Apr. 21, 2005

Cell cycle control (mammalian)Basic mechanisms & protein componentsCheckpoints

Chap. 21, by Lodish et al.,5th ed. 2004Chap. 17, by Alberts et al.,4th ed. 2002

鍾明怡

mychung@vghtpe.gov.tw

Advanced readings (not all required)

By mechanismSCF

Nat Rev MCB 2004;5:739-751.Nat Rev MCB 2005;6:9-20.

APCOncogene 2005;24:314-325.Oncogene 2005; Ralph Wasch

& Dirk Engelbert (publishedonline ahead of print).

Polo-like kinasesNat Rev MCB 2004; 5:429-

440.Oncogene 2005; 24:277-286.

Role of translationNat Rev MCB 2005; 6:69-78.

By checkpointDNA damage checkpoint

Nat Rev MCB 2004; 5:792-804.Nat Rev MCB 2005;6:44-55.

Spindle checkpointChromosome Research

2004;12:599-616.

OthersConnecting proliferation and apoptosis

in development and disease. Nat RevMCB 2004; 5:805-815.

Cancer genes and the pathways theycontrol. Nat Med 2004; 10:789-799.

Haploinsufficiency for tumor suppressorgenes: when you don’t need to goall the way. Biochimica et BiophysicaActa 2004;1654:105-122.

Mammalian cell cycle control

The basic organization of cellcycle and its control systemare essentially the same in alleukaryotic cells.

Why discuss mammalsseparately?Differences between yeastand mammals.

Cell division in multicellularorganisms

In addition to availability of nutrients, itmust also receive stimulatoryextracellular signals for a multicellularorganism to divide.Mitogens vs. growth factorsProliferation vs. growthe.g. PDGF

Cell cycle control and human diseases.

Cell growth andcell cycle control

Only cycle control

both

N/C ratio↑

N/C ratio↓↓

A simplified modelof one way thatmitogensstimulate celldivision.

•••

Study of cell cycle inmammalian systems

Cell-culture based. Normal tissues, e.g. fibroblast. Complication:

replicative cell senescence after 25-40 divisions. Tumor tissues

Reveal characteristics of normal cells. Diploid. Anchorage dependence. Contact inhibition. Replicative cell senescence.

Cell cycle controlwhy & how?

Basic programs: timed events in thecorrect order; triggered only once ineach cycle; irreversible; must completeprior to initiation of the next event.Sensor.Adaptability: adjust to cell type,environment, …etc.Robustness: backup mechanisms.

Cell cycle checkpoints

The two most important things in a cell cycle:1.2.

Mechanisms of cell cycle control (1)

Generally operate through negativeintracellular signals. Default setting: “STOP”. Gradual decrease in the

negative signal to zero results in “GO”. Inactivation of checkpoints results in progression of

cell cycle without surveillance.

Based on cyclically activated protein kinases,cdks. Cyclin-dependent kinase (cdk). Regulated by a bunch of enzymes and proteins, e.g.

cyclin.

Cyclins and cdks in vertebrates

Cyclins activate cdks; alsodirect their targets.

Another level of regulation:accessibility of the substrate,or target site on the substrate.

B-type cyclinsTarget of APC

Mechanisms of cell cycle control (2)

Transcriptional regulation.Translational regulation.Post-translational regulation. Activating and inhibitory phosphorylation. Inhibitory proteins.Protein degradation. SCF mediated. APC mediated.Subcellular localization.

Transcriptional regulation

Level of cyclin isregulated at the level oftranscription,translation anddegradation. In embryonic cell cycles, M-cyclin

is synthesized at a constant rate;M-cyclin accumulates due todecreased degradation.

In most cells, however, M-cyclinincreases during G2 and M byincreasing transcription.

Will be discussed later.

G0 G1

Translational regulation←Basal cellhyperplasia inprostate

Normal basal cell

Webpathology.comhttp://www.webpathology.com/case.cfm?case=18

JBC 1998;273:29864-29872.

eIF4

cyclins and cdks

(Cdk7-cyclin H-Mat1)

amountvs.

activity

Cannot activate oneself via autophosphorylation

Cdk7/cyclin HMat1

Cdk7(Ser170 Thr176)/cyclin HCdk7/cyclin H/Mat1

“Active CAK”Cdk2/cyclin A Cdk2 (Thr160) /cyclin A

Cdk2 (Tyr15 Thr160) /cyclin A

Cdc25AWee1

Activating and inhibitoryphosphorylations of M-Cdk

Cyclin A/Cdk2 Cyclin A/Cdk2

Cyclin B/Cdk1 Cyclin B/Cdk1

Cdc25A

Cdc25B

Cdk inhibitory proteins (CKIs)

Two classes of Cdk inhibitorsCdk Inhibitory proteins (CIP/KIP)Inhibit all Cdk1,2,4,6 complexes.p21, p27, p57.

