APC = anaphase-promoting complex

1. The different cyclins are degraded by two different E3 ligases e.g. cyclin B, the G2/M cyclin is degraded by APC = anaphase-promoting complex Not to be confused with: APC (adenomatous polyposis coli) APC (antigen-presenting cell)

2. How does APC function? M APC M The cyclin must be degraded in order for anaphase to take place

3. Protein Level cyclin A cyclin B Time M M M Remember? cyclins must be removed for mitosis to be completed

4. Negative feedback generates a repeating oscillator

5. CDKs are positively regulated by cyclins A Cyclin promotes synthesis of the nextcyclin that in turn, promotes destruction of the previous one These regulatory activities are indirect

6. active inactive CDK CDK + p21 p21 Cyclin Cyclin Mechanisms of CDKs regulation 1. Abundance of cyclins 2. CDK phosphorylation 3. Binding to CKIs (inhibitory proteins)

7. Activating phosphorylation is catalyzed by Cdk-Activating Kinases (CAKs). However, they are abundant and not regulated 3 1 Cdk Phosporylation of Thr by CAK 2 4 Cyclin Substrate binding to the kinase

8. Inhibitory phosphorylation is also involved in CDKs regulation M-CDK - e.g. Phosphorylation by Wee1 Tyr kinase blocks the CDK’s active site - e.g. Cdc25 is a phosphatase that removes this inhibitory block - The Wee1/Cdc25 switch event is regulated by substrates and extrinsic signals

9. active inactive CDK CDK + p21 p21 Cyclin Cyclin Mechanisms of CDKs regulation 1. Abundance of cyclins 2. CDK phosphorylation 3. Binding to CKIs (inhibitory proteins)

10. Cdk inhibitor proteins (CKIs) - Discovered by asking : “what binds to CDKs”? - The INK4 family proteins (e.g. p16) bind to CDK4/6, blocking cyclin D binding - The Cip/Kip family proteins (e.g. p21, p27) bind to blocking active site of multiple CDKs - CKIs normally regulate entry into S phase

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

12. p16 is Frequently Mutated in Human Tumors

13. Chemical structures of small molecular cdk inhibitors (none approved so far) Senderowicz, A. M. et al. J Natl Cancer Inst 2000;92:376-387

14. Summary - The cell cycle is controlled by Cdks, activated by cyclins and CAKs, and inhibited by CKIs - Cyclins are positively and negatively regulated by cyclin-Cdks complexes - Any process in the cell cycle is dependent on the previous one - The cell cycle progresses in the right order

15. Cyclins regulate other cyclins, both negatively and positively But, it cannot be just an intrinsic cell cycle clock…

16. Mitogensstimulate the onset of the cell cycle In this case, we are very different from yeasts

17. Mitogens control cyclin D expression

18. Mitogens control cyclin D expression - Mitogens act by activating the D-Cdk4/6 complexes - Mitogens act by inhibiting CKIs - Mitogen signaling is correlated with growth, answering the question: “have I grown enough?”

19. We actually have: 3 D-type cyclins 2 E-type cyclins 2 A-type cyclins 3 B-type cyclins

20. cyclin D and growth w/o growth signals, sub-threshold levels of enzymes will lead to quiescence (G0) START/Restriction point

21. - Activated D-Cdk4/6 initiates transcription of cyclin E and activation of E-Cdk2 - Activated E-Cdk2 allows progression through START - From here on, it’s a cell cycle clock game

22. In the next lectures we will focus on the molecular machinery that acts at the START point in normal and cancer cells