phosphorylation of cdk targets changes their activity
DESCRIPTION
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 PresentationTRANSCRIPT
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Phosphorylation of CDK Targets Changes Their Activity
Now performsa cell cycle function
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How are CDK’s Regulated?
1. By cyclin synthesis and destruction
2. By phosphorylation
3. By binding to CDK inhibitory proteins (CKIs)
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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!
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Cyclin Synthesis and DestructionDrives the Early Embryonic Cell Cycle
sea urchin!sea urchin!
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Cyclin Destruction is Controlled by Ubiquitination
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But is cyclin abundance the only
way to control CDK activity?
The Cell Cycle According to Cyclin Abundance
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How are CDKs Regulated?
Isolate mutants that phenocopy cdc2- alleles
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CDKs are Regulated by Phosphorylation
is a kinase is a phosphatase
CAK(CDKActivatingKinase)
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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
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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?
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and, cln1 and cln2 !!
The Identification of G1 Cyclins in S. cerevisiae
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How Does High Copy Suppression Work?
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The S. cerevisiae Cell Cycle
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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)
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Sic1 is Destroyed by Ubiquitination
(Cdc4,Cdc53)
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Once and Only Once Replication is Controlled by CDKs
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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
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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
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The Human Cell Cycle
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Cyclin Dependent Kinase Inhibitors (CKIs)
Cyclin
CDKp21
Cyclin
CDK4
Cyclin
CDK
CDK4p16
p16
p21
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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
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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
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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
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CKIs Regulate the G1-S Transition
(p16)
(p21, p27)
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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