Topic 2A Regulation of the Cell Cycle

1. Topic 2ARegulation of the Cell Cycle

2. Please note: • These slides replace slides 36 to end of topic 2

4. Checkpoints • Restriction • G2-M checkpoint • Anaphase checkpoint • Mitotic spindle checkpoint • DNA replication checkpoint

5. Nobel Prize Physiology 2001

6. Major players • Cyclin • Cyclin-dependent kinases (Cdk)

7. Cyclin • Concentrations change during cell cycle

8. Cyclin • Serine-threonine kinases • # different types of cyclin • Cyclin A, D and E—G1S transition • Cyclin B used in G2M transition • Cyclin D is also called G1 cyclin • Not expressed cyclically • Synthesized by growth factor stimulation

9. More on cyclins • Cyclins for G2M transition = mitotic cyclins • Cyclins for restriction point = G1 cyclins • S phase checkpoints use S cyclins

11. Cyclin-dependent kinases • Enzymes that phosphorylate other proteins • Require cyclin binding to become active enzymes

12. G2-M transition • Masui’s experiment—maturation-dependent factor present in oocytes

14. Maturation Promoting Factor

15. Further Refinements • Isolation and purification of protein components • Maturation promoter factor • Consists of cyclin and cyclin-dependent kinase (CdK)

16. S. pombe studies • If small cells—too many divisions • Wee-1 • Encodes inhibitory kinase • Big yeast cells—not enough cell divisions • Cdc-25 • Activating phosphatase

17. Cdc25 and Wee-1 • Wee-1 phenotype caused by mutation in cdc2 gene • Phosphorylates inhibitory tyrosine molecule • Cdc25 • Protein phosphatase—dephosphorylates tyrosine

18. Activation of Cdks • Cyclin necessary but not sufficient • Must get a specific set of phosphorylations Cdk25 Wee-1 CAK

21. G2/M Checkpoint • Uses Cdk/cyclin complex and M-Cdk • If everything ready for mitosis, then: • M-cyclin binds Cdk • Wee-1 phosphorylates inhibitory sites • Cdk-activating kinase phosphorylates activation phosphorylation site • Cdc-25 phosphatase removes inhibitor phosphates

22. Role of Activated Mitotic Cdk-cyclin • Phosphorylates lamin proteins • Phosphorylates condensin • Phosphorylates MT-associated proteins

23. Anaphase Promoting Complex 

24. APC • Mitotic Cdk-cyclin complex • Activates APC • Ubiquitin ligase

26. Ubiquitin ligase • Adds ubiquitin to a protein

27. Addition of ubiquitin • Protein targeted for destruction

30. APC activities • Ubiquitinates • Securin • Mitotic cyclin

31. Ubiquitin-mediated degradation of cyclins • Exit from mitosis

32. Restriction Checkpoint • Influenced by: • Growth factors • Nutrients • Cell Size • DNA damage • TOR • Passage thru this checkpoint commits cell to DNA replication

33. mTOR • Mammalian Target of rapamycin • Central role in signalling network that controls cell size • Coordinates cell cycle progression • Activated in presence of nutrients and growth factors • Facilitates entry into S phase

34. Inhibition of mTOR • Prevent cyclin-dependent kinase (Cdk) activation • Inhibits Rb • Accelerates turnover of cyclin D • Together-- G1 phase arrest

35. Regulation of Restriction • G1 cdk-cyclin complex • Phosphorylates Rb (retinoblastoma protein) • Release of Rb • Activation of E2F transcription factor

37. Mitotic Spindle Checkpoint • Ensures all chromosomes attached to the mitotic spindle • Interacts with APC pathway • Errors led to aneuploidy

39. When kinetochores not attached

40. DNA Replication Checkpoint • Monitors the state of DNA replication • Occurs between G2 and M • Inhibition of DNA replication • Phosphatase that catalyzes final step in mitotic Cdk-cyclin pathway is inhibited

43. Should be able to: • Define the cell cycle phases • Describe the purpose of mitosis • Provide details on each stage of mitosis • Describe how the protein motors move the chromosomes • Explain how cytokinesis works in animal cells • Describe the checkpoints for the cell cycle