Transcript by AKT - Scheid Signalling Lab @ York University

CDCA7
A CASE STUDY IN CELLULAR REGULATION
January 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
• 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
• 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
Growth
Factors
Receptor
Tyrosine
Kinase
P
P
P
P
PIP3
PIP2
PI3K
PDK1
P
P
AKT
CDCA7 P
P
14-3-3
TOR
rictor
14-3-3
P
14-3-3
Cytoplasm
AKT
P
14-3-3
14-3-3
AKT
CDCA7 P
14-3-3
P
CDCA7
Myc
Myc
Transcription
Pro-apoptotic
Genes ?
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
CDCA7 | CONSERVATION
AKT consensus site
CDCA7 T163
>90% conserved
human
monkey
dog
mouse
chicken
frog
zebrafish
24
49
69
78
112
190
R X R X X T/S F/L
R P R R R T
F
261
363
AKT kinase 0.005%
1
371
T163
humCDCA7
zinc finger
261
361
CDCA7 IS PHOSPHORYLATED AT T163
• Many ways to prove phosphorylation
• Custom made antibody against phospho-T163
Radioactivity and mutational analysis
T163A
T163A + CIP
CDCA7
Vector
CDCA7+ CIP
WT
Phosphotase
a-FLAG
T163A
a-P-T163
Vector
CDCA7 IS PHOSPHORYLATED AT T163
0
5
15
45
120
360
PDGF (min)
a-P-T163
a-FLAG
Treatments
w/ growth factors
Merge
1.0
T= 0’
2.2
3.6
4.7
T= 20’
4.0
3.9
Ratio P-T163/
Total CDCA7
T= 30’
Immunohistochemistry
T= 40’
T= 50’
T= 60’
CDCA7 IS PHOSPHORYLATED AT T163 BY AKT
Inhibitors
Vector
CDCA7
Akt inh VIII
IP: a-FLAG
Blot: a-P-T163
IP: a-FLAG
Blot: a-FLAG
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 | CONSERVATION
>90% conserved
human
monkey
dog
mouse
chicken
frog
zebrafish
24
49
69
78
112
1
190
261
363
371
T163
humCDCA7
NLS?
zinc finger
NLS?
261
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
361
WHERE IS CDCA7 FOUND?
a-Flag
CDCA7
T163A
CDCA7
DAPI
CDCA7 CONTAINS AN NLS
• Passive diffusion into nucleus <45 KDa
SV40
SV40 KE
157-188
R171E
R171/176E
157-188 CDCA7
157-167 CDCA7
R176E
167-188 CDCA7
157-188 (T163A)
R176/184E
R184E
157-RRPRRRTFPGVASRRNPERRARPLTRSRSRIL-188
PHOSPHORYLATION ALTERS LOCALIZATION
a-CDCA7
Unstimulated
PDGF
PDGF + LY
Nuclei
Merge
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 | CONSERVATION
>90% conserved
human
monkey
dog
mouse
chicken
frog
zebrafish
24
49
69
78
112
1
190
261
363
371
T163
humCDCA7
NLS?
zinc finger
NLS?
261
157-186
361
RRPRRRTFPGVASRRNPERRARPLTRSRSR
14-3-3 consensus binding site
R-[S/F/Y]-X-pS/T -X
cdcA7 T163
Mekk2 T283
R
G
R
R
R
K
T
T
F
F
-P
P
P
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 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
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
CDCA7 BINDS14-3-3 AND IS PHOSPHO DEPENDENT
P165A
X pT X P
T163A
F164A
- R
R161A
R162A
-
R158A
P159A
R160A
Vector
Wildtype
14-3-3 consensus site
S/F/Y
Western blots
Blot:
a-FLAG
a-P-T163
a-14-3-3
14-3-3 ALTERS CDCA7 LOCALIZATION
a-Flag
DAPI
CDCA7
T163A
CDCA7
R161A
CDCA7
R161A/
T163A
CDCA7
Is 14-3-3 masking the NLS within the T163 region?
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
D(170-370) CDCA7
D(153-370) CDCA7
D(230-370) CDCA7
D(260-370) CDCA7
T163A CDCA7
D(112-137) CDCA7
D(1-146) CDCA7
D(1-172) CDCA7
D(1-202) CDCA7
D(1-234) CDCA7
WT CDCA7
Co-immunoprecipitation
CDCA7
WT
T163A
D(112-137)
D(1-146)
His-Myc Pulldown
Blot: a-FLAG
D(1-172)
D(1-202)
D(1-234)
D(260-370)
D(230-370)
D(170-370)
D(153-370)
Input
Blot: a-FLAG
+
+
+
+
+
+
+
-
SO HOW DOES CDCA7 AFFECT PHENOTYPE?
APOPTOSIS
PROLIFERATION
14-3-3/CDCA7 BINDING INFLUENCE MYC-INDUCED TRANSFORMATION
Colony formation assay
14-3-3/CDCA7 BINDING INFLUENCE MYC-INDUCED APOPTOSIS
Rat1
Trypan blue exclusion
Myc-Rat1
Sh1-Myc-Rat1
Growth
Factors
Receptor
Tyrosine
Kinase
P
P
P
P
PIP3
PIP2
PI3K
PDK1
P
P
AKT
CDCA7 P
P
14-3-3
TOR
rictor
14-3-3
P
14-3-3
Cytoplasm
AKT
P
14-3-3
14-3-3
AKT
CDCA7 P
14-3-3
P
CDCA7
Myc
Myc
Transcription
Pro-apoptotic
Genes ?
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