MOLECULAR CELL BIOLOGY SIXTH EDITION MOLECULAR CELL BIOLOGY SIXTH EDITION Copyright 2008 © W. H. Freeman and Company CHAPTER 20 Regulating the Eukaryotic Cell Cycle CHAPTER 20 Regulating the Eukaryotic Cell Cycle

Chapter 20 Regulating the Eukaryotic Cell Cycle Checkpoint surveillance mechanism Master controllers Regulated degradation of proteins In this chapter

20.1 Overview of the Cell Cycle and Its Control I.An Ordered Series of Events Leading to Cell Replication *1. G 1 (first gap) phase *2. S phase *3. G 2 (second gap) phase *4. M phase a. Prophase b. Metaphase c. Anaphase d. Telophase *5. Chromosome 의 number 변화 *6. Cell cycle 에 걸리는 시간

II. Regulated Protein Phosphorylation and Degradation Control Passage (1)G 1 Cyclin-CDKs (2) S-phase Cyclin-CDKs (3) Mitotic Cyclin-CDKs (4) Ubiquitin-Protein Ligase (5) Checkpoint Mechanisms a. Checkpoints b. Cell division 의 정확성을 위해 중요 c. Cyclin-CDKs 의 활성을 조절

III. Diverse Experimental Systems to Identify and Isolate Cell-Cycle Control Proteins (1) Cell-fusion experiment with cultured mammalian cells a. Mitotic cell 과 G 1 -phase cell 의 fusion b. G 1 -phase cell 과 S-phase cell 의 fusion (2) Temperature-sensitive experiment with cultured yeast cells a. Saccharomiyces cerevisie (budding yeast) b. Saccharomiyces pombe (fission yeast) c. Temperature-sensitive mutant d. Wild type transformation

20.2 Control of Mitosis by Cyclin and MPF Activity Xenopus oocyte 의 cell division I. Maturation-Promoting Factor (MPF) stimulates Meiotic Maturation in Oocytes and Mitosis in Somatic Cells (1) Meiotic maturation 을 promoting 하는 diffusible factor a. G 2 -arrested Xenopus oocyte 의 분리 b. MPF (Mitosis promoting factor) c. Mammalian cell 에서 MPF 효과

II. Mitotic Cyclin Was First Identified in Early Sea Urchin Embryos (1) Cyclin Component 확인 (2) Cyclin B III. Cyclin B Level and Kinase Activity of MPF Change Together (1) MPF activity 의 cycling 과 Mitotic event 와의 상관관계 (2) Kinase activity of MPF a. H1 kinase activity b. MPF activity assay c. Mitosis entry and exit

IV. Anaphase-Promoting Complex (APC/C) Control Degradation of Mitotic Cyclins and Exit from Mitosis *1 B-type cyclins *2 Polyubquitination *3 Degradation by proteasome (1) Ubiquitin-protein ligase a. B-type cyclins or other target protein 에 ubiquitination b. Destruction box 를 recognition (2) Regulation of mitotic cyclin levels a. APC/C specificity factor = Cdh1 b. Phosphorylation of Cdh1 by G 1 cyclin-CDK c. Dephosphorylation of Cdh1 by Cdc14

20.3 Cyclin-Dependent Kinase Regulation During Mitosis I. MPF Component Are Highly Conserved (1) cdc2+ gene (2) Cdc 13 (S. pombe) (3) Cdc 2 antibody II. Phosphorylation of the CDK Subunit Regulates the Kinase activity of MPF (1) Temperature-sensitive cdc25 gene (2) wee1 gene

(3) Regulation of kinase activity of MPF a. Mitotic cyclin 과 CDK 가 결합한 MPF 는 매우 낮은 activity b. Wee1: Protein-tyrosine kinase c. CAK: CDK activating kinase d. Cdc25: Phosphatase e. Y15 를 F15 로 교체 시 III. Conformational Changes Induced by Cyclin Binding and Phosphorylation Increase MPF Activity (1) Free CDK2 (human) (2) Cyclin-CDK2 Binding 시 (3) Phosphorylation 과 dephosphorylation 에 의한 conformational change

20.4 Molecular Mechanisms for Regulating Mitotic Events I.Phosphorylation of Nuclear Lamins and Other Proteins (1) Promotes Early Mitotic Events (2) Nuclear Lamina a. Lamin 구조 b. Lamin filament c. MPF 에 의한 phosphorylation d. Lamin A/C gene 의 mutation 과 질병과의 관계 (3) Nuclear envelope proteins phosphorylated by MPF (4) SMC proteins (5) Phosphorylated condensin (6) Other proteins

II. Unlinking of Sister Chromatids (1) Cyclin-destruction box a. Polyubiquitination site b. Reaction mixture 에 농도를 높여가며 첨가 c. Decondensation 과 segregation 이 delay (2) Cohesin a. Linkage of sister chromatids b. SMC protein family c. Antibody treatment d. Phosphorylation by protein kinase (3) Regulation of cohesin cleavage a. Securin b. Separase c. Cdc20 d. 작동 mechanism

III. Chromosome Decondensation & Reassembly of the Nuclear Envelope Depend on Dephosphorylation of MPF Substrate (1) Cdc 14 a. Phosphatase b. MPF antagonist c. Dephosphorylation of Cdh1 d. Other substrate ( 2) Reassembly of the Nuclear Envelope (telophase) a. Extension of ER b. Reassembling of NPCs by dephosphorylation c. Ran-GEF d. Karyomere e. Dephosphorylation of lamin A and C

20.5 Cyclin-CDK & Ubiquitin-Protein Ligase Control of S-phase I.A CDK is Critical for S-phase Entry *1 S-phase entry *2 Critical size *3 START (1) cdc28 temperature sensitive mutant a. Wild-type: CDC28 b. cdc28 ts mutant cell (2) Yeast CDK (Highly homologous) a. S. cerevisiae 에서 cell cycle control : G 1 to S transition b. S. pombe 에서 cell cycle control : G 2 to M transition

II. S-Phase Promoting Factor (SPF) & Three G 1 Cyclins (1)Identification of G 1 -Cyclins (2) CLN gene products a. CLN 1,2,3 b. 약 100 amino acids 가 high homologous region : CDK interaction domain c. No destruction box d. 영양 상태에 따른 CLN gene 발현 (3)Cdh1 : Late-G1 cyclin-CDK subtrate (4)Early S-phase cyclins

III. Degradation of the S-phase inhibitor (Sic1) Triggers DNA Replication (1)Sic1 (S-Phase inhibitor) (2)Proteasomal degradation 에 의한 major transition 의 조절 (3)SCF Ubiquitin-protein ligase IV. Multiple Cyclins Regulate the Kinase Activity during Different Cell-cycle Phases