cell proliferation and its regulationbiochemistry.ucsf.edu/programs/ptf/m3 links/cell proliferation...

Hiten Madhani, MD, PhD

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Cell Proliferation and Its Regulation(Lecture)

OBJECTIVES• Listthekeypropertiesofstemcells.• DescribethetwokeyfunctionsofthecellcyclewithrespecttoDNA.• Listthefourphasesofthecellcycleanddescribewhathappensineach

phase.• Namethefourcyclin-Cdkcomplexesthatdrivethehumancellcycle.• DiagramthepathwaybywhichtheG1CdkactivatestheG1/SCdk.Describe

themoleculareventsthattakeplaceateachstepofthepathway.• NamethetwoclassesofCdkinhibitorsandthecyclin-Cdkcomplexesthey

inhibit.• Diagramthepathwayleadingfromepidermalgrowthfactor(EGF)tothe

activationofthecyclinDgene.Describethemoleculareventsthattakeplaceateachstepofthepathway.

• DescribetheWntsignalingpathwayanditseffectoncellproliferation.• DescribehowvariationintheexpressioinoftheAPCgenecontrolscellular

responsivenesstoWntsignaling.• DescribeinmoleculartermshowTGFßinhibitscelldivision.• Describehowapoptosiscanbetriggeredbyeitherextracellularor

intracellularsignals.• ListthemembersoftheBcl-2proteinfamilyanddescribehowtheyeither

promoteorinhibitapoptosis.• Listthetwomajorcaspasesinvolvedininitiatingapoptosis,andcontrast

theirdifferencesinmechanismofactivation.• Describehowp53isactivatedinresponsetoDNAdamage.• Describehowp53causescellcyclearrestandapoptosis.• Describethespindleassemblycheckpointandthemolecularfunctionofthe

Mad2protein.

KEY WORDSanaphase mitogenactivatedproteinkinased(MAPK)antimitogen mitoticspindleAPC(adenomatouspolyposiscoli) MYCapoptosis p16ß-catenin p21Bcl-2 p27caspase p53cellcycle progenitorcellcentrosome PUMAcheckpoint Rafchromosomesegregation Rascyclin Rbcyclin-dependentkinase(Cdk) receptortyrosinekinaseCdkinhibitor SH2domain

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KEY WORDS,continuedcytochromeC mitogencytokinesis sisterchromatidE2F sisterchromatidcohesionepidermalgrowthfactor(EGF) SosGrb2 stemcellgrowthfactor survivalfactorkinetochore terminaldifferentiationMad2 transforminggrowthfactorß(TGFß)MEK Wnt

I. INTRODUCTIONUncontrolledcellproliferationisahallmarkofcancer.Asdescribedintheoverviewlectureofthisblock,multiplemutationscausecancercellstodivideunchecked.Inthislecturewewillfocusonthenormalmechanismsofcelldivision,withanemphasisonthosedirectlyrelevanttocancer.

Innormaltissues,cellproliferationisgenerallyrestrictedtocellsthatreplenishthetissue.Most,ifnotall,tissuesarethoughttocontainstem cells thathavethisreplenishmentfunction(Figure1).Stemcellsareself-renewingcellsthatcandivideasymmetricallytoyieldanewstemcellandaprogenitor cell.Progenitorcellsmayormaynotundergofurtherdivisions,ultimatelyleadingtoterminal differentiation.Oncecellshaveterminallydifferentiated,theyhaveaspecializedfunctionandarenolongerdividing.Mosttissuesaremadeupofsuchnon-dividingcells.Thusproliferationisnormallytightlycontrolledsothatonlyparticularcellsinthebodyaredividing.

Programmedcelldeathorapoptosis,istheprocessbywhichexcessordamagaedcellsinthebodyareremoved.Thebalancebetweentheproductionofnewcellsandcelldeathmaintainstheappropriatenumberofcellsinatissue,whichisreferredtoashomeostasis.Apoptosisisalsoakeymechanismbywhichcancer-pronecellsareeliminated.

Figure 1. StemCells.Stemcellsareself-renewingcells.Theycandivideasymmetricallytoproduceanewstemcell(indicatedbyacircle)andaprogenitorcell.Progenitorcellsdividetoproducecellsthatundergoterminaldifferentiationtoproducethematurecellsthatmakeupatissueororgan.

