causes polyploidy.
Purified spindles demonstrate a ring of O-GlcNAc modified proteins and localization with tubulin near the spindle poles.
O-GlcNAc modified proteins are enriched at the midbody and at the nascent nuclear envelope
Sup TP Sup TP
IB: O-GlcNAc
GFP OGT Sup TP Sup TP GFP OGT
IB: MPM-2
Sup TP Sup TP GFP OGT
Coomassie Blue
F.
250 kDa
160 kDa
105 kDa
75 kDa
50 kDa
250 kDa
160 kDa
105 kDa
75 kDa
50 kDa
250 kDa
160 kDa
105 kDa
75 kDa
50 kDa
G. H.
O-GlcNAc is enriched in the mitotic taxol pellet and OGT over-expression elevates protein GlcNAcylation.
Mitotic phosphorylation is disrupted after OGT over-expression.
OGT over-expression alters protein expression in the taxol pellet samples.
Molec. Biol. Cell 1 9, 41 30- 41 4
Chad Slawson
TP=taxol pellet
CAD
ETD
ETD is a Breakthrough for O-GlcNAc:
MS performed by Hunt et al. Univ. Virginia
Synthetic C-Myc O-GlcNAc peptide
The O-GlcNAc Ion Suppression Problem: Ionization of O-GlcNAc peptide is suppressed in MALDI and ESI by naked peptides
Solutions: 1. Top-down MS
1. High mass accurancy instrument 2. Need large amount pure protein.
Probably not effective for complex samples
2. Enrich O-GlcNAc peptides
1. Direct enrichment of O-GlcNAc peptides not effective
2. Enzymatic/chemical derivatization available
NH
O
HN
S CO
N
H
OO
NH
C
O
C
O
NH
NO2
CHH3C O
CO O N
O
O
NHS-ester reaction with primary amine; (Ambergen)
NH
O
HN
S CO
N
H
OO
NH
C
O
CO
N
HNO2
CHH3C O
CO NH
H2C C CH
+ NH2H2C C CH
DMF, RT
Propylargylamine; (Aldrich)
Alkyne for Click Chemistry
Method to Quantify O-GlcNAc Site Occupancy: Photocleavable Biotin Alkyne – 1 Step Synthesis
from Commercial Compounds:
Molecular and Cellular Proteomics (2010) 9: 1 53- 1 60.
Olejnik et al. (1995) Proc. Natl. Acad. Sci. USA 92, 7590-7594.
O
OH
OHO
NHACO
OHOHO
NHO
OH
N
NH
O
HN
S CO
NH
OO
NH
C
O
CO
NH
NO2
CHH3C O
CO NH
H2C
NN
365 nm UV
NH
O
HN
S CO
NH
OO
NH
C
O
CO
NH
NO2
CH3C O
+ CO2
+ O
OH
OHO
NHACO
OHOHO
NHO
OH
N
NH2
H2C
NNAdds Pos. Charge
Photocleavable Biotin Tag for O-GlcNAc Site Mapping by ETD:
Attach to GalNAz-GlcNAc by Click Chemistry
Affinity Purify - Streptavidin
O-GlcNAc-peptides
Release by U.V cleavage
Molecular and Cellular Proteomics (2010) 9: 1 53- 1 60.
Recently: I.D. 274 O-GlcNAcylated proteins & Map 458 O-GlcNAc sites in Brain (Feng et al PNAS in Press 2012)
Combinatorial Proteomic Approach to Identify Mitotic GlcNAcylation and Phosphorylation Sites
GFP OGT
Purify MB, digest w/ trypsin/Lys-C
UDP-GalNAz GalT1, O/N
C18 trap X-biotin-alkyne Cycloaddition
SCX
Avidin chromatography
FT TiO2 chromatography
p-peptides
Orbitrap-CID FT-ETD
Naked peptides FT
Protein level quantification
Glyco-peptides
SILAC labeling
UV-cleavage
Quantification Site-mapping ID and quantification
elution
Orbitrap-CID 1. Easy release from avidin beads (w/o harsh condition) 2. Avoiding biotin fragmentation issue 3. Reduced precursor ion m/z 4. Adds Positive Chg for ETD; Small tryptics. 5. Diagnostic Fragment Ions: CID=peptide, not site.
Better tag technology
The chemoenzymatic approach for O-GlcNAc enrichment Method
CID: Diagnostic Fragment Ions
U.V.Cleavage
Spindle Midbody: Quantified: >700 Proteins; >320 PhosphoSites by CID Orbitrap. >~450 GlcNAc Mass Pair:Peptides; High Res.Orbitrap CID): ~150 O-GlcNAc Sites by FT-ETD
O
OH
OHO
NHACO
OHOHO
NHO
OH
N
NH2
H2C
NN
Click-Chemistry
Science Signaling 2010
Elevating OGT 2-Fold Dramatically Affects the CDK1 Circuit:
Science Signaling 2010
Aurora Kinase Polo Kinase circuits Also Affected in a Similar Manner
DIBO-Alkyne based click chemistry for the enrichment of O-GlcNAc peptides
DIBO-Alkyne (Reagent 1)
Reactive group to –N3
Linker arm (Cleavable)
Handle group (biotin)
Dr. Junfeng Ma
• Copper Free • No TBTA • Fewer steps • Reagent more stable and cheaper
Conclusions: ♥ O-GlcNAc is a Major Regulatory Post-Translational Modification in all multicellular
eukaryotes - Plants & Animals & Viruses (some bacteria).
♥ O-GlcNAc is Required for Life at All Levels in Mammals and Plants.
♥ Crosstalk or Interplay Between O-GlcNAcylation & Phosphorylation is Extensive and Involved in Many Cellular Processes.
♥ O-GlcNAc is Important to Transcription:Regulates Pol II & is Part of the Histone Code where Some Sites are at Contact Regions with the DNA of the Nucleosome.
♥ O-GlcNAc - “Metabolic Sensor” That Modulates Signaling & Transcription in Response to Cellular Status.
♥ Many Toxic Affects of Hyperglycemia Likely Result From Dysregulation of the Balance Between O-GlcNAc and Phosphorylation = Glucose Toxicity.
♥ O-GlcNAcylation is Elevated in Nearly All Cancers Examined and it Likely Plays a Role in Molecular Processes Leading to Cancer.
Disclosures: Supported by NIH R01CA42486, R01DK61671; N01-HV-00240; P01HL107153, R24DK084949 and Dr. Hart is ”The Beth W. and A. Ross Myers Scholar” of the Patrick C. Walsh Prostate Cancer Research Fund. Dr. Hart receives a share of royalty received by the university on sales of the CTD 1 1 0. 6 antibody, which are managed by JHU.
Acknowledgements Hart Lab
Hunt Lab
Donald F. Hunt Namrata D. Udeshi Univ. Virginia
For tools to study O- GlcNAc (eg. antibodies, plasmids, protocols): email: gwhart@jhmi. edu
Genaro A. Ramirez-Correa, Weidong Gao, and Anne M. Murphy Department of Pediatrics/Division of Cardiology Brian Lewis, NIH
Rick Huganair