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MOLECULAR CELL BIOLOGY
SIXTH EDITION
CHAPTER 20
Regulating the Eukaryotic
Cell Cycle
Copyright 2008 © W. H. Freeman and Company
Chapter 20 Regulating
the Eukaryotic Cell
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Checkpoint surveillance mechanism
Master controllers
Regulated degradation of proteins
In this chapter
Cycle
20.1 Overview of the Cell Cycle and Its Control
I.
An Ordered Series of Events Leading to Cell
Replication
*1. G1(first gap) phase
*2. S phase
*3. G2(second gap) phase
*4. M phase
a. Prophase
b. Metaphase
c. Anaphase
d. Telophase
*5. Chromosome의 number변화
*6. Cell cycle에 걸리는 시간
II. Regulated Protein Phosphorylation and
Degradation Control Passage
(1) G1 Cyclin-CDKs
(2) S-phase Cyclin-CDKs
(3) Mitotic Cyclin-CDKs
(4) Ubiquitin-Protein Ligase
(5) Checkpoint Mechanisms
a. Checkpoints
b. Cell division의 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 cell과 G1-phase cell의 fusion
b. G1-phase cell과 S-phase cell의 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
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Xenopus oocyte의 cell division
I. Maturation-Promoting Factor (MPF) stimulates
Meiotic Maturation in Oocytes and Mitosis in Somatic Cells
(1) Meiotic maturation을 promoting하는 diffusible factor
a. G2-arrested Xenopus oocyte의 분리
b. MPF (Mitosis promoting factor)
c. Mammalian cell 에서 MPF효과
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 activity의 cycling과 Mitotic event와의 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 protein에 ubiquitination
b. Destruction box를 recognition
(2) Regulation of mitotic cyclin levels
a. APC/C specificity factor = Cdh1
b. Phosphorylation of Cdh1 by G1 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 cyclin과 CDK가 결합한 MPF는 매우 낮은 activity
b. Wee1: Protein-tyrosine kinase
c. CAK: CDK activating kinase
d. Cdc25: Phosphatase
e. Y15를 F15로 교체 시
III. Conformational Changes Induced by Cyclin Binding and
Phosphorylation Increase MPF Activity
(1) Free CDK2 (human)
(2) Cyclin-CDK2 Binding 시
(3) Phosphorylation과 dephosphorylation에 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. MPF에 의한 phosphorylation
d. Lamin A/C gene 의 mutation과 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 mixture에 농도를 높여가며 첨가
c. Decondensation과 segregation이 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. cdc28ts mutant cell
(2) Yeast CDK (Highly homologous)
a. S. cerevisiae 에서 cell cycle control : G1 to S transition
b. S. pombe 에서 cell cycle control : G2 to M transition
II. S-Phase Promoting Factor (SPF) & Three G1
Cyclins
(1) Identification of G1-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 degradation에 의한 major transition의 조절
(3) SCF Ubiquitin-protein ligase
IV. Multiple Cyclins Regulate the Kinase Activity
during Different Cell-cycle Phases