Plant Molecular and Cellular Biology Lecture 9: Mechanisms of Cell Cycle Control Gary Peter

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Plant Molecular and Cellular Biology Lecture 9: Mechanisms of Cell Cycle Control Gary Peter 9/10/ Learning Objectives Explain why a cell cycle was selected for during evolution Explain the conceptual
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Plant Molecular and Cellular Biology Lecture 9: Mechanisms of Cell Cycle Control Gary Peter 9/10/2008 1 Learning Objectives Explain why a cell cycle was selected for during evolution Explain the conceptual basis for cell cycle regulation Explain the structure and function of the proteins that mediate cell cycle progression Explain the molecular mechanisms of the complex feedback loops Explain the status of knowledge of G1 control and the plant cell cycle 9/10/2008 PMCB Lecture 9: G. Peter 2 Xenopus Cell Free Extracts for Studying DNA Replication and Mitosis 9/10/2008 PMCB Lecture 9: G. Peter 3 ge/portal/0ba528148b1d8a26e04012ac0e017a42&h=327&w=472&sz=28&hl=en&start=12&um=1&tbnid=du53hf007ltj6m:&tbnh=89&tbn w=129&prev=/images%3fq%3dxenopus%2bcell%2bcycle%26svnum%3d10%26um%3d1%26hl%3den%26client%3dfirefoxa%26rls%3dorg.mozilla:en-us:official%26sa%3dn Purification of Maturation Promoting Factor from Xenopus A unit of MPF was defined as the minimum amount need to induce 20% of the pronuclei to enter M-phase a after 2h reaction 9/10/2008 PMCB Lecture 9: G. Peter 4 Lohka et al PNAS 85: Purification of Maturation Promoting Factor from Xenopus Most purified preparations of MPF Contains histone H1 kinase and autophosphorylation activities Two main polypeptides co-elute with activity Cyclin dependent kinase and cyclin 9/10/2008 PMCB Lecture 9: G. Peter 5 Lohka et al PNAS 85: Biochemical Approach to Dissection Immunodepletion and add back experiments Cyclin depletion Engineered proteins used to show ubiquitin degradation of cyclin B in mitosis 9/10/2008 PMCB Lecture 9: G. Peter 6 Effect of Cyclins on Chromosome Duplication in Xenopus Early Embryo Cycles A) Variation in cyclin A, B and E B) cyclin E enters the nucleus and triggers duplication cyclin B does not enter the nucleus unless it is engineered with a nuclear localization signal 9/10/2008 PMCB Lecture 9: G. Peter 7 Murray 2004, Cell 116: Regulating Cyclin Destruction Two major proteolytic systems regulate the cell cycle- both are E3 complexes that facilitate the transfer of Cyclin SCF APC ubiquitin from the active site of an E2 G1 +++ enzyme to a substrate that is thus marked for destruction by the proteasome. SCF is active throughout the cell cycle, and the stability of individual substrates is regulated by their phosphorylation and the level of the accessory proteins that recognize them. APC is activated at the onset of anaphase and ubitquitinates its substrates as cells exit from mitosis APC appears to recognize substrates directly and its activity is regulated during the cell cycle as a result of phosphorylation by Cdk1 and other kinases. M +++ 9/10/2008 PMCB Lecture 9: G. Peter 8 Murray 2004, Cell 116: Major Events at Cell Cycle Stages G1 cell growth and centrosome replication (only in animals) Cyclin D/E S- DNA synthesis Cyclin A G2 Cell growth and gap in cell cycle to insure that DNA replication is complete M Chromosome condensation and separation 9/10/2008 PMCB Lecture 9: G. Peter 9 Cyclin B Budding Yeast Cell Cycle: Control of G1 Progression Upon exiting M phase G1-cdk accumulates, but needs to be off for growth and extracellular signals to induce the next cell cycle Hct1, a close relative of cdc20, binds APC to keep M-cyclin level low in G1 Hct1-APC activity is high late in mitosis Sic1 is a CKI for M-cdk