Lecture 2: Regulation of MPF Cdk activity

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Outline for Fridays Lectures
Lecture 1 Understanding cell cycle
mechanisms (Or Discovery of MPF)
Lecture 2 Regulation of MPF (Cdk) activity
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Table 17-1 Some Eucaryotic Cell-Cycle Times
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---------------------------------------------- Tab
le 17-1 Some Eucaryotic Cell-Cycle Times
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----------------------- Cell Type Cell-Cycle
Times --------------------------------------------
---------------------------- Early frog embryo
cells 30 minutes Yeast cells 1.5-3
hours Intestinal epithelial cells about 12
hours Mammalian fibroblasts in culture about 20
hours Human liver cells about 1
year ---------------------------------------------
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Important questions in cell cycle research

• How do S-phase and M-phase occur
• mechanistically?

• How are these events regulated?

• How are these events co-ordinated?

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• Lasker Award Winners
• 2001 Nobel Prize Winners

Tim Hunt
In recognition of their contributions toward
understanding cell division mechanisms
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How are questions relating to cell cycle
control addressed experimentally?
A) Biochemically using oocyte systems,
especially Xenopus laevis (the frog)
B) Genetically with the yeasts a) budding
yeast, Saccharomyces cerevisiae b) fission
yeast, Schizosaccharomyces pombe

• Developmental considerations with Drosophila
• melanogaster and C. elegans

D) For growth control, mammalian cells
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Before Xenopus and yeasts
Paradigms gleaned from simple mammalian cell
fusion experiments
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From mammalian cells to frogs, or
From the phenomena to a biochemical explanation
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Interphase arrest
maturation
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before progesterone
MPF activity
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Progesterone
MPF activity
Fertilization
High
Interphase arrest
Metaphase arrest
Low
Meiosis I
Meiosis II Arrest
Fertilized egg
Fully grown oocyte
Second mitosis
First mitosis
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Normal Development
Fertilized egg
Protein synthesis inhibited
Fertilized egg
Protein synthesis inhibited
Fertilized egg
MPF injected
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Preparation of cell free extract
Activated frog eggs
Remove buffer
Withdraw cytoplasm with syringe
Crush eggs in centrifuge
Lipid
Yolk
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Using frog egg cell free extracts, Lohka and
Maller were able to purify a dimer with MPF
activity

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Now, from a brute force biochemical approach to
The elegance of genetics
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Budding Yeast Life Cycle
Meiosis and sporulation
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Fission Yeast Life Cycle
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G2
Spindle formation
M
METAPHASE
ANAPHASE
Spindle pole duplication
S
Bud emergence
DNA replication
START
cytokinesis
G1
cell growth
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SPB duplication
Chromosome condensation
spindle formation
M
cytokinesis
G1
START
G2
S
DNA replication
cell growth
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MUTAGENIZE CELLS
Conditional mutants
not defective in growth
heterogeneous phenotypes
(grow under one set of conditions but not
another)
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In both budding and fission yeasts,
conditional (grow under one set of conditions
but not another) mutants defective in cell cycle
progression were obtained
These are called cdc for cell division cycle
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Isolation of temperature-sensitive cdc mutants
in budding yeast
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G2
Spindle formation
M
METAPHASE
ANAPHASE
Spindle pole duplication
S
Bud emergence
DNA replication
START
cytokinesis
cdc28
G1
cell growth
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MUTAGENIZE CELLS
Fission yeast
not defective in growth
heterogeneous phenotypes
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The first class of cdc2 mutants
Conclusion cdc2 activity is required
for cell cycle progression
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The second class of cdc2 mutants
Conclusion cdc2 activity is rate-limiting
for cell cycle progression
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What is cdc2?
Is cdc2 function related to MPF?
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Cloning of S. pombe cdc2 by
complementation
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Sequence alignment of S. pombe cdc2 with
S. cerevisiae Cdc28p
Putative protein kinases
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Protein kinase??????
Phosphorylates substrates to alter their function
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Cell Cycle Control Points
Does a human homolog exist?
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Transform temperature-sensitive cdc2 mutant with
human cDNA library
Cloning of human CDC2 by complementation
Made antibodies To CDC2
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What is this protein?
This protein is cdc2!
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Cyclic accumulation of two proteins in clam eggs
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This protein is a cyclin!
Cdc2
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- Abs to cyclin recognized the other MPF component
- cyclin box is responsible for binding cdc2
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MPF (maturation promoting factor) is
1) cdc2/CDC28
2) mitotic cyclin
Cdc2 was the first recognized CDK or
Cyclin-Dependent Kinase
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G1 Cyclins
Mitotic Cyclins
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Cyclins

• higher eukaryotes
• D early G1 phase
• E late G1 phase, S phase
• A S and G2 phase
• B S and G2 phase, after A

• S. cerevisiae
• 3 CLNs G1 phase
• 6 CLBs S and G2 phases

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CDKs (cyclin dependent kinase)
Cdc2 (CDC28 in S. cerevisiae) CDK1 - required
in all organisms for entry into mitosis -
required in both yeasts for S phase
CDK2 - S phase role in higher eukaryotes (not
essential)
CDK4 - correlated with entry into S phase in
higher eukaryotes
There also are CDK3, CDK5, CDK6, CDK7 and CDK8 -
CDK3 and 6 have not been studied extensively -
CDK5 activity is involved in neuronal function -
CDK7 CAK - CDK8 is involved in regulating
RNA polymerase II
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Model of eukaryotic cell cycle as CDK-cyclin
engine
CDK1 cyclin B/A
CDK4/6 cyclin D
CDK2 cyclin E
CDK2 cyclin A
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End of lecture 1