Upload
tanner-farley
View
29
Download
1
Tags:
Embed Size (px)
DESCRIPTION
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 - PowerPoint PPT Presentation
Citation preview
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
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
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
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