Cell Cycle Regulation and Checkpoints

1. Cell Cycle Regulation and Checkpoints

2. Cell division cycle has 4 general steps Interphase G1 S G2 Mitosis

3. Cell Cycle Overview • G1 phase • S Phase • G2 • Mitosis • Each of these steps has multiple levels of regulation to prevent errors from occuring What regulates these steps? 3

4. Cell Cycle Regulation

5. Cyclin Dependent Kinases • Cyclical changes in Cdk activity • Serine/Threonine kinases • Very tightly regulated

6. Cyclin Dependent Kinases (CDKs) • Small kinases • Activity rises and falls • Cyclical changes

7. Cyclin Dependent Kinases • Yeast have one CDK: CDK1 • Mammals have that four are used during the cell cycle • G1 CDKs • G1/S CDKs • S CDKs • Mitotic CDKs

8. CDK Activity

9. CDKs • CDKs are dependent on Cylins • Contain a flexible T loop that can block the ATP binding site 9

10. CDKs

11. Cyclins • Proteins that bind to CDKs to activate their function • All cyclins contain a 100 aa sequence called the cyclin box • G1, G1/S, S, Mitotic Cyclins • G1 cyclins: • G1/S cyclins:

12. Cyclins, continued • S phase cyclins: • Mitotic Cyclins

13. Cyclin Expression

14. Cyclins and CDKs

15. Regulation of CDKs • CDK must be phosphorylated for activation • CDK inhibitors

16. CKIs • CKIs p21, p27 and p57 inhibit late G1 cyclin-CDK and S phase cyclin-CDK activity 16

17. Ink4 family • Ink4s (inhibitors of kinase 4) specifically control mid G1 phase • p15 and p16 17

18. CKIs

19. Regulation of Cell Cycle: E2F • E2F factors are encoded by delayed response genes 19

20. Regulation of Cell Cycle: E2F • When bound to Rb, it functions as a transcriptional repressor

21. Regulation of Cell Cycle: E2F • As they accumulate, S phase cyclin-CDKs and mitotic cyclin-CDKs maintain Rb phosphorylation through S, G2, and early M 25

22. Cell cycle kinase inhibitor p16 regulates Restriction Point

23. Cdk/cyclin activity modify Rb transcriptional repression Overexpression of p16 results in cell cycle arrest p16 binds to and inhibits cdk4/cyclinD complex during early G1 Cdk4/cyclinD1 phosphorylate Rb Rb is a transcriptional repressor that inhibits transcription of genes essential for entry into the cell cycle Rb repression is released by cdk/cyclin phosphorylation High levels of p16 therefore lead to less cell cycle progression by maintaining Rb-mediated repression Mid G1 Late G1

24. Yeast Metazoans

25. G1 Cyclins regulate DNA replication Experimental Design: Stimulate cell to divide Microinject antibody against cyclin D (G1 cyclin) that block its function Add BrdU (nucleotide analog that labels newly synthesized DNA and can be distinguished from endogenous DNA) Compare BrdU incorporation into the DNA of cells with or without the blocking G1-cyclin antibody

26. G1 Cyclins regulate DNA replication Blue bars- cells injected with control antibody; set to 100% for each time point Red bars- cells injected with anti-cyclin D antibody BrdU levels measured over time Blocking G1-cyclin function delays entry into S-phase

27. Origins of Replication • DNA replication is initiated from prereplication complexes assembled at origins during early G1 • S-phase cyclin-CDK complexes simultaneously trigger initiation from prereplicationcomplexes 27

28. Loading of complexes at origins of replication

29. Degradation S-Phase Inhibitor Triggers DNA Replication • S-phase cyclin-CDK heterodimers accumulate in late G1

30. Degradation S-Phase Inhibitor Triggers DNA Replication • Sic1 is degraded and the S-phase cyclin-CDK complexes induce DNA replication • Permits the sudden activation of large numbers of complexes • Prevents a gradual increase in kinase activity 30

31. SCF and APC/C • Proteasomaldegradation is mediated by two ubiquitin-protein ligase complexes • SCF and APC/C • Control three major transitions in the cell cycle: 31

32. SCF and APC/C • APC/C directed to polyubiquitinylate the anaphase inhibitor securinby Cdc20 • Cdh1 targets the APC/C to remaining cyclin B

33. SCF and APC/C • SCF ubiquitin-protein ligase is not regulated by phosphorylation of specificity partners • Phosphorylation of substrate

34. SCF and APC/C

35. Ubiquitin-mediated protein degradation in Anaphase Anaphase Promoting Complex is an E3 ubiquitin ligase APC interacts with distinct proteins to target degradation of different substrates Specificity factor-substrate Early Anaphase: Cdc20- securin Late Anaphase: Cdh1- M-cyclin Degradation of proteins dictates the forward movement of the cell cycle

36. MPF MPF is a complex of 2 proteins: mitotic cyclin and cdk Regulation of both proteins dictates MPF activity Mitotic Cyclin levels increase due to increased synthesis which peaks at metaphase Mitotic Cyclin levels precipitously decrease at late anaphase due to destruction of the protein MPF activity is inhibited by phosphorylation of M-CDK by Wee1 MPF activity is activated by removal of phosphate by cdc25

37. Regulation of MPF activity • How is MPF activity regulated? • Control the stability of cyclins • Addition layers of regulation also exist 37

38. Regulation of MPF • Cyclin and cdk form complex that has low kinase activity • Wee1: protein tyrosine kinase that phosphorylates an inhibitory residue in CDK

39. Regulation of MPF • Conformational changes in the cyclin-CDK2 complex increases its affinity for protein substrates

40. Regulation of MPF –Inhibitory phosphorylation sites

41. Cell Cycle Checkpoints • Each step must take place and be completed before the next stage can occur • DNA replication and chromosome segregation must occur with complete accuracy • Cells have many levels of regulation to control these events

42. Cell Cycle Regulation • Once a cell passes the restriction point in late G1, it is committed to passing through S phase

43. Checkpoints and Cancer • Elevated cyclin D1 is common in cancer • Loss of Rb is common in cancers that arise later in life, including carcinomas of the breast, lung, bladder 43

45. Cell Cycle Regulation

46. Cell Cycle Regulation • Major steps in the cell cycle--Step 1 • Cylin-CDK is absent in early G1 • Hypophosphorylated DNA replication initiation factors are free to bind ORC complexes at origins • generate pre-replication complexes that are inactive until phosphorylation by S-phase cyclin-cdk 46

47. Cell Cycle Regulation • In mid G1, mid G1 cyclin-cdks are expressed • Mitotic cyclins have been targeted to the APC/C by the specificity factor Cdh1 to inactivate • allow new Cyclin B to accumulate (step 2) • Mid G1 cyclin-cdks also phosphorylate transcription factors to activate late G1 and S phase cyclins(step 3) 47

48. Cell Cycle Regulation • When Cyclin B is expressed, they are immediately bound by inhibitors • When cyclin B-cdk activity peaks, they phosphorylate the inhibitors (step 4) to mark them for poly-ubiquitination by SCFub-protein ligase (Step 5) • Degradation of S phase cyclin-cdk inhibitors releases S phase cyclin-cdkactivities to phosphorylate key regulatory sites in prereplication complexes, stimulating initiation of replication (step 6) • Mitotic cyclin-cdks are expressed in late S phase and G2

49. Cell Cycle Regulation • After DNA replication finishes, mitotic cyclin-cdks are activated by Cdc25 phosphatase • These events promote early mitosis

50. Phosphorylation of Nuclear Lamins