Regulating the Eukaryotic Cell CycleIntroduction of Cell Cycle Four phases: Four phases: GG1 1, S, G, S, G2 2, M, M CyclinsCyclins and and CDKsCDKs DNA replication DNA replication and chromosome and chromosome segregationsegregationControl of Cell CycleCheckpointsCheckpointsRegulation of Cell Cycle Protein Phosphorylation and DegradationProtein Phosphorylation and Degradation2001 Nobel PrizeLeland H. Hartwell Leland H. Hartwell 1970s1970s “Checkpoint”“Checkpoint” Yeast geneticsYeast genetics 100 CDC genes100 CDC genes Start geneStart geneTim Hunt Tim Hunt 1980s1980s CyclinsCyclins Sea UrchinsSea UrchinsPaul M. NursePaul M. Nurse 1970s1970s CDKsCDKs yeast yeast “for their discoveries of key regulators of the cell cycle”“for their discoveries of key regulators of the cell cycle”Premature Chromosome CondensationM-phase Promoting Factor M-phase Promoting Factor (MPF)(MPF)Assay for MPF in a Xenopus oocyteHistory of MPFPCC M-phase Trigger MPFP32 and P45, kinase XenopusXenopus (1988) (1988)Cdc gene found in yeastyeast(1970s) (1970s) Cyclin found in urchinurchin (1980s) (1980s)MPF include CdkCdk and and cyclincyclin P32 = P34cdc2 = CDK1 P45 = P56cdc13 = cyclin BCDK and CyclinCyclinsCyclinsCDKsCDK CDK kinasekinase domain domainStructure of Human CDK2Structure of Human CDK2CDKsCDKs and CyclinsStructural Models of Human CDK2Inactive Inactive T loop blocksT loop blocksUnphosphorylatedUnphosphorylated but but T loop displacedT loop displacedPhosphorylation opens Phosphorylation opens the structure morethe structure moreActivation of CDKsActivation of Cdc2/CDK1Thr 161Tyr15CAK and CKI CAK (CDK activating kinase): CAK (CDK activating kinase): Cyclin HCdk7 Mat1CAK and CKI CAK (CAK (CdkCdk activating kinase) activating kinase) CKI (CKI (CdkCdk inhibitor): inhibitor):CIP/KIP family (p21cip1, p27kip1, p57kip2)INK4 family (p16INK4a, p15INK4b, p18INK4c, p19INK4d)CAK and CKIFunction of MPFMitosis of HeLa CellsG1G2 SM (12hrs)anaphasetelophaseLate telophaseprophaseprometaphasemetaphaseinterphaseCdc2/cyclin BCdc2/cyclin BUbiquitin-mediated Proteolysis The cell cycle is a series of degrading The cell cycle is a series of degrading events.events. Cell cycle is regulated by destruction of Cell cycle is regulated by destruction of cyclinscyclins. . There may be There may be cyclincyclin-specific -specific ubiquitinubiquitin- - conjugating enzymes that may be active conjugating enzymes that may be active only at certain times of the cell cycle.only at certain times of the cell cycle.Discovery of Cyclin B Discovery of Cyclin B UbiquitinationUbiquitinationIP of 35S-Labeled Cyclin BNormalExposureOverExposureCyclin BCyclin B-Ub Ladder2004 Nobel PrizeIrwin RoseIrwin Rose College of Medicine, College of Medicine, University of University of California, Irvine, USA California, Irvine, USA AvramAvram HershkoHershko RappaportRappaport Institute, Institute, TechnionTechnion Israel Israel Institute of Institute of Technology Haifa, Technology Haifa, Israel Israel Aaron Aaron CiechanoverCiechanover RappaportRappaport Institute, Institute, TechnionTechnion Israel Israel Institute of Institute of Technology Haifa, Technology Haifa, Israel Israel “ “for the discovery of for the discovery of ubiquitinubiquitin-mediated protein degradation“ -mediated protein degradation“ UbiquitinProteins are usually tagged for selective destruction in proteolytic complexes called proteasomes by covalent attachment of ubiquitin, a small, compact, highly conserved protein (76 amino acids). Ubiquitination of Protein SubstratesAPC and SCF APC (Anaphase-promoting complex)APC and SCF APC (Anaphase-promoting complex) SCF (Skp1- Cul1- F- box protein)APC and SCFCell Cycle Regulation M-phase regulation G1-phase regulation S-phase regulation DNA damage regulationMitotic Spindle CheckpointM-phase RegulationCyclin BCyclin BD90TubulinH2BMergeG1-phase RegulationG1-phase Rb InhibitionS-phase Regulation ORC (Origin Recognition Complex) ARC (Autonomously Replication Sequence) Cdc6 pre-RC (prereplicative complex) MCM (minichromosome maintenance proteins) DNA helicase (“licensing” factor)Critical function of Cdc6 in G1-SCritical function of Cdc6 in G1-SDNA damage checkpointDNA damage checkpointFundamental processes in the eukaryotic cell cycleFundamental processes in the eukaryotic cell cycleOverview of checkpoint controls in the cell cycleOverview of checkpoint controls in the cell cycleCell Cycle and DiseasesCell Cycle and DiseasesCell Cycle and DiseasesCell Cycle and DiseasesCell Cycle and DiseasesCell Cycle and DiseasesCell Cycle and DiseasesCell Cycle and DiseasesCell Cycle and Diseases