1. Phosphorylation of CDK Targets Changes Their Activity Now performs a cell cycle function

2. Yeasts have one CDK and several cyclins Humans have 4 CDKs and 4 cyclins

3. CDK/Cyclin complex regulation controls the cell cycle - Cyclin-Cdk complexes function in different phases - G1/S-Cdk complexes commit the cell to a new cell cycle - S-Cdk complexes promote S phase (and block it in G2!) - M-Cdk complexes trigger entry into mitosis - M-Cdk complexes are inactivated before anaphase X

4. Example: cycB-Cdk1 appears in mitosis, phosphorylates lamin and leads to nuclear envelope breakdown during early mitosis cycB-Cdk1 will be destroyed during mitosis to allow formation of a new nuclear envelop breakdown during telophase

5. How are CDK’s Regulated? 1.By cyclin synthesis and destruction 2.By phosphorylation 3.By binding to CDK inhibitory proteins (CKIs)

6. 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!

7. Cyclin Synthesis and Destruction Is Essential for Cell Cycle Progression sea urchin!

8. Cyclin Destruction is Controlled by Ubiquitylation But what is ubiquitylation??!!

9. Ubiquitylation: a post-translational protein modification Proteasome: the cellular garbage can E1, E2, and E3 enzyme cascade E3 ubiquitin ligase is a protein complex that confers specificity: i.e. which protein to target

10. Not to be confused with: APC (adenomatous polyposis coli) APC (antigen-presenting cell) An E3 ubiquitin ligase called the Anaphase Promoting Complex (APC) destroys mitotic cyclins (and other things)

11. How does the APC function? APC Cyclin and securin must be destroyed in order for anaphase to take place M M

12. Negative feedback generates a repeating oscillator APC Activity changes during the cell cycle

13. A cyclin promotes synthesis of the next cyclin that in turn, promotes destruction of the previous one The Cell Cycle According to Oscillating Cyclin/CDK and APC Activity

14. Once and Only Once S phase is Controlled by CDKs

15. But is cyclin abundance the only way to control CDK activity? NO! The Cell Cycle According to Oscillating Cyclin/CDK and APC Activity

16. How else are CDKs Regulated? Genetic studies in fission yeast.

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

18. 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

19. Figure 8.8 The Biology of Cancer (© Garland Science 2007) How else are CDKs Regulated? Biochemical studies in mammalian cells.

20. CDKs are regulated by Cyclin Dependent Kinase Inhibitors (CKIs) Cyclin CDK p21 Cyclin CDK4 Cyclin CDK CDK4 p16 p21

21. The Discovery of p21 and p16: what binds to CDKs? 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

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

23. Figure 8.13b The Biology of Cancer (© Garland Science 2007) Russo et al. (1996) Nature 382:325 Jeffrey et al. (1995) Nature 376:313

24. 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

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

26. p16 is Frequently Mutated in Human Tumors

27. Hanahan and Weinberg, Cell 144:646 (2011) Therapeutic Targeting of the Hallmarks of Cancer

28. Senderowicz, A. M. et al. J Natl Cancer Inst 2000;92:376-387 Chemical structures of small molecular cdk inhibitors

29. 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 inhibition40-200 Apoptosis100-1000 Cell cycle arrest100-300 Cyclin D1 depletion100-300 Differentiation100-300 VEGF depletion50-100 Sensitization to standard chemotherapies100-300 Epidermal growth factor receptor tyrosine kinase inhibition21 000 Protein kinase A inhibition122 000 Senderowicz, A. M. et al. J Natl Cancer Inst 2000;92:376-387

30. 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, h11.6588 Senderowicz, A. M. et al. J Natl Cancer Inst 2000;92:376-387

31. http://www.clinicaltrials.gov/ 51 studies for CDK inhibitors