CDCA7 A CASE STUDY IN CELLULAR REGULATIONJanuary 23rd, 2014

Tim Gabor | Dr. Michael Scheid| York University

LECTURE KEY WORDS- Cell cycle- Transcription factors- Phosphorylation- Heterodimerization- Immunohistochemistry- Immunprecipitation- Growth factors- Apoptosis- Colony formation- Nuclear localization - Consensus sequences- Motifs

CDCA7 | A CASE STUDY IN CELLULAR REGULATION

• Cell cycle control is the endgame of cellular regulation

- critical balance between proliferation and apoptosis CANCER

CDCA7 | A CASE STUDY IN CELLULAR REGULATION

• Cell cycle control is the endgame of cellular regulation

- critical balance between proliferation and apoptosis CANCER

• Modes: -phosphorylation, -subcellular localization - heterodimerization

CDCA7 | WHAT IS KNOWN

• Novel member of cell division cycle-associated gene family

• Myc and E2F target gene with peak expression at G1-S

• Frequently overexpressed in human tumors

• JPO2 binds Myc and promotes Myc dependent transformation• JPO2 and CDCA7 share cysteine rich C-term which may bind DNA

• Not known if CDCA7 interacts with Myc

MYC | JUST THE FACTS• Discovered in Burkitt’s lymphoma

patients• Member of bHLH-LZ family of transcription factors

• Requires heterodimerization with Max to transactivate

• Regulates the expression of ~10-15% of genes

• Role in development, cell division, cell growth, metabolism, angiogenesis

• Driving force of cell cycle and malignant transformation

• Active in 70% of human cancers

• ~100,000 cancer deaths per year in the US due to changes in Myc

• Early response gene induced by growth factors, levels peak at G0-G1

14-3-3

P

P P

PPIP2

PIP3PI3K

rictor

TOR

PDK1P

AKT

PAKT

P

P

14-3-3

Transcription Pro-apoptotic Genes ?

AKT

P

P

14-3-3

14-3-3

14-3-3

Myc

CDCA7

Myc

14-3-3

CDCA7

CDCA7

Growth Factors

ReceptorTyrosineKinase

P

P

Cytoplasm

Nucleus

CDCA7 | A CASE STUDY IN CELLULAR REGULATION

• Cell cycle control is the endgame of cellular regulation

- critical balance between proliferation and apoptosis CANCER

• Modes: -phosphorylation-subcellular localization -heterodimerization

humCDCA7

1 371

361261zinc finger

AKT consensus site R X R X X T/S F/LCDCA7 T163 R P R R R T F

T163

AKT kinase 0.005%

24 49

>90% conservedhuman monkeydogmousechickenfrogzebrafish

69 78 112 261 363190

CDCA7 | CONSERVATION

CDCA7 IS PHOSPHORYLATED AT T163

WT

T163A

CD

CA

7

CD

CA

7+

CIP

T1

63A

T1

63A

+ C

IP

Vector

a-FLAG

a-P-T163

• Custom made antibody against phospho-T163

• Many ways to prove phosphorylation

Radioactivity and mutational analysis

Phosphotase

a-FLAG

a-P-T163

Vector

0 5 15 45 120 360 PDGF (min)

Merge

Ratio P-T163/Total CDCA7

1.0 2.2 3.6 4.7 4.0 3.9

T= 0’ T= 20’

T= 30’

T= 40’

T= 50’

T= 60’

CDCA7 IS PHOSPHORYLATED AT T163

Treatmentsw/ growth factors

Immunohistochemistry

Vect

or CDCA7

Akt inh VIII

IP: a-FLAGBlot: a-P-T163

IP: a-FLAGBlot: a-FLAG

CDCA7 IS PHOSPHORYLATED AT T163 BY AKT

Inhibitors

CDCA7 | A CASE STUDY IN CELLULAR REGULATION

• Cell cycle control is the endgame of cellular regulation

- critical balance between proliferation and apoptosis CANCER

• Modes: -phosphorylation-subcellular localization -heterodimerization

humCDCA7

1 371

361261zinc finger

T163

24 49

>90% conservedhuman monkeydogmousechickenfrogzebrafish

69 78 112

NLS?

NLS?

261 363190

157-186 RRPRRRTFPGVASRRNPERRARPLTRSRSR

CDCA7 | CONSERVATION

How do we test for a nuclear localization signal?Isolate region in question and test its ability to target

an innocuous protein to the nucleus

T163A CDCA7

CDCA7

a-Flag DAPI

WHERE IS CDCA7 FOUND?

