1. CDCA7 A case study in cellular regulation
January 23rd, 2014
Tim Gabor | Dr. Michael Scheid| York University

3. Lecture key words Cell cycle Transcription factors Phosphorylation
Heterodimerization
Immunohistochemistry
Immunprecipitation
Growth factors
Apoptosis
Colony formation
Nuclear localization
Consensus sequences
Motifs

4. CDCA7 | a case study in cellular regulation
Cell cycle control is the endgame of cellular regulation - critical balance between proliferation and apoptosis  CANCER

5. 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

6. 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

7. 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

8. TOR rictor Growth Factors PI3K PIP2 PIP3 PDK1 Receptor Tyrosine Kinase
14-3-3 P P
Cytoplasm
AKT P
14-3-3
AKT P
CDCA7 P P
14-3-3
rictor
TOR
14-3-3 P
CDCA7 P
AKT
14-3-3
14-3-3 P
CDCA7
Myc
Myc
Transcription
Pro-apoptotic
Genes ?
Nucleus

9. 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

10. CDCA7 | Conservation AKT consensus site R X R X X T/S F/L
CDCA7 T R P R R R T F
>90% conserved
human
monkey
dog
mouse
chicken
frog
zebrafish 24 49 69 78
112
190
261
363
AKT kinase 0.005% 1
T163
371
humCDCA7
zinc finger
261
361

11. CDCA7 is phosphorylated at t163
Many ways to prove phosphorylation
Custom made antibody against phospho-T163
Radioactivity and mutational analysis
Phosphotase
CDCA7
CDCA7+ CIP
T163A
T163A + CIP
Vector
a-FLAG
a-P-T163 WT
T163A

12. CDCA7 is phosphorylated at t163
a-FLAG
a-P-T163
Vector 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
Treatments
w/ growth factors
T= 0’
T= 20’
T= 30’
Immunohistochemistry
T= 40’
T= 50’
T= 60’

13. CDCA7 is phosphorylated at t163 by AKT
Inhibitors
Vector
CDCA7
Akt inh VIII
IP: a-FLAG
Blot: a-P-T163
Blot: a-FLAG

14. 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

16. CDCA7 | Conservation How do we test for a nuclear localization signal?
>90% conserved
human
monkey
dog
mouse
chicken
frog
zebrafish 24 49 69 78
112
190
261
363 1
T163
371
humCDCA7
NLS?
NLS?
zinc finger
261
361
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

17. Where is cdca7 found?
T163A
CDCA7
a-Flag
DAPI

18. CDCA7 contains an NLS Passive diffusion into nucleus <45 KDa
SV40
SV40 KE
R171E
CDCA7
CDCA7
R176E
R171/176E
CDCA7
(T163A)
R176/184E
R184E
157-RRPRRRTFPGVASRRNPERRARPLTRSRSRIL-188
Supplementary Figure 2

19. Phosphorylation alters localization
a-CDCA7
Nuclei
Merge
Unstimulated
PDGF
PDGF + LY
Supplementary Figure 3

20. 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

21. CDCA7 | Conservation 157-186 RRPRRRTFPGVASRRNPERRARPLTRSRSR
>90% conserved
human
monkey
dog
mouse
chicken
frog
zebrafish 24 49 69 78
112
190
261
363 1
T163
371
humCDCA7
NLS?
NLS?
zinc finger
261
361
RRPRRRTFPGVASRRNPERRARPLTRSRSR
consensus binding site R-[S/F/Y]-X-pS/T -X -P
cdcA7 T R R R T F P
Mekk2 T G R K T F P

22. | Just the facts
Large family of highly conserved, small, acidic polypeptides of kDa
Seven different isoforms in humans, σ directly implicated in cancer
Binds to protein ligands at defined phospho-serine/threonine motif RSXpS/TXP
Over 200 known ligands
regulates process relevant to cancer biology: cell-cycle progression, apoptosis and mitogenic signaling

23. | Modes of influence
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 motifs = high affinity
Adapted from Hermeking, 2005
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

24. CDCA7 binds14-3-3 and is phospho dependent
Western blots
S/F/Y
consensus site R X pT X P
Wildtype
Vector
R158A
P159A
R160A
R161A
R162A
T163A
F164A
P165A
Blot:
a-FLAG
a-P-T163 a

25. 14-3-3 alters CDCA7 localization
R161A
CDCA7
T163A
R161A/
a-Flag
DAPI
Is masking the NLS within the T163 region?

26. 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

27. CDCA7 binds the transcription factor Myc
Co-immunoprecipitation
His-Myc Pulldown
Blot: a-FLAG
Input
WT CDCA7
T163A CDCA7
D( ) CDCA7
D(1-146) CDCA7
D(1-172) CDCA7
D(1-202) CDCA7
D(1-234) CDCA7
D( ) CDCA7
D( ) CDCA7
D( ) CDCA7
D( ) CDCA7 WT
T163A
D( )
D(1-146)
D(1-172)
D(1-202)
D(1-234)
D( )
D( )
D( )
D( ) + -
CDCA7

28. So how does cdca7 affect phenotype?
Apoptosis
proliferation

29. 14-3-3/CDCA7 binding influence Myc-induced transformation
Colony formation assay

30. 14-3-3/CDCA7 binding influence Myc-induced apoptosis
Rat1
Trypan blue exclusion
Myc-Rat1
Sh1-Myc-Rat1

31. TOR rictor Growth Factors PI3K PIP2 PIP3 PDK1 Receptor Tyrosine Kinase
14-3-3 P P
Cytoplasm
AKT P
14-3-3
AKT P
CDCA7 P P
14-3-3
rictor
TOR
14-3-3 P
CDCA7 P
AKT
14-3-3
14-3-3 P
CDCA7
Myc
Myc
Transcription
Pro-apoptotic
Genes ?
Nucleus

32. summary
CDCA7 is a novel target of AKT required for Myc-dependent apoptosis
Phosphorylation of T163 inhibits CDCA7/Myc apoptosis by:
Promoting binding
Disruption of Myc binding
Shuttling to the cytoplasm
Potential for medical intervention in Myc tumors where AKT is dysregulated