Stem Cell (self-renewing)

Progenitor (Dividing)

Terminally DifferentiatedCells (Non-Dividing)


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II. ThE CELL CYCLECell division occurs in defined stages, which together comprise the cell cycle.Intermsofthegeneticmaterial,cellsmustreplicatetheirchromosomalDNAonceeverycellcycleandsegregatethesister chromatidsproducedbyDNAreplicationtoyieldtwogeneticallyidenticaldaughtercells(Figure2).Recallthathumancellsarediploid,containingtwocopiesofeachchromosome(homologs).DuringDNAreplication,cohesionproteinsattachthereplicatedsisterchromatidstoeachother,holdingthemtogether.Thissisterchromatidcohesioniscriticalforthesubsequentsegregationofchromatidsintothedaughtercells.

Thecelldivisioncycleisbrokenupintofourstages:G1,S1,G2andM(Figure3).DNAreplicationoccursduringS(“synthesis”)phase.Chromosomesegregationandcelldivision(cytokinesis)occursinM(mitosis).G1isthegapbetweenMandS.CellgrowthisoneoftheimportanteventsofG1.ThetransitionfromG1toSrepresentsacriticalcontrolpointinthecellcycle.G2isthegapbetweenSandM.Mitosisitselfhasseveralsteps.Threekeystepsaredescribedbelow.

A. Assembly of the mitotic spindle (Figure 4). AtthebeginningofMphase,themicrotubulesarereorganizedtobuildamitotic spindle.Itspurposeistoaccuratelysegregatethetwosisterchromatidstooppositepolesofthecell.Sisterchromatids,whichareattachedtoeachotherthroughsisterchromatidcohesion,becomelinkedtomicrotubulesviaattachmentsitesoneachchromatidcalledkinetochores.Kinetochoresareprotein-DNAcomplexesin which proteins that can capture microtubules are complexed with specific DNAsequencesatthecentromeres.Theotherendofeachmicrotubuleis

Figure 2. Thechromosomecycle.Akeypurposeofcelldivisionistheduplicationofthegeneticmaterialcarriedonchromosomesanditsaccuratesegregationsuchthateachdaughtercellacquiresonecopyofeachchromatid.

SisterChromatids

Chromosome

Replication

Segregation

Figure 3. Thecellcycle.ReproducedwithpermissionfromAlbertsetal.Molecular Biology of the Cell.GarlandPublishing,2002.

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Figure 5.Anaphase.Onlyonepairofsisterchromatidsisshown;however,thisoccurssimultaneouslyforall23pairsofhumanchromosomes.

Figure 6.Cytokinesis.Afterthetwosisterchromatidsaresegregatedtooppositepoles,cellsundergocytokinesis.

Figure 4.Themitoticspindle.

attachedtothecentrosome(alsocalledthespindlepolebodyormicrotubuleorganizingcenter).Thetwokinetochoresofeachpairofsisterchromatidsareattachedtooppositecentrosomes,andthereforewillbepulledtooppositepoles.

B. Anaphase is the step in mitosis when sister chromatid cohesion is rapidly dissolved. Asaresult,thepullingforcesofthemicrotubulescausethetwosisterchromatidstomovetotheoppositepoles(Figure5).

C. Cytokinesis is the step in which cells physically divide into two daughter cells (Figure 6). Forareviewofmitosis,seethemitosisandmeiosisonlinemoduleoniROCKET.

Figure 4

Figure 5


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III. CELL CYCLE CONTROL: TWO ISSUESHowisthecellcyclecontrolled?Theproblemcanbebrokendownintotwoparts:First,howisthecellcycleregulatedsothatthedifferentphasesoccurinthecorrectorder?Second,howdoextracellularsignalsactivateorinhibitthecellcycle?

A. Cyclin Dependent Kinases: The core of the cell cycle control systemThe events that occur in each part of the cell cycle are carried out by specific proteins,andtheseproteinsmustbesynthesizedoractivatedatthecorrecttimeinthecycle.Forexample,beforeDNAsynthesiscanbegin,theenzymesthatproducethenucleotidesusedinDNAsynthesismustbeactivated.ThisoccurslateinG1phase.(RememberNucleotideMetabolism?SeelecturefromM&N.)

