1. Cell Cycle Sequential series of events from one mitosis to the next Regulated at critical transition points: G1/S and G2/M Regulation is essential for growth control and for genetic integrity

2. Cell Cycle - Overview

3. The G2/M Transition Occurs when cell cycle events are “complete” Requires intact genome, replicated chromosomes, accumulation of critical factors mediating exit from interphase - prophase and entry to M phase. MPF (“metaphase-promoting factor,” a.k.a., “mitosis-promoting factor,” a.k.a. “maturation- promoting factor”) is universal regulator of this important transition

4. End-point of G2/M = Metaphase

5. How do we know that MPF is essential? Xenopus oocyte “meiotic maturation” - a model system MPF in other cells M-phase regulation revealed by yeast mutants Saccharomyces cerevisiae - CDC28 Schizosaccharomyces pombe - cdc2 “Intersecting cell cycles” - topic for journal critique, Tuesday 16 April

6. Xenopus - Meiotic Maturation

7. MPF Activity Cycles

8. Further Evidence for Universal “MPF”

9. Budding Yeast Cell Division Cycle (cdc) Mutants

10. Fission Yeast cdc Mutants

11. Evidence for “Cyclin” Activity Sea urchin (yet another model!): a specific protein cycles in quantity during cleavage divisions of the embryo This was designated “cyclin” Subsequently, it was shown that this protein co-purifies with MPF activity, but that it is not the sole component of MPF MPF = cyclin B + p34 (CDC28/cdc2)

12. Phosphorylation and Dephosphorylation Regulate MPF Activity Y15 - inhibitory phosphorylation by wee1/mik1 kinases Active site for ATP binding T161 - phosphorylated by Cdk7/CycH (CAK) This residue occludes substrate-binding site until it is phosphorylated Y15 - active dephosphorylation by Cdc25 protein phophatase

13. Kinases and Phosphatases

14. Missing Pieces: MPF/Cdk Substrates Dramatic increase in phosphorylated proteins - MPM2 antigens Autophosphorylation of cdc2 *Histone H1 phosphorylation assay (physiological significance?) Histone H3 phosphorylation on ser10 may be more significant Spatially and temporally correlated with chromosome condensation Nuclear lamins - phosphorylation leads to solubilization (breakdown of nuclear envelope)

15. How do cells get out of metaphase? Via anaphase by action of the anaphase promoting complex (APC) Injection into Xenopus oocytes of RNA encoding non-degradable cyclin B leads to sustained high MPF and metaphase arrest Ubiquitination of chromosome cohesins and cyclin targets them to the proteosome

16. Synthesis - Destruction Cycle

17. Other Cell Cycle Kinases MPF - Cdk1 = “maestro of M phase NIMA (“never in mitosis”) family of kinases (Aspergillus nidulans) Polo family kinases (Drosophila melanogaster) Aurora family kinases All implicated in control of centrosome and spindle poles

18. Multiple Cdk-Cyclin Complexes Regulate Cell-Cycle Transitions G1 inactivation of APC G1/S G2/M

19. Models for Cdk Regulation