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Phosphorylation of CDK Targets Changes Their Activity

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

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Phosphorylation of CDK Targets Changes Their Activity

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

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

  3. Generation of a “Cycling” Frog Egg Extract • Inject females with hormones • so that they lay eggs 2. Pack eggs into a centrifuge tube and spin 4. Add sperm chromatin and away you go! 3. Remove Cytoplasmic Extract

  4. Cyclin Synthesis and Destruction Drives the Early Embryonic Cell Cycle sea urchin! sea urchin!

  5. Cyclin Destruction is Controlled by Ubiquitination

  6. The Cell Cycle According to Cyclin Abundance But is cyclin abundance the only way to control CDK activity?

  7. How are CDKs Regulated? Isolate mutants that phenocopy cdc2- alleles

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

  9. Conformational Changes Associated with CDK Phosphorylation Free CDK CDK + Cyclin T161 phosphorylation The T-loop blocks substrate access Binding of cyclin moves the T-loop Phosporylation moves the T-loop more

  10. How does the G1-S Transition Work? cdc2 mutants arrest in G2 cdc28 mutants arrest in G1 but… S. pombe cdc2+ can subsitute for S. cerevisiae cdc28 and vice versa Why?

  11. The Identification of G1 Cyclins in S. cerevisiae and, cln1 and cln2 !!

  12. How Does High Copy Suppression Work?

  13. The S. cerevisiae Cell Cycle

  14. Cln3/Cdc28 Clb5/Cdc28 Clb6/Cdc28 G1 S SIC1 Cln1/Cdc28 Cln2/Cdc28 (cyclin/cdk) make a bud The G1-S Transition in S. cerevisiae Growth Signal

  15. Sic1 is Destroyed by Ubiquitination (Cdc4, Cdc53)

  16. Once and Only Once Replication is Controlled by CDKs

  17. Human cdc2 (cdk1) Rescues cdc2 Mutant Transformation with human cDNA library expressed with SV40 viral promoter cdc2 mutant cells at 25C cdc2 transformed mutant cells at 35C cdk1 cdk1

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

  19. The Human Cell Cycle

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

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

  22. active inactive CDK CDK + p21 p21 Cyclin Cyclin p27Kip1 CDK2 Cyclin A The p21 Family of CDK inhibitors (p21CIP1/WAF1, p27KIP1, p57KIP2) Cyclin A CDK2 Jeffrey et al. (1995) Nature 376:313 Russo et al. (1996) Nature 382:325

  23. active inactive CDK4/6 CDK4/6 INK4 INK4 + + Cyclin D Cyclin D Russo et al. (1998) Nature 395:237 Brotherton et al. (1998) Nature 395:244 The INK4 Family of CDK inhibitors (p16INK4a, p15INK4b, p18INK4c, p19INK4d)

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

  25. p16 is Frequently Mutated in Human Tumors

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