cell cycle regulatory and programmed cell death

7/29/2019 Cell Cycle Regulatory and Programmed Cell Death

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Cell Cycle Regulatory Proteins

Sri Widyarti

[email protected] revised 211009
mailto:[email protected]:[email protected]


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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
http://ajprenal.physiology.org/cgi/content/full/278/4/F515/F2http://ajprenal.physiology.org/cgi/content/full/278/4/F515/F2


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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)
http://ajprenal.physiology.org/cgi/content/full/278/4/F515/F3


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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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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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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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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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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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cell cycle regulatory and programmed cell death

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