Phosphorylation of CDK Targets Changes Their Activity Now performs a 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 hormones so that they lay eggs 2. Pack eggs into a centrifuge tube and spin 3. Remove Cytoplasmic Extract 4. Add sperm chromatin and away you go!

Cyclin Synthesis and Destruction Drives the Early Embryonic Cell Cycle 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?

CDKs are Regulated by Phosphorylation is a kinase is a phosphatase CAK (CDK Activating Kinase)

Conformational Changes Associated with CDK Phosphorylation The T-loop blocks substrate access Free CDKCDK + CyclinT161 phosphorylation Binding of cyclin moves the T-loop Phosporylation moves the 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/Cdc28 Cln2/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 cdk1 cdc2 mutant cells at 25C Transformation with human cDNA library expressed with SV40 viral promoter cdc2 transformed mutant cells at 35C

Human cyclin E Rescues cln1,2,3 Mutant cyc E cln1,2,3 triple mutant cells at 25C Transformation with human cDNA library expressed with yeast promoter cln1,2,3 transformed mutant cells at 35C

Figure 8.8 The Biology of Cancer (© Garland Science 2007)

Figure 8.9 The Biology of Cancer (© Garland Science 2007)

Figure 8.10 The Biology of Cancer (© Garland Science 2007)

Cyclin Dependent Kinase Inhibitors (CKIs) Cyclin CDK p21 Cyclin CDK4 Cyclin CDK CDK4 p16 p21

The Discovery of p21 and p16 Cultured cells Adding 35 S[Met] Metabolic labeling Lysis cells midly Immunoprecipitate Add anti-CDK4 antibody Add protein A-agarose beads SDS-PAGE Autoradiography Xiong et al. (1993) Genes & Dev. 7:1572 Cyclin CDK4 p21 CDK4 Cyclin D p21 p16 Cell Line NormalTransformed Normal Competing peptide  -CDK4

The p21 Family of CDK inhibitors (p21 CIP1/WAF1, p27 KIP1, p57 KIP2 ) CDK Cyclin active p21 + inactive CDK Cyclin p21

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 (p16 INK4a, p15 INK4b, p18 INK4c, p19 INK4d ) INK4 + CDK4/6 Cyclin D active CDK4/6 Cyclin D INK4 + inactive Russo et al. (1998) Nature 395:237 Brotherton et al. (1998) Nature 395:244

CKIs Regulate the G1-S Transition (p16) (p21, p27)

p16 is Frequently Mutated in Human Tumors

Senderowicz, A. M. et al. J Natl Cancer Inst 2000;92: Chemical structures of small molecular cdk inhibitors

Table 1. Pharmacologic effects of flavopiridol * IC 50 = concentration that inhibits growth or activity by 50%; NCI = National Cancer Institute; DTP = Developmental Therapeutics Program; VEGF = vascular endothelial growth factor. EffectIC 50, nM Growth inhibition, NCI DTP screen66 cdk inhibition Apoptosis Cell cycle arrest Cyclin D1 depletion Differentiation VEGF depletion Sensitization to standard chemotherapies Epidermal growth factor receptor tyrosine kinase inhibition Protein kinase A inhibition Senderowicz, A. M. et al. J Natl Cancer Inst 2000;92:

Table 2. Phase I trials with cdk modulators * Free = concentration of UCN-01 in saliva. Flavopiridol (96)UCN-01 (131) Schedule72-h continuous infusion every 2 wk72-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/m 2 per day for 3 days) Hypotension (78 mg/m 2 per day for 3 days) Nausea/vomiting, hyperglycemia, and hypoxemia (42.5 mg/m 2 per day for 3 days) Other toxic effectsAnorexia, proinflammatory syndromeHeadache, myalgias Suggestion of activityNon-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/m 2 per day for 3 days) 344 nM (78 mg/m 2 per day for 3 days) Total = 36.4 µM (42.5 mg/m 2 per day for 3 days) Free * = 111 nM (42.5 mg/m 2 per day for 3 days) Plasma half-life, h Senderowicz, A. M. et al. J Natl Cancer Inst 2000;92: