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Phosphorylation of CDK Targets Changes Their Activity. Now performs a cell cycle function. How are CDK’s Regulated? By cyclin synthesis and destruction By phosphorylation By binding to CDK inhibitory proteins (CKIs). Generation of a “Cycling” Frog Egg Extract. Inject females with hormones - PowerPoint PPT Presentation
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Phosphorylation of CDK Targets Changes Their Activity
Now performsa cell cycle function
How are CDK’s Regulated?
1. By cyclin synthesis and destruction
2. By phosphorylation
3. By binding to CDK inhibitory proteins (CKIs)
Generation of a “Cycling” Frog Egg Extract
1. Inject females with hormonesso that they lay eggs
2. Pack eggs into acentrifuge tube and spin
3. RemoveCytoplasmic Extract
4. Add sperm chromatinand away you go!
Cyclin Synthesis and DestructionDrives the Early Embryonic Cell Cycle
sea urchin!sea urchin!
Cyclin Destruction is Controlled by Ubiquitination
But is cyclin abundance the only
way to control CDK activity?
The Cell Cycle According to Cyclin Abundance
How are CDKs Regulated?
Isolate mutants that phenocopy cdc2- alleles
CDKs are Regulated by Phosphorylation
is a kinase is a phosphatase
CAK(CDKActivatingKinase)
Conformational Changes Associated with CDK Phosphorylation
The T-loop blockssubstrate access
Free CDK CDK + Cyclin T161 phosphorylation
Binding of cyclinmoves the T-loop
Phosporylation movesthe T-loop more
How does the G1-S Transition Work?
but…S. pombe cdc2+ can subsitute
for S. cerevisiae cdc28 and vice versa
cdc2 mutants arrest in G2
cdc28 mutants arrest in G1
Why?
and, cln1 and cln2 !!
The Identification of G1 Cyclins in S. cerevisiae
How Does High Copy Suppression Work?
The S. cerevisiae Cell Cycle
The G1-S Transition in S. cerevisiae
Growth Signal
Cln3/Cdc28
Cln1/Cdc28Cln2/Cdc28
make a bud
SIC1
G1
S
Clb5/Cdc28
Clb6/Cdc28
(cyclin/cdk)
Sic1 is Destroyed by Ubiquitination
(Cdc4,Cdc53)
Once and Only Once Replication is Controlled by CDKs
Human cdc2 (cdk1) Rescues cdc2 Mutant
cdk1cdk1
cdc2 mutantcells at 25C
Transformation with humancDNA library expressedwith SV40 viral promoter cdc2 transformed
mutant cells at 35C
Human cyclin E Rescues cln1,2,3 Mutant
cyc Ecyc E
cln1,2,3 triple mutantcells at 25C
Transformation with humancDNA library expressed
with yeast promoter cln1,2,3 transformed mutant cells at 35C
The Human Cell Cycle
Cyclin Dependent Kinase Inhibitors (CKIs)
Cyclin
CDKp21
Cyclin
CDK4
Cyclin
CDK
CDK4p16
p16
p21
The Discovery of p21 and p16
Cultured cells
Adding 35S[Met]
Metabolic labeling
Lysis cells midly
Immunoprecipitate
Add anti-CDK4 antibody
Add protein A-agarose beads
SDS-PAGE
Autoradiography
CDK4p16
Xiong et al. (1993) Genes & Dev. 7:1572
Cyclin
CDK4p21
CDK4
Cyclin D
p21
p16
Cell Line
No
rma
l
Tra
ns
form
ed
Tra
ns
form
ed
No
rma
l
Competing peptide - -+ +
-CDK4
The p21 Family of CDK inhibitors(p21CIP1/WAF1, p27KIP1, p57KIP2)
CDK
Cyclin
active
p21+
inactive
CDK
Cyclinp21
p27Kip1
Cyclin ACDK2
Russo et al. (1996) Nature 382:325
Cyclin A
CDK2
Jeffrey et al. (1995) Nature 376:313
The INK4 Family of CDK inhibitors(p16INK4a, p15INK4b, p18INK4c, p19INK4d)
INK4+CDK4/6
Cyclin D
active
CDK4/6Cyclin D
INK4+
inactive
Russo et al. (1998) Nature 395:237Brotherton et al. (1998) Nature 395:244
CKIs Regulate the G1-S Transition
(p16)
(p21, p27)
p16 is Frequently Mutated in Human Tumors
Tumor t ype L ines (n) Del etion s (n) D eleti ons (%) Astrocytoma 17 14 82 Bladder 15 5 33 Breast 10 6 60 Colon 20 0 0 G lioma 35 25 71 L eukemia 4 1 25 L ung 59 15 25 Melan oma 99 57 58 N euro blast oma 10 0 0 O steosarcoma 5 3 60 O vary 7 2 29 Renal 9 5 56 Total 29 0 13 3 46
Table 1. D eleti ons i n tumor cells and p rimary tumors.
See Kamb et al. (1994) Science 264: 436; Nobori et al. (1994) Nature 368:753 for detail
9p21