M267 Lecture 4, Cell Cycle March 14, 2005

1. M267 Lecture 4, Cell Cycle March 14, 2005

2. G1 M G2 S

3. Cyclin D1 cdk4 Cyclin B G1 M cdc2 G2 Cyclin A cdk2 S

4. p p p p p p p p p cdk cdk WEE1 cyclin Phosp. Thr 161 cyclin WEE1 Dephosp. Thr 161 p cdc25 p cdk cdc25 cyclin ACTIVE CDK Degrade cyclin CKI CKI cdk cdk p cyclin

5. G2 S G1

6. Cohesins cyclin deg. cdc2/28 G2 Prophase Metaphase Anaphase Telophase G1

7. Proteolysis Rules

8. Cyclin B degradation In mitosis

9. -p34cdc2cyB 0 15 30 50 0 15 30 50 min interphase

10. -p34cdc2cyB +p34cdc2cyB 0 15 30 50 0 15 30 50 min min interphase mitotic

11. D PROTEIN A

12. D PROTEIN A 125I Ig BEAD

13. M extract I extract buffer

14. M extract I extract buffer Time 0 6 12 20 25 30 35 40 60

15. Protein Turnover: Protein Degradation and Turnover: Ubiquitin-ATP dependent proteolysis takes place in 26S Proteasomes: Proteasomes are hollow cylinders w/ cap structures on each end. Proteolysis occurs inside!

16. cyclin B cyclin B sythesis

17. cyclin B cdc2 p p p cyclin B cyclin B sythesis

18. cyclin B cyclin B cdc2 p cdc2 p cdc25 p p p p ACTIVE cdc2/ cyclin B KINASE cyclin B cyclin B sythesis

19. cyclin B cyclin B cdc2 p cdc2 p cdc25 p p p p cyclin B cyclin B + cdc2 cyclin B sythesis

20. cyclin B cyclin B cdc2 p cdc2 p cdc25 p p p p cyclin B cyclin B + cdc2 p cdc2 cyclin B sythesis p p

21. cyclin B cyclin B cdc2 p cdc2 p cdc25 p p p p cyclin B cyclin B + cdc2 p cdc2 cyclin B sythesis p p

22. cyclin B cyclin B cdc2 p cdc2 p cdc25 p p p p cyclin B cyclin B + cdc2 p cdc2 cyclin B sythesis p p

23. cyclin B protein X cyclin B cdc2 p cdc2 p cdc25 p p p p protein X p p cyclin B cyclin B + cdc2 p cdc2 cyclin B sythesis p p

24. cyclin B protein X cyclin B cdc2 p cdc2 p cdc25 p p p p protein X p p cyclin B cyclin B protease + cdc2 p cdc2 cyclin B sythesis p p

25. cyclin B protein X cyclin B cdc2 p cdc2 p cdc25 p p p p protein X p p cyclin B cyclin B protease + cdc2 p cdc2 cyclin B sythesis p p

26. Cyclin B Cyclin B Cyclin B p anaphase promoting complex Ub Ub p Ub Ub Ub Ub

27. Ub Ub Ub Ub p p proteasome Cyclin B Cyclin B Cyclin B Ub Ub Ub Ub APC

28. Cln1- Cln2- Cln3- G1 M G2 S

29. Cln1- Cln2- Cln3- G1 M G2 S Met-cln2 (met repressible) Gal-clb2 (gal inducible)

30. Minus methionine Cln1- Cln2- Cln3- G1 M G2 S Met-cln2 (met repressible) Gal-clb2 (gal inducible)

31. Cyclin B RNA

32. Cyclin B RNA These cells do not express any G1 cyclins (clns). Consequently, they arrest in G1. They can accumulate cyclin B mRNA in response to galactose.

33. Cyclin B RNA Cyclin B protein These cells do not express any G1 cyclins (clns). Consequently, they arrest in G1. They can accumulate cyclin B mRNA in response to galactose. Although cyclin B mRNA can be made, cyclin B protein cannot be made – cyclin B protein continues to be degraded.

34. Cyclin B RNA Cyclin B protein H1 kinase activitry These cells do not express any G1 cyclins (clns). Consequently, they arrest in G1. They can accumulate cyclin B mRNA in response to galactose. Although cyclin B mRNA can be made, cyclin B protein cannot be made – cyclin B protein continues to be degraded. Since no cyclin is available to activate cdc28, no CDK activity (e.g. histone phosphorylation activity) is present.

35. Cyclin B RNA Cyclin B protein H1 kinase activitry If one removes methionine, Cln2 (the G1 cyclin) is made. Now cyclin B protein can be made.

36. Cyclin B RNA Cyclin B protein H1 kinase activitry If one removes methionine, Cln2 (the G1 cyclin) is made. Now cyclin B protein can be made. Now that cyclins are made, the CDK catalytic subunit can be activated, and CDK activity (histone phosphorylation) can occur

37. G1 G1 S S M M G1 G1 S S M M Finish Finish Start Start Start Start CLNs CLNs CLNs CLNs CLBs CLBs CLBs CLBs Cyclin Oscillations in yeast CLN1, CLN2 and CLN3 associated kinases are absent in early G1, reach peak levels at the G1-S transition, and decline as cells enter G2. These kinases not only promote budding and DNA synthesis, but also inactivate CLB cyclin proteolysis. Activation of the mitotic kinase during G2 causes repression of CLN1, CLN2 and CLN3 synthesis and onset of M phase and ultimately leads to the reactivation of proteolysis.

38. APC mediated destruction machinery for cyclin B Cyclin Oscillations in yeast CLN1, CLN2 and CLN3 associated kinases are absent in early G1, reach peak levels at the G1-S transition, and decline as cells enter G2. These kinases not only promote budding and DNA synthesis, but also inactivate CLB cyclin proteolysis. Activation of the mitotic kinase during G2 causes repression of CLN1, CLN2 and CLN3 synthesis and onset of M phase and ultimately leads to the reactivation of proteolysis. Cyclin B proteolysis then persists during G1 until the reactivation of CLN cyclins

39. Cyclin B RNA Cyclin B protein H1 kinase activitry What would happen to CLB2 synthesis if the cyclosome was “broken”?

40. Cyclin B RNA Cyclin B protein H1 kinase activitry What would happen to CLB2 synthesis if the cyclosome was “broken”? Gal promoter CLB2 protein b-gal protein

41. pGAL-CLB2 -GAL +GAL WT

42. pGAL-CLB2 -GAL +GAL WT Cdc16-123 Cdc23-304 Cse1-22

43. Cse1 cdc16 cdc27 cdc23 The cyclosome, the “machine” that ubiquitinates cyclin B, and leads to cyclin B desruction

44. Degradation of non-cyclin proteins by the proteasome

45. Express a non-degradable Cyclin B

46. Express a non-degradable Cyclin B Cells do not arrest at anaphase, even though cyclin B is present.

47. Express a non-degradable Cyclin B Cells arrest here Cells do not arrest at anaphase, even though cyclin B is present.

48. Inhibit the activity of the cyclosome/APC Cells arrest at anaphase

49. Inhibit the activity of the cyclosome/APC APC directed degradation of some other protein – before cyclin is degraded – must be necessary for sister chromatid separation; i.e., for the metaphase to anaphase transition to occur. ? Cells arrest at anaphase

50. Cut 2 accumulates in anaphase in APC mutants