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Cell Cycle Regulatory Proteins
Sri Widyarti
[email protected] revised 211009
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Introduction
Cell proliferation is ultimately a nuclear event and
is divided into differentphases in what is now
called the cell cycle. The cell cycle entails an ordered series of
macromolecular events that lead to cell division.
Regulation of the cell cycle is critical for thenormal development of multicellular organisms.
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The master controllers of
these events are a small
number ofheterodimeric
protein kinases thatcontain :
Regulatory subunit
(cyclin), and
Catalytic subunit(cyclin-dependent
kinase)
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The kinases regulate the activities of
multiple protein involved in DNA
replication and mitosis by phosphorylatingthem at specific regulatory sites, activiting
some and inhibiting others to coordinate
their activities
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Overview of the Cell Cycle
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The Phase of Cell Cycle
G0 phasequiescent cells
G1phase prepares cell for DNA synthesis
S phase DNA replication G2phase cells prepare for mitosis (M
phase)
M phase subdivided into prophase,metaphase, anaphase,and telophase, whichis followed by cytokinesis (cell division).
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Transition of Cell Cycle Phase
Transition from one cell cycle phase to another is
a coordinated,sequential, and synchronized
process that occurs with precise
timing in a well-defined order to ensure that the cellular machinery
is ready for DNA replication, that DNA
replication occurs onlyonce in each cycle, that
DNA replication is completed before mitosisstarts, and that chromosomes are replicated into
identical setsin daughter cells.
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Restriction Point
After passing a "restriction point"in late G1,
cells are no longer responsive to
extracellular signalsand complete the cellcycle under the control of specific cellcycle
regulatory proteins.
The average cell cycle time is 12 hfor G1,6 h for S, G2 lasts 6 h, and mitosis is
completed in 30min
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G1 is the phase that varies most among celltypes.
Mammalian cell that have stopped dividingare almost always arrested during G1.
The process of cell division in cultured cellscan stop or slow down by allowing the cellsto run out of either nutrients or space or byadding inhibitors protein synthesis.
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Restriction Point
The point in late G1
where passage through
the cell cycle becomesindependent of
mitogens is called the
restriction point
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The Control System
Ensure that the events associated with each phase
of the cell cycle are carried out at the appropriate
time and in the sequence Sure that each phase of the cell cycle has been
properlycompletedbefore the next phase is
initiated
Able torespond to external conditions that
indicate the need for cell growth and division
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Cell Cycle Regulatory Proteins
Cell cycle progression is controlled by :
positive [cyclins and cyclin-dependent
kinases (cyclin-CDK)] cell cycleregulatory proteins and
negative (CDK inhibitors) cell cycle
regulatory proteins.
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Various Ways to Studied Cell-
Cycle progression Labeling S-phase cells (3H-thymidine or
bromo-deoxyuridine (BrdU)
Visualized by autoradiography
Staining with anti-BrdU antibodies
Analysis of DNA content with flow
cytometer
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A. The3
H-thymidine-labeled cells have been visualized byautoradiography
B. An immunofluorescence micrograph of BrdU-labeled
glial precursor cells in culture
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Microinjection
experiments with anti-
cyclin D antibodydemonstrate that
cyclin D is required
for passage through
the restriction point
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A simpified view of the core of
the cell-cycle control system
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The structural basis of Cdk
activation
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Proliferation, Hypertrophy and
Apoptosis Injury can result in proliferation,
hypertrophy, or apoptosis, which we believe
are linked at the level of the cell
cycle. Proliferation requires normal progression
throughthe cell cycle.
Hypertrophy occurs when cells engage thecellcycle but cannot progress beyond lateG1 (G1/S arrest).
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Proliferation, Hypertrophy and
Apoptosis Apoptosisis associated with exit from the
cell cycle, which typically occursin G1.
Thus these processes may share commonpathways and mayexplain why certain cell
populations undergo proliferation and
apoptosis, whereas proliferation andhypertrophy are independentevents.
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Cyclins and CDK
Cell cycle regulatory proteins are localizepredominantly in the nucleus.
The kinase activity is composedof two subunits:
cyclins and their partner, cyclin-dependent kinases
(CDK).
Cyclins have very short half-lives(
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CDK Inhibitors
CDK inhibitors negatively regulate cell
cycle progressionby inhibiting cyclin-CDK
complexes, resulting in cell cycle arrest.
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Entry of Quiescent Cells Into the
Cell Cycle Entry of quiescent cells (G0) into early G1 requires
D-type cyclins (D1, D2, D3), which are expressedin a cell-type-specificmanner.
D-cyclins are transcriptionally regulated, and
levels are increased by specific mitogens such asgrowth factors.
D-cyclin levels decrease on mitogen withdrawal
Growth inhibitors such as interferon, transforming
growth factor- (TGF- ) suppress D-type cyclin
transcription.
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Cyclin D
D-type cyclins associate with and activate CDK4
and 6 in G1.