Thecellturnsproteinactivityonandoffatappropriatetimesinthecellcycleusingcyclin-dependent kinases(Cdks).Likeotherproteinkinases,cdksturnproteinsonoroffbyphosphorylatingthem.Eachcyclin-dependentkinasehastwosubunits-akinasesubunitandacyclinsubunit(Figure7).

Cyclins were first identified as key cell-cycle regulators when it was observed thattheyundergoacycleofsynthesisanddestructionduringeachcellcycle.ThereareseveraldifferentCdksandanumberofcyclins.Thekinasesubunitsarepresentthroughoutthecellcycle,whilethecyclinsubunitsare produced at specific times in the cell cycle, thus providing temporal regulationofthecyclin-CdKcomplex.Asthecyclinsubunitisproduced,itbindstothekinasesubunitandactivatesit.Thecyclinsubunitalso

Figure 7.Cyclin-dependentkinases(Cdks).Cdksarethekeyregulatorsofthecelldivisioncycleinorganismsasdiverseasbaker’syeastandhumans.Cyclin-dependentkinaseshavetwosubunits,thekinase(oftensimplycalledtheCdk)andaregulatoryproteincalledacyclin.

TableI.Thefourkeycyclin-Cdkstatdrivethecell

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targets its kinase partner to specific protein substrates. The key cyclin-Cdk complexesthatdrivethecellcyclearelistedinTableI.

ThecellcyclecanbeviewedasaCdkcycle(Figure8).AsthecellentersG1phaseattheendofmitosis,cyclinDisproducedandbindstoitstwopartnercdks–cdk4andcdk6.Thesecyclin-cdkcomplexesarebothreferredtoasG1-Cdks.ActivationoftheG1-CdksturnsontheeventsthatoccurintheearlyphaseofG1.OneoftheeventsthatoccursinearlyG1issynthesisofcyclinE.Asitismade,itbindstoCdk2,formingG1/S-Cdk.AstheG1/S-Cdkactivityaccumulatestoacriticalthreshold,ittriggersthetransitionfromlateG1intoSphase.CyclinAismadeinSphase.Italsobindstocdk2,formingS-Cdk.ThelatterisrequiredforDNAsynthesis.CyclinBismadeduringSphaseandG2.Asitismade,itbindstoCdk1formingM-Cdk.WhenM-Cdkreachesathresholdactivity,ittriggersthetransitionfromG2intomitosis.

B. how the G1-Cdk turns on the G1/S CdkThetransitionbetweenearlyG1andlateG1illustratesonewaythatcyclin-dependentkinasesregulatetheprogressionofthecellaroundthecellcycle.Thisisacrucialcontrolpointwhichisoftenderegulatedincancer.

InordertomovefromearlyG1tolateG1,thecellmustsynthesizecyclinE.TranscriptionofthecyclinEgenerequiresatranscriptionfactorcalledE2F.IncellsthatarenotproliferatingandincellsthatareinearlyG1,theE2FtranscriptionfactorisboundtothepromoterforthecyclinEgene,butitisinhibitedbyaproteincalledRb. (RbstandsforRetinoblastoma,achildhoodtumoroftheretina–moreonthisinthetumorsuppressorandoncogenelecture!).WhenG1-CdkactivityincreasesnearthemiddleofG1,G1-CdkphosphorylatestheRbprotein(Figure9).ThisreleasesRbfromE2FandallowstranscriptionofthecyclinEgenetotakeplace.ThecyclinEproteinbindstotheCdk2kinasetoformtheG1/S-Cdk.E2Falsotranscribes

Figure 8.ThecellcycleasaCdkcycle.Differentphasesofthecellcyclearedrivenbydifferentcyclin-Cdkcomplexes.Inthissimplified view, there exists aG1-Cdk,aG1/S-Cdk,aS-CdkandaM-CdkthatactinsequencetoprogramtheeventsofG1,theG1-Stransition,Sphase,andMphase.


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anumberofothergenesimportantforSphase,includingthegenesforDNApolymeraseandthymidylatesynthase.