Hct1-APC, Sic1, and reduced transcription of cyclins act together to ensure early G1 has virtually no cdk activity 9/10/2008 PMCB Lecture 9: G. Peter 10 Regulation of the G1/S Transition Extracellular signals modulate the activation of CDK4 & CDK6 associate with the D- cyclins and phosphorylate prb releasing E2F and DP1 transcription factors that induce the expression of genes responsible for G1/S transition and S 9/10/2008 PMCB Lecture 9: G. Peter 11 progression Regulation of the G1/S Transition CDK2/cyclin E complex is responsible for the G1/S transition and regulation of centrosome duplication in animal cells Cyclin A expression drives the start of DNA synthesis 9/10/2008 PMCB Lecture 9: G. Peter 12 Regulation of S/G2 Transition Little is known about this transition Late in S CDK1 associates with cyclin A to trigger the transition Check points important here 9/10/2008 PMCB Lecture 9: G. Peter 13 Cell Cycle Checkpoints: Sensors for Preventing Disastrous Premature Cell Cycles Unfavorable environments Unreplicated/incomplete DNA synthesis Damaged DNA Spindle unattached 9/10/2008 PMCB Lecture 9: G. Peter 14 DNA Replication Checkpoints: Prevention of Mitosis Before DNA Synthesis is Complete Unfinished forks are somehow sensed and send a negative intracellular signal that inhibits cdc25 protein phosphatase which blocks activation of M-cdk and arrests the cell cycle to permit time for completion of DNA replication prior to mitosis 9/10/2008 PMCB Lecture 9: G. Peter 15 DNA Damage Two checkpoints Late G1 Late G2 Late G2 checkpoint functions similarly to the checkpoint for incomplete DNA replication by blocking activation of M-cdk Late G1 checkpoint, DNA damage induces p53 which in turn stimulates transcription of CKI (p21) which binds to and inhibits G1/S-cdk and S-cdk blocking entry into S phase 9/10/2008 PMCB Lecture 9: G. Peter 16 Regulation of G2/M Transition Late in G2 CDK1/cyclin B appears and triggers the G2/M transition by phosphorylating a large number of subunits 9/10/2008 PMCB Lecture 9: G. Peter 17 Mitosis Phosphorylation of APC by CDK1/cyclin B is required for transition to anaphase 9/10/2008 PMCB Lecture 9: G. Peter 18 Control of Mitosis by M-CDKs Dual phosphorylation in late G2 of cdc25 protein phosphatase by polo kinase and M-cdk turns on all of the M-cdk that has accumulated in G2 Positive feedback loop model M-cdk phosphorylation activates chromosome condensation (condesin complex) and microtubule rearrangements and other events leading to mitotic spindle assembly 9/10/2008 PMCB Lecture 9: G. Peter 19 Spindle-Attachment Checkpoint: Prevention of Mitosis Before DNA Synthesis is Complete This checkpoint insures that all chromosomes are properly attached to the spindle before sister chromatid separation occurs Any kinetochore (region of chromosome that attaches spindle microtubules) that is not properly attached sends out a negative signal blocking cdc20-apc activation and sister chromatid separation 9/10/2008 PMCB Lecture 9: G. Peter 20 Regulation of the M/G1 Transition The mitotic CDK/cyclin B activity is in activated by a CKI (sic1-yeast) Hct-APC target ubiquitination and proteolysis of M CDK CKI sic1 and Hct are activated by dephosphorylation 9/10/2008 PMCB Lecture 9: G. Peter 21 Summary Specific cyclin expression and activation of CDK s drive progression through the cell cycle Extracellular and intracellular signals are critical for cell cycle progression Control of G1/S is regulated by cyclin D expression which is controlled by hormones and other signals Checkpoints to insure complete DNA synthesis, no DNA damage and spindle attachment are critical go no go points 9/10/2008 PMCB Lecture 9: G. Peter 22
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