CDCA7 CONTAINS AN NLS

157-167 CDCA7

167-188 CDCA7157-188 (T163A)

157-188 CDCA7

SV40 SV40 KE 157-188 R171E

R176E

R184ER176/184E

R171/176E

157-RRPRRRTFPGVASRRNPERRARPLTRSRSRIL-188

• Passive diffusion into nucleus <45 KDa

PHOSPHORYLATION ALTERS LOCALIZATION

a-CDCA7 Nuclei Merge

Unstimulated

PDGF

PDGF + LY

CDCA7 | A CASE STUDY IN CELLULAR REGULATION

• Cell cycle control is the endgame of cellular regulation

- critical balance between proliferation and apoptosis CANCER

• Modes: -phosphorylation-subcellular localization -heterodimerization

humCDCA7

1 371

361261zinc finger

14-3-3 consensus binding site R-[S/F/Y]-X-pS/T -X -P

cdcA7 T163 R R R T F PMekk2 T283 G R K T F P

T163

24 49

>90% conservedhuman monkeydogmousechickenfrogzebrafish

69 78 112

NLS?

NLS?

261 363190

157-186 RRPRRRTFPGVASRRNPERRARPLTRSRSR

CDCA7 | CONSERVATION

14-3-3 | JUST THE FACTS• Large family of highly conserved, small, acidic

polypeptides of 28-33 kDa• Seven different isoforms in humans, 14-3-3σ

directly implicated in cancer • Binds to protein ligands at defined phospho-

serine/threonine motif RSXpS/TXP

• 14-3-3 regulates process relevant to cancer biology: cell-cycle progression, apoptosis and mitogenic signaling

• Over 200 known ligands

14-3-3 | MODES OF INFLUENCE• 14-3-3 exists as a dimer and offers two binding

sites for phospho-S/T motifs• Can function as adaptor protein for:a) two proteins that would otherwise not

associateb) one protein with two 14-3-3 motifs = high

affinity

• Affects change by:• Alteration of enzymatic activity – maintains

RAF1 in inactive state• Alteration of DNA-binding activity – increases

p53 DNA-binding after DNA damage• Sequestration - BAD, FKHRL1, HDAC5 and

CDC25C are localized to cytoplasm• Altering protein-protein interactions - reduced

affinity of CDC25A to CDC2• Adaptor protein functions – bridging of RAF1 to

BCR

• Sequestration • Altering protein-protein interactions

Adapted from Hermeking, 2005

CDCA7 BINDS14-3-3 AND IS PHOSPHO DEPENDENT

a-FLAG

a-P-T163

a-14-3-3

Vector

P165A

F164A

T163A

R162A

R161A

R160A

P159A

R158A

Wildtype

Blot:

14-3-3 consensus site - - R X pT X PS/F

/Y

Western blots

14-3-3 ALTERS CDCA7 LOCALIZATION

R161A CDCA7

T163A CDCA7

R161A/T163A CDCA7

CDCA7

a-Flag DAPI

Is 14-3-3 masking the NLS within the T163 region?

CDCA7 | WHAT IS KNOWN

• Novel member of cell division cycle-associated gene family

• Myc and E2F target gene with peak expression at G1-S

• Frequently overexpressed in human tumors

• JPO2 binds Myc and promotes Myc dependent transformation• JPO2 and CDCA7 share cysteine rich C-term which may bind DNA

• Not known if CDCA7 interacts with Myc

WTT163A

D(112-137)

D(1-146)

D(1-172)D(1-202)

D(1-234)

D(260-370)

D(230-370)

D(170-370)

D(153-370)

++++

+++-

---

CDCA7

His-Myc PulldownBlot: a-FLAG

InputBlot: a-FLAG

WT C

DC

A7

T1

63

A C

DC

A7

D(1

12

-13

7)

CD

CA

7D

(1-1

46

) C

DC

A7

D(1

-17

2)

CD

CA

7

D(1

-20

2)

CD

CA

7D

(1-2

34

) C

DC

A7

D(2

60

-37

0)

CD

CA

7

D(2

30

-37

0)

CD

CA

7

D(1

70

-37

0)

CD

CA

7D

(15

3-3

70

) C

DC

A7

CDCA7 BINDS THE TRANSCRIPTION FACTOR MYC

Co-immunoprecipitation

SO HOW DOES CDCA7 AFFECT PHENOTYPE?

APOPTOSIS

PROLIFERATION

14-3-3/CDCA7 BINDING INFLUENCE MYC-INDUCED TRANSFORMATION

Colony formation assay

Rat1

Myc-Rat1

Sh1-Myc-Rat1

14-3-3/CDCA7 BINDING INFLUENCE MYC-INDUCED APOPTOSIS

Trypan blue exclusion

14-3-3

P

P P

PPIP2

PIP3PI3K

rictor

TOR

PDK1P

AKT

PAKT

P

P

14-3-3

Transcription Pro-apoptotic Genes ?

AKT

P

P

14-3-3

14-3-3

14-3-3

Myc

CDCA7

Myc

14-3-3

CDCA7

CDCA7

Growth Factors

ReceptorTyrosineKinase

P

P

Cytoplasm

Nucleus

SUMMARY• CDCA7 is a novel target of AKT required for

Myc-dependent apoptosis• Phosphorylation of T163 inhibits CDCA7/Myc apoptosis by:• Promoting 14-3-3 binding• Disruption of Myc binding

• Shuttling to the cytoplasm

• Potential for medical intervention in Myc tumors where AKT is dysregulated