C. Brakes on the cell cycle: Cdk inhibitors.TheRbproteincanbeviewedasa“brake”onthecellcyclebecauseitpreventsthetranscriptionofthegeneforcyclinEbyinhibitingE2F.Threeotherproteinsthatactas“brakes”onthecellcyclearetheCdkinhibitorsp21,p16,andp27.Whyisthecellcyclecontrolledbybothactivators(e.g.cyclins)andinhibitors?Aswewillsee,itissothatcellscanrespondtobothgrowthactivatingandgrowthinhibitingsignals.

Cdk inhibitors fall into two classes: general and specific (Figure 10). General Cdkinhibitors,suchasp21andp27,bindtomultipleCdk-cyclincomplexes,shutting off the cell cycle at multiple points. p16 is a specific Cdk inhibitor whichtargetsCdk4-cyclinD(G1Cdk).

Aswillbediscussedintheoncogeneandtumorsuppressorlecture,theseinhibitorproteinsplayanimportantroleincancer.

IV. CONTROLLING PROLIFERATION: ThE ROLE OF MITOGENS AND ANTI-MITOGENSNon-dividingcellsexistinphasecalledG0(Gzero).G0cellscanre-enterthecellcycleinG1whenstimulatedbymitogens,whichareextracellularproteinsthatstimulatecellproliferation.Mitogensarealsocalled growth factors. Conversely,cellscanbearrestedinG1viatheactionofanti-mitogens (proteinsthatinhibittheactivityofmitogens).Manygrowthfactorsandinhibitorsare

Figure 9.HowtheG1-CdkactivatestheG1/S-Cdk.TheG1-Cdk(Cdk4-cyclinD)phosphorylatestheRb(Retinoblastoma)proteinreleasingitfromtranscriptionfactorE2F.E2FcannowactivatetranscriptionofcyclinE,whichinturnresultsintheproductionofcyclinEproteinandformationoftheG1/SCdk.

Cdk4-cyclin D (G1-Cdk)

Rb

E2F

cyclin E

Cdk2

Cdk2-cyclin E (G1/S-Cdk)

promoter

cyclin E gene

OFF

E2F

Rb

cyclin E gene

ON

E2F

RbP

G1-Cdk OFF

G1-Cdk ON

A. B. Control RelationshipsMolecular Events

promoter

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known.Wewilldiscussoneexampleofeach:themitogenepidermal growth factor(EGF)andtheanti-mitogentransforming growth factor ß(TGFß).

Whatarethenormalfunctionsofthesefactors?EGFfunctionstopromotewoundhealing. After a wound is formed, epidermal and inflammatory cells secrete EGFandothergrowthfactors.Itsignalscellsatthemarginsofthewoundtoproliferatesothatthewoundmaybehealed.TGFßactsasabraketothisprocesssothattheproliferationoccursataratethatisnottoofasttocoordinatewithotheraspectsofwoundhealing.

A. The EGF Signaling PathwayThispathwayallowsEGFontheoutsideofthecelltoultimatelycontrolthecellcycleontheinsideofthecell.Itinvolvesmanystepsandcomponents.Thiscomplexityisthoughttoexistfortworeasons.First,evolutionoftenproceedsbyadaptingexistingpathwaysandcombiningpathwaystocarryoutnewfunctions.Theresulting“newpathway”maybequitecomplexbecauseofitshistory.Second,thereisevidencethathavingapathwaywithmultiplesteps filters noise and allows many opportunities for regulatory control. Such apropertymakessenseinthecontextofthecellcyclewherecellsareeitherproliferatingorarrestedinG0.

EGFfunctionsbybindingtotheextracellulardomainofEGFreceptor,acellsurfaceproteinwithasingletransmembranedomain(Figure11).ThecytoplasmicdomainofthereceptorisaproteintyrosinekinasethustheEGFreceptoriscalledareceptor tyrosine kinase.WhenEGFbindstoitsreceptor,thereceptorformsadimerinwhichonesubunitphosphorylatestheother(transphosphorylation)onparticulartyrosineresiduesinthecytoplasmicpartofthereceptor.Thesephosphorylatedtyrosinesserveasbindingsitesforothercytoplasