Signal Transduction and Cell Cycle

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MPHY616
Signal Transduction and Cell Cycle Paul Shapiro,
Room 222 Pharmacy Hall pshapiro_at_rx.umaryland.edu O
ffice 6-8522 Lab (Rm 269) 6-2980
Objectives A. Overview cell cycle. B. Define
major mitotic structural changes. C. Define
major G2/M-phase signaling events.
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Somatic Cell Cycle
G0
R
R restriction point
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Mitosis
Prophase Metaphase Anaphase Telophase
Entry Chromosome condensation Spindle pole
formation Nuclear envelope breakdown Other
structural changes
Exit Chromosome congression Microtubu
le attachment Chromosome
segregation Nuclear envelope reassembly Other
structural reassembly
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Regulation of CDK activity.

• Cyclin synthesis and binding
• CDK phosphorylation
• CDK dephosphorylation

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Cdc25A
Cell Cycle G1-Phase
Cdk4/6
CyclinD
()

1. Cdk 4/6 activated through interaction with cyclin
   D and dephosphorylation.
2. Rb phosphorylation relieves Txn repression.
3. Expression of genes required for DNA synthesis.

Cdk2
()
CyclinE
Rb
Inactive
Active
pRb
Rb
HDAC
X
Transcription
E2F
E2F
DP-1
DP-1
OFF
ON
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Cell Cycle G1-Phase

• G1 progression Dependent on mitogen
  availability.
• Increased expression of cyclin D, immediate early
  genes
• (Fos, Myc). Dependent on ERK MAP kinases
  (TCF/Elk-1).
• Activation of protein translation Indirect
  phosphorylation
• of eIF4E by ERK. Enhances mRNA binding and
  protein
• expression.

• G1 inhibition G1/S checkpoint
• (No mitogens, senescence, contact inhibition,
  DNA damage).
• A. Increased expression of Cdk inhibitors
  (CdkI)
• -p15/16 INK4 target Cdk4/6
• -p21CIP / p27KIP target Cdk2
• ERK and p53 (DNA damage) up-regulate p21CIP
• expression. p53 regulation by
  phosphorylation
• B. Increased cyclin D degradation GSK3b
  phosphorylation
• Decreased Cdc25A expression TGF-b mediated.

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G1 DNA damage checkpoint.

1. DNA damage / recognition.
2. Phosphorylation
3. Stabilization of p53
4. Expression of p21
5. Inactivation of CDK

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Cell Cycle SPhase / DNA synthesis

• 1. Accurately replicate each chromosome.
• (avg. 40mm DNA / Chromosome)
• Replication initiated at multiple
• sites on chromosome.
• Reassemble into proper structure.
• Nucleosomes / Scaffolds.
• Dependency of M-phase on S-phase.
• Unreplicated or damaged DNA.
• Dependency of S-phase on M-phase.
• Prevent changes in cell ploidy.
• Endoreduplication / endomitosis
• Successive S-phases without M-phase.

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Cell Cycle G2-Phase progression
Goal Prevent transmission of DNA replication
errors or DNA damage and prepare for mitosis.
Myt1 and Wee1
Cdc25B/C
()
(-)
Cdc2 CyclinB
Tyr15 (-)
()
G2-Phase
Mitosis
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Cell Cycle G2-Phase (DNA damage checkpoint)
DNA damage Sensors Effectors and
co-effectors
DNA-PK
ATM/ATR
Chk1/2
p53
p21CIP, GADD45
Cdc25B/C
(-)
Cdc2/CyclinB
G2 progression blocked
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Stages of mitosis
Start
? 4 meters of DNA in 46 chromosomes per G2 cell.
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The Microtubule Organizing Center A. Animal
cells centrosome B. Yeast spindle pole body.
Major Components 1) Centriole pair surrounded by
pericentriolar material. 2) g-tubulin - acts as
tether for a and b tubulin and microtubule
nucleation ( and - ends). 3) Motor proteins
Dynein and kinesin. ( accessory proteins)
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Centrosomes and Centrioles 1) Duplication at
G1/S boundary is distinct from maturation during
G2/M transitions. A) Control of duplication
Protein kinases Mps1p (S. cerevisiae), Cyclin
E and A/cdk2. B) Centrosome maturation
growth in size during G2/M. -Recruitment of
cytoplasmic proteins. -Nucleation of
microtubules. Centrosome proteins Maybe
50, including signaling proteins, chaperones,
motor proteins, structural proteins.
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Centrosome maturation role of phosphorylation.
A. Regulation by protein kinases Polo-like
kinases (Plk). -Human homologue to Drosophila
polo gene product. -Plk active during M-phase
and may regulate entry and exit. B. Plk
regulation of G2/M transitions Microinjection
studies showed profound effects on centrosome
maturation and bipolar spindle
formation. Differences between normal diploid
cells and tumor cells suggested the presence of
a centrosome checkpoint. C. Plk is regulated by
upstream kinases D. Potential target
proteins -Centrosome? -Cdc25 -Anaphase
promoting complex (APC) (3 proteins
Cdc16,27, and Tsg24)
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Spindle Assembly Checkpoint (also called
mitotic or kinetochore-attachment
checkpoint) Function Inhibit progression into
anaphase in order to prevent chromosome
segregation errors.
Questions 1) What senses proper MT attachment
or tension? 2) How does one unattached
chromosome inhibit anaphase in the attached
chromosomes?
Figure from W. Wells (1996) Trends in Cell
biology, Vol 6 p228.
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Kinetochore
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Spindle checkpoint proteins
MAD (mitotic arrest-deficient) BUB (budding
uninhibited by benzimidazole) MAD1/2/3 - All
interact with anaphase promoting complex
proteins (at least in 2-hybrid screens) and are
thought to inhibit. MAD1-phosphoprotein (by
Mps1?) (pre-meta kinetochores). MAD2-cointeracts
with MAD1 (pre-meta kinetochores). MAD3-unknown
function. BUB1/2/3 - BUB1 - S/T kinase
(pre-meta kinetochores). BUB2 - unknown
function. BUB3 - associates with BUB1, BUB1
substrate (pre-meta kinetochores).
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Mad2 at unattached kinteochores
Red unattached White just attached
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Nuclear Envelope Breakdown (NEB) during mitosis

• Nuclear envelope versus nuclear membrane
• Envelope consists of
• Nuclear pore complex
• Outer membrane
• Inner membrane
• Lamina
• -(A/C and B-type lamins)
• -lamina-associated
• polypeptides (LAPs)
• -lamin B receptor (LBR)

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B. Rapid NEB at the end of prophase. C. NEB
regulated by phosphorylation of lamins and other
lamina proteins Not simply due to increases in
cdc2/cyclin B activity. Other lamin kinases
PKC, Arg-Ser (RS) kinase, PKA D. Reassembly of
nuclear envelope following dephosphorylation Pro
tein Phosphatase 1 (PP1) targeting of lamin B
involved.
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NEB
Regulation may involve functional nuclear pores
and lamina. A. Lamin B and p56 (inner membrane
protein and putative lamin B receptor)
interactions weakened by hyperphosphorylation. B
. Intact nuclear pore complexes may be
required for hyperphosphorylation. C. Similarly,
nuclei disassembly does not occur without
lamina. D. Pores likely provide access to lamin
kinases.
() mitotic extracts
From Collas (1998) J. Cell Science, Vol.1111293.
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Organelle disassembly during mitosis Golgi
fragmentation
Warren (Yale) Cdc2 mediated GM130
phosphorylation and p115 interactions. Malhotra
(UCSD) Raf-1 and MEK1 activity. Targets?
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Mitotic Exit Anaphase Promoting Complex (APC) /
Cyclosome.
1) Composed of at least 8 subunits in
vertebrates. 2) Catalyzes the transfer
(ligation) of ubiquitins to a N-terminus 9 amino
acid destruction box (D-box) sequence on
cyclins and other proteins. (RXALGXIXN) E1
ubiquitin-activating enzyme. E2 ubiquitin
carrier. E3 ubiquitin protein ligase. 3)
Ubiquitinated proteins targeted for
degradation. 4) APC activity regulated, in part,
by phosphorylation. (Kinases involved and
mechanisms not well defined) 5) Protein-protein
interactions involved in APC activation. (ATP-ind
ependent)
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Regulators of APC activity during metaphase
WD40 repeat proteins - Humans hCDC20 (p55CDC)
and hCDH1 S. Cer. (Cdc20p, Cdh1p/Hct1p) D.
mel. (fizzy, fzy and fizzy-related, fzr) CDC20
and CDH1 bind APC and activate cyclin
ubiquitination activity. CDC20 regulates APC in
mitosis and confers a strict D-box dependence on
APC, CDH1 is not strictly dependent on
D-box. CDC20 protein levels increase in G2/M,
whereas, CDH1 levels are relatively constant
throughout the cell cycle. CDH1 may facilitate
G1 degradation of proteins lacking D-box (eg.
Plk).
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Model for cell cycle regulation of APC
From Fang et al., Mol. Cell (1998) Vol.2163 (M.
Kirschner-Harvard)
Degradation
Cyclin B/Cdc2 Other kinases?
Mitotic Checkpoint
CDC20 CDH1
CDC20 and P
CDC20/CDH1 APC-P
CDH1 APC
APC
APC-P
S/G2
Metaphase
Early Anaphase
Late Mitosis/G1
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Inhibitors of mitosis