Inhibitors of kinase 4 (INK4)Inhibit Cdk4&6.p16, p15.

Proteolysis in cell cycle control (1)–SCF mediated

Named after its three subunits.Regulation of G1/S-cyclins and CKI levels.SCF activity is constant throughout cell cycle (?).Controlled by phosphorylation of the targetprotein.

p27 p27Cyclin ECdk2

SCF ubiquitin ligases

Skp1-Cul1-F box (Skp2) = SCFSkp2

E2

Substrateinteraction

E2/E3interaction

Nat Rev Mol Cell Biol 2004;5:739-751.

Proteolysis andCDK activity

3

Nat Rev Mol Cell Biol 2004;5:739-751.

Proteolysis in cell cycle control (2)–APC mediated

Anaphase-promoting complex.Degrades M-cyclins and otherproteins at mitosis.

Oncogene (2005)24, 314–325

APC/C (cyclosome)APC/C is a multisubunit E3 ubiquitin ligase with atleast two alternative forms, one activated byCdc20 (APC/CCDC20) and the other by Cdh1(APC/CCDH1). Cdh1=Hct1.The homolog of the largest subunit of APC, Apc1,in fission yeast has also been described as aconstituent of the 20S cyclosome.Required for Entry into anaphase (APC/CCDC20). Exit from mitosis (APC/CCDH1). Maintenance of low Cdk activity in early G1 (APC/CCDH1).

Subunits of APC

Oncogene (2005) 24, 314–325

Role ofAPC incell cycle

Oncogene (2005)24, 314–325

active inactive inactive inactive active active

4

Checkpoints(by cell-cycle phases; by specific aims)

Molecular signaling cascades that promote cell-cycle delay or arrest in response tounfavorable conditions, e.g. DNA damage,thereby providing more time to overcome theunfavorable condition or to pursue analternative.

-- Adapted from Nat Rev Mol Cell Biol 2004;5:792-804.

Lodish, et al. 5th ed.2

Passage through “restriction point”:G1 progression & initiation of S phase

Growth factor → cyclin D (↑txc) =>active G1-Cdk

(cyclin D:cdk4/6)

(cyclin E:cdk2)

Restriction point

Intracellular control of cell cycleRe-replication block

Cells at S phasecontain signals forDNA replication andinitiate DNA synthesisin G1 cell after fusion.

S + G2 => no DNAreplication in G2 =>DNA can be replicatedonly once in a cycle.

G2 + G1 => cannotinduce G1 cells into Sphase.

Control of entry into S phase (1)

Origin of replication.ORC. Associated with Ori throughout

the cell cycle.

Regulatory proteins Cdc6.

Transient in early G1. Mcm (Minichromosome

maintenance ) proteins, … etc. Form “pre-replicative complex”.

Ready for DNA replication.

Control of entry into S phase (2)Activation of cyclin A/Cdk2in late G1 triggers S phase. Phosphorylation of ORC =>

initiate DNA replication. phosphorylation of cdc6 =>

degradation by SCF. Phosphorylation of Mcm =>

export from the nucleus. “geminin”occupies Ori,

cannot re-initiate untilcycle is completed.

cannot reassemble pre-RC.

How to initiate DNA synthesisin the next cycle?

DNA damagecheckpoint

Cdc25ASer126

(inactive)(degradation)

G1, G2 arrest

G1/Sblock

DNA repairApoptosis

ActivatedATM Chk2

(active)

ATM= ataxiatelangiectasiamutated.

ATR= ataxiatelangiectasiaand RADrelated

CHK1/2=checkpointkinase 1/2

Nat Rev Mol Cell Biol2005;6:44-55.

DNA damagecheckpoint

Replicationcheckpoint

DSB, or

Cdc25C(inactive)

G2 arrest

Role of p53

Overexpression of a mutated form of thep53 tumor suppressor gene in ahepatic metastasis in colorectalcancer. The brown stain(immunohistochemistry, PAP)shows that the mutated p53localizes to the nuclei of the cancercells where it fails to transduce theapoptotic signal. This renderscancer cells refractory to classicalradio- or chemotherapy.

http://www.rrk-berlin.de/igtlab/eu_network/application.html

Both Rb and p53 are “tumorsuppressor genes”and show“dominant”inheritance in affectedfamilies. What are the mechanismsfor these phenomena?

How about p27Kip1?

DNA replication checkpoint

Late S/G2.Sensor mechanisms are not fully understood. Unreplicated DNA (?). Unfinished replication fork (?). Final target: the pathway for M-Cdk activation.

Inhibits DNA synthesis

high doses

DNA replication checkpoint

RPA = replicationprotein A

Cdc25C (inactive)

G2 arrestp53p21CIPG2 arrest Repair

Apoptosis

Nat Rev Mol Cell Biol2005;6:44-55.

Summary ofS phasecheckpoints

Nat Rev MCB2004;5:792.

Nat Rev MCB2004;5:792.

Control of entry into M-phase

MitosisProphasePro-metaphaseMetaphaseAnaphaseTelophase

Cytokinesis

S

M

~30min/cyclefairly synchronous

Control of M-Cyclin quantity

G1 allows Cell growth Regulation by extracellular signals.

M-cyclin transcript

(transcriptand protein)

Control of M-Cdk activity

Polokinase

Cyclin Bconcentration

The roles of M-CdkChromosome condensation (condensin complex;phosphorylated by M-cyclin => change DNAcoiling => initiating condensation.; MCB).Induces the assembly of mitotic spindle.Ensures attachment of chromosomes to mitoticspindle.Nuclear envelop breakdown.Actin rearrangement.Re-organization of Golgi and ER.

Details of these events are not all fullyunderstood.

Cohesion of sister chromatidsSister chromatids are “joined”together by “cohesins”,deposited along the each sister chromatid as DNA isreplicated.Cohesion of sister chromatids are broken at anaphase.

Chrosome condensationM-Cdk phosphorylates condensin subunits, triggeringthe assembly of condensin complex and theprogressive condensation of the chromosomes.

Blue: DNARed: anti-condensin

Prometaphase Metaphase

Nuclear envelope breakdownin mitosis

Assembly of mitotic spindle

M-Cdk phosphorylates MAP andpromotes stabilization of microtubule.

Sister chromatid separation

APC-mediated degradation of M-cyclin=> inactivate M-Cdk => initiation ofanaphase.

txc

-

Unattached kinetochore

Spindlecheckpoint

Spindle checkpoint

Sensor monitors the status of kinetochore.Mechanism not yet clear. Mad2 at unattached kinetochore sends out (-)

signal; (-) APC activation. Defective spindle checkpoint => earlier anaphase

initiation. Spindle checkpoint is not required in frog embryo

and yeast; some other mechanism(s) is controllingthe timing of anaphase.

Mad2- tubulinDNA

(A) Just after nuclearenvelope breakdown.(B) Early prometaphase.(C) Mid-prometaphase.(D) Late prometaphase.(E) Metaphase.(F) Anaphase.

Mad2 and spindle checkpoint

Chromosome Research 2004;12:599-616.

Chromosome Research 2004;12:599-616.

Non-disjunction

Cytokinesis

Contractile ring: composed of actin andmyosin filaments, formed around theequator of the cell.Control mechanisms to assure thatmitosis occurs prior to cytokinesis.

Exit from mitosis

Degradation of M-cyclin => inactivationof M-Cdk => decrease of kinases activation of phosphatases.EventsDisassembly of mitotic spindle.Decondensation of chromosomes. Formation of nuclear envelope.

S

M

~30min/cyclefairly synchronous

Control of M-cyclin quantity

G1 allows Cell growth Regulation by extracellular signals.

M-cyclin transcript

(transcriptand protein)

Suppressed Cdk activities in early G1CKI: Sic1 in yeast; p21/p27/p57 in mammals;transcription.G1 cyclins are not inactivated by APC/CCDH1 and CKI.

S-cyclinTxc

w/ cdc20

InactiveTxc factor

activeTxc factor

CKI

Cdh1 Cdh1 Cdh1

CKI CKI CKI

CKI

CKI Cdh1

Cdh1

(1)

(2)

1

Try to give an organized generaloverview of mammalian cell cyclecontrol with updated information.Some of the details are not yet fullyexplored.Please do not be misled by the cartoons.