A critical substrate for cyclin D-CDK is the 110-kDaprotein product of the retinoblastoma gene
(pRb), whichregulates G1/S transition.
pRb is hypophosphorylatedduring G0 and early G1
and is growth restrictive by sequesteringthetranscription factor E2F.
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Phosphorylation
ofretinoblastoma
(Rb) gene
protein
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. Cyclin D phosphorylates someof the sites on pRb,
but only to the point in which pRb is still
hypophosphorylated, active, and growthrestrictive.
Cyclin E phosphorylatesthe remaining sites, sothat pRb is now hyperphosphorylated pRb,
inactive, and growth permissive and releases E2F,which bindsto the promoter regions of severaltarget genes essential forfurther cell cycleprogression, including immediate early genes,
thymidine kinase, and dihydrofolate reductase. Increased cyclin E activates CDK2, which also
phosphorylates Rb.
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G1/S Transition
The transition from late G1
into the S phase
determines the cell's growth characteristics.
G1 arrest resultsin antiproliferation orhypertrophy
G1 exit is associatedwith apoptosis.
G1/S transition results in DNA synthesisandproliferation.
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Cyclin E on G1/S Transition
Cyclin E levels increase in late G1,where it associates
with and activates CDK2, thereby playing apivotalrole in G1/S transition.
Cyclin E inductionis less dependent on exogenousgrowth factors and is regulatedby intrinsic factors ofthe cell cycle such as E2F.
Cyclin E-CDK2also phosphorylates pRb and a
positive cyclin E-synthesis
feedback loop existsthrough the phosphorylation of pRb, leadingto releaseof E2F.
Recently, a novel cyclin E2 was cloned,which
associates with CDK2.
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Cyclin A on G1/S Transition
Cyclin A levels peak in late G1, aremaximal during the S phase, and persist
through G2. Cyclin A activates CDK2 whichis essential
for DNA synthesis.
Forced overexpression ofcyclin A inducesDNA synthesis, and reducing cyclin Alevels preventscell proliferation.
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Cell Cycle Protein on Mitosis
The first cyclin identified was cyclinB,
which is required for mitosis.
Cyclin B levels fluctuatedue to synthesisand degradation, whereas its partner, cdc2
(formerlycalled CDK1), does not.
Cyclin B-cdc2 activity, similarto CDK2,depends on its phosphorylation status.
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Phosphorylation of cyclin B-cdc2
in Mitosis (1)
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Phosphorylation of cyclin B-cdc2
in Mitosis (2) Synthesis of cyclin B during interphase and
binding to cdc2 induces phosphorylation on3 sites: phosphorylation on threonine 14 (T14) and
threonine 161 (T161) by Wee1 (Y15) and Myt1(T15, Y15) is inhibitory, and phosphorylation ontyrosine 14 (Y14) by CDK-activating kinase isactivating.
Dephosphorylation of inhibitory sites by dual-specific cdc25 phosphatase leads to activation ofcyclin B-cdc2.
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Phosphorylation of cyclin B-cdc2
in Mitosis (3) A positive feedback loop (not shown) leads to
cyclin B-cdc2-mediated phosphorylation ofcdc25with further generation of active cyclin B-cdc2
complexes. Inhibition of Wee1/Myt1 through phosphorylation
of kinases prevents deactivation of cyclin B-cdc2.
Autophosphorylation of cyclin B leads toubiquitin-mediated degradation of cyclin Bprotein. Finally, cdc2 is dephosphorylated bykinase-associated phosphatase (KAP).
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Phosphorylation of cyclin B-cdc2
in Mitosis (4) Monomeric cdc2 is unphosphorylated and
inactive.
cdc2 undergoes a conformational change onbinding to cyclin B, which resultsin thephosphorylation on threonine 14 (Thr 14), tyrosine15 (Tyr15), and Thr 161 amino acid residues oncdc2.
Phosphorylationof Thr 14 and Tyr 15 by thekinases Wee1 and Myt1 are growth inhibitory,
which dominates over Thr 161 phosphorylation,which is growthactivating.
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Phosphorylation of cyclin B-cdc2
in Mitosis (5) Consequently, the triple phosphorylated cdc2-
cyclinB heterodimer is inactive.
Dephosphorylation of Thr 14 and Tyr
15 by thedual-specific phosphatase cdc25 is essential for
entryinto mitosis.
Active cdc2-cyclin B phosphorylates substrates
(H1histone, laminins, nucleolin) required forchromosome condensation,nuclear envelope
breakdown, and formation of the mitotic spindle.
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Phosphorylation of cyclin B-cdc2
in Mitosis (6) On completion of mitosis, cyclin B is
degraded via theubiquitin-proteasome
pathway, leading to the dissociation andinactivation of the complex, and cdc2 is
finally dephosphorylatedby kinase-
associated
phosphatase.
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CDK Inhibitors: Negative
Regulators of the Cell Cycle (1) Cyclin-CDK complexes are negatively regulated
by cell cycle proteins called CDK inhibitors that
bind to specific cyclin-CDKcomplexes and in so
doing inhibit their activity.
There are twofamilies of CDK inhibitors, which
are based on the target cyclin-CDKthey inhibit,
and on shared homologous sequences. Individual CDKinhibitors are named according to
their molecular weight.
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CDK Inhibitors: Negative
Regulators of the Cell Cycle (2) TheINK4 family only inhibit cyclin D-CDK
complexes and share an ankyrinrepeat.
The Cip/Kip family are more promiscuous andinhibit CDK2, 4, and 6, and share a CDK2-
binding domain.
The molecular mechanisms whereby CDK
inhibitors inhibit cyclin-CDKcomplexes are stillincompletelyunderstood.
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CIP/KIP Family of CDK
Inhibitors p21. The CDK inhibitor p21Cip1, WAF1, SDI1 (p21), a
21-kDa protein, is the founding member of the
Cip/Kip family.
p27. The 27-kDa protein p27Kip1 (p27) is widely
expressed in nonproliferating (quiescent) cells.
p57. The CDK inhibitor p57Kip2 (p57) is expressed
in many differentiated cells and in many adulttissues.
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p21
p21 is transcriptionally regulatedin a p53-dependent and a p53-independent manner.
p21 expression can also be modulated through
posttranslationalmechanisms.
In addition to binding CDKs, p21 can alsoassociatewith PCNA throughthe COOH-terminaldomain, which may be sufficient for G
1
arrest.
p21 expressionincreases during proliferation andthat p21 remains bound to CDKcomplexes inproliferating cells.
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p21
p21 may therefore actas a scaffold to facilitate theassembly of cyclins and CDKs requiredfor DNAsynthesis.
The role of p21 is check-point controlof G1/S-phase transition rather than withdrawal from thecellcycle and differentiation.
p21 inhibits G2/M phase of the cell cycle and thus
may also regulate mitosis, which furtherdistinguishes it fromother members of the Cip/Kipfamily of CDKinhibitors.
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p27
p27 expression is postranscriptionally regulatedbychanges in protein translation and degradationthrough theubiquitin proteolytic pathway and is
also posttranslationallymodified byphosphorylation.
p27 is not regulated by p53.
p27 regulates growth arrest in responseto TGF-,rapamycin, cAMP, and contact inhibition.
p27 can be both an inhibitor anda substrate forcyclin E-CDK2.
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p27
Cyclin E-CDK2 may be inhibitedin G0 by p27.
However, after growth factor-mediated activation
of cyclin D-CDK4, p27 preferentially binds to
these complexesand redistribution of p27 to cyclin
D-CDK4 results in activationof cyclin E-CDK2
with subsequent phosphorylation of p27, which
enhances p27 degradation. The final result may beactivationof more cyclin E-CDK2, which
facilitates G1 progression.
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Low levelsof p27 have been shown in a variety of
tumors, and p27 levelsare critical in renal cell
differentiation, apoptosis, proliferation,and
hypertrophy.
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p57
p57 binds CDK2, 3,and 4, and overexpression
leads to G1 arrest.
Further studies
suggest a close cooperation of p27and p57 to control proliferationand differentiation
in multiple tissues during development.
However, the precise function of p57 in cell cycle
regulation,and in particular in the kidney, remainsto beelucidated.
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INK4 Family of CDK Inhibitors
The INK4 family (p15, p16, p18, p19, p20)
consist of four or more ankyrin repeats and inhibit
CDK4 and CDK6 complexes in G1
.
INK4 members are tumorsuppressor genes, where
p15 and p16 are deleted and mutatedin a variety
of tumors, and the selected disruption of p19 in
mice predisposes to tumor development.
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TGF--mediated inductionof p15 blocks
activation of cyclin D-CDK4 complexes by
displacementof p27 from these complexes to
downstream binding and inhibitionof cyclin E-
CDK2 heterodimers.
The expression of INK4 CDKinhibitors may be
required to maintain quiescent cells in G0.
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Hypertrophy
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Cell hypertrophy is defined as cell enlargement
due to an increasein protein and RNA content
without DNA replication and can be due to cell
cycle-dependent or -independent mechanisms.
The presentparadigm suggests that hypertrophy is
an active process requiringentry into the cell
cycle, without progression through the S phase.
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Hypertrophy can also be defined asG0/G1-phase
arrest, which explains why hypertrophy and
proliferation are exclusive at a single cell level.
Thus certaingrowth factors, hormones,
extracellular matrix, mechanical forces,and
hyperglycemia that induce hypertrophy facilitate
entry into
the cell cycle. In contrast, tubular cellhypertrophy can be cellcycle independent due to
the inhibition of protein degradation.
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http://ajprenal.physiology.org/cgi/content/full/278/4/F515/F6