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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)
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!
But is cyclin abundance the only
way to control CDK activity?
The Cell Cycle According to Cyclin Abundance
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?
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)
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 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
Figure 8.13b The Biology of Cancer (© Garland Science 2007) Russo et al. (1996) Nature 382:325
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
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
Senderowicz, A. M. et al. J Natl Cancer Inst 2000;92:376-387
Chemical structures of small molecular cdk inhibitors
Table 1. Pharmacologic effects of flavopiridol*
•IC50 = concentration that inhibits growth or activity by 50%;
•NCI = National Cancer Institute; DTP = Developmental Therapeutics Program; VEGF = vascular endothelial growth factor.
Effect IC50, nM
Growth inhibition, NCI DTP screen 66
cdk inhibition 40-200
Apoptosis 100-1000
Cell cycle arrest 100-300
Cyclin D1 depletion 100-300
Differentiation 100-300
VEGF depletion 50-100
Sensitization to standard chemotherapies 100-300
Epidermal growth factor receptor tyrosine kinase inhibition 21 000
Protein kinase A inhibition 122 000
Senderowicz, A. M. et al. J Natl Cancer Inst 2000;92:376-387
Table 2. Phase I trials with cdk modulators
* Free = concentration of UCN-01 in saliva.
Flavopiridol (96) UCN-01 (131)
Schedule 72-h continuous infusion every 2 wk 72-h continuous infusion every 4 wk (cycle 1) followed by 36-h continuous infusion every 4 wk (cycles 2 or higher)
Dose-limiting toxicity (maximal tolerated dose)
Diarrhea (50 mg/m2 per day for 3 days)
Hypotension (78 mg/m2 per day for 3 days)
Nausea/vomiting, hyperglycemia, and hypoxemia (42.5 mg/m2 per day for 3 days)
Other toxic effects Anorexia, proinflammatory syndrome Headache, myalgias
Suggestion of activity Non-Hodgkin's lymphoma and renal, colon, gastric, or prostate cancer
Melanoma, non-Hodgkin's lymphoma, or leiomyosarcoma
Median plasma concentration at maximal-tolerated dose
271 nM (50 mg/m2 per day for 3 days) 344 nM (78 mg/m2 per day for 3 days)
Total = 36.4 µM (42.5 mg/m2 per day for 3 days) Free* = 111 nM (42.5 mg/m2 per day for 3 days)
Plasma half-life, h 11.6 588
Senderowicz, A. M. et al. J Natl Cancer Inst 2000;92:376-387