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CDCA7 A case study in cellular regulation. January 23 rd , 2014 Tim Gabor | Dr. Michael Scheid| York University. Lecture key words. Cell cycle Transcription factors Phosphorylation Heterodimerization Immunohistochemistry Immunprecipitation Growth factors Apoptosis Colony formation

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cdca7 a case study in cellular regulation

CDCA7 A case study in cellular regulation

January 23rd, 2014

Tim Gabor | Dr. Michael Scheid| York University

lecture key words
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 regulation1
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 regulation2
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
CDCA7 | What is known
  • Myc and E2F target gene with peak expression at G1-S
  • Novel member of cell division cycle-associated gene family
  • 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
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
  • Early response gene induced by growth factors, levels peak at G0-G1
  • 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
slide8

Myc

PI3K

Myc

AKT

AKT

AKT

P

P

P

P

P

P

P

P

P

P

P

P

rictor

TOR

Growth

Factors

PIP2

PIP3

Receptor

Tyrosine

Kinase

14-3-3

Cytoplasm

PDK1

14-3-3

CDCA7

14-3-3

14-3-3

CDCA7

14-3-3

14-3-3

CDCA7

Pro-apoptotic

Genes ?

Transcription

Nucleus

cdca7 a case study in cellular regulation3
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
slide10

CDCA7 | Conservation

AKT consensus site RXRXXT/SF/L

CDCA7 T163 RPRRRTF

>90% conserved

human

monkey

dog

mouse

chicken

frog

zebrafish

112

24

49

69

78

190

261

363

AKT kinase 0.005%

371

T163

1

humCDCA7

zinc finger

261

361

cdca7 is phosphorylated at t163
CDCA7 is phosphorylated at t163
  • Many ways to prove phosphorylation
  • Custom made antibody against phospho-T163

WT

CDCA7+ CIP

T163A + CIP

Vector

T163A

CDCA7

T163A

Radioactivity and mutational analysis

Phosphotase

a-FLAG

a-P-T163

cdca7 is phosphorylated at t1631
CDCA7 is phosphorylated at t163

Treatments

w/ growth factors

T= 0’

T= 20’

T= 30’

Immunohistochemistry

T= 40’

T= 50’

T= 60’

Vector

360

15

45

120

5

0

PDGF (min)

a-P-T163

a-FLAG

Merge

3.9

3.6

4.7

4.0

2.2

1.0

Ratio P-T163/

Total CDCA7

cdca7 is phosphorylated at t163 by akt
CDCA7 is phosphorylated at t163 by AKT

Inhibitors

IP: a-FLAG

Blot: a-P-T163

CDCA7

Aktinh VIII

Vector

IP: a-FLAG

Blot: a-FLAG

cdca7 a case study in cellular regulation4
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
slide16

CDCA7 | Conservation

>90% conserved

human

monkey

dog

mouse

chicken

frog

zebrafish

112

24

49

69

78

190

261

363

371

T163

1

humCDCA7

zinc finger

NLS?

NLS?

261

361

157-186 RRPRRRTFPGVASRRNPERRARPLTRSRSR

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

where is cdca7 found
Where is cdca7 found?

a-Flag

DAPI

CDCA7

T163A

CDCA7

cdca7 contains an nls
CDCA7 contains an NLS
  • Passive diffusion into nucleus <45 KDa

SV40

SV40 KE

R171E

157-188

R171/176E

R176E

157-188 CDCA7

157-167 CDCA7

157-188 (T163A)

167-188 CDCA7

R184E

R176/184E

157-RRPRRRTFPGVASRRNPERRARPLTRSRSRIL-188

Supplementary Figure 2

slide19

Phosphorylation alters localization

Nuclei

Merge

a-CDCA7

Unstimulated

PDGF

PDGF + LY

Supplementary Figure 3

cdca7 a case study in cellular regulation5
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
slide21

CDCA7 | Conservation

>90% conserved

human

monkey

dog

mouse

chicken

frog

zebrafish

112

24

49

69

78

190

261

363

371

T163

1

humCDCA7

zinc finger

NLS?

NLS?

261

361

157-186 RRPRRRTFPGVASRRNPERRARPLTRSRSR

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

cdcA7 T163 RRR T F P

Mekk2 T283 G R K T F P

14 3 3 just the facts
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
  • Over 200 known ligands
  • 14-3-3 regulates process relevant to cancer biology: cell-cycle progression, apoptosis and mitogenic signaling
14 3 3 modes of influence
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 associate
  • b) 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
CDCA7 binds14-3-3 and is phospho dependent

Western blots

S/F/Y

14-3-3 consensus site - - R X pT X P

Wildtype

P159A

R158A

P165A

R160A

R162A

T163A

F164A

R161A

Vector

Blot:

a-FLAG

a-P-T163

a-14-3-3

14 3 3 alters cdca7 localization
14-3-3 alters CDCA7 localization

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

a-Flag

DAPI

CDCA7

T163A

CDCA7

R161A

CDCA7

R161A/

T163A

CDCA7

cdca7 what is known1
CDCA7 | What is known
  • Myc and E2F target gene with peak expression at G1-S
  • Novel member of cell division cycle-associated gene family
  • 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
cdca7 binds the transcription factor myc
CDCA7 binds the transcription factor Myc

Co-immunoprecipitation

D(230-370) CDCA7

D(170-370) CDCA7

D(260-370) CDCA7

D(153-370) CDCA7

D(112-137) CDCA7

D(1-234) CDCA7

D(1-146) CDCA7

D(1-172) CDCA7

D(1-202) CDCA7

T163A CDCA7

CDCA7

WT CDCA7

+

WT

+

T163A

+

D(112-137)

+

D(1-146)

-

D(1-172)

-

D(1-202)

His-Myc Pulldown

Blot: a-FLAG

-

D(1-234)

+

D(260-370)

+

D(230-370)

+

D(170-370)

-

D(153-370)

Input

Blot: a-FLAG

14 3 3 cdca7 binding influence myc induced apoptosis
14-3-3/CDCA7 binding influence Myc-induced apoptosis

Rat1

Trypan blue exclusion

Myc-Rat1

Sh1-Myc-Rat1

slide31

Myc

PI3K

Myc

AKT

AKT

AKT

P

P

P

P

P

P

P

P

P

P

P

P

rictor

TOR

Growth

Factors

PIP2

PIP3

Receptor

Tyrosine

Kinase

14-3-3

Cytoplasm

PDK1

14-3-3

CDCA7

14-3-3

14-3-3

CDCA7

14-3-3

14-3-3

CDCA7

Pro-apoptotic

Genes ?

Transcription

Nucleus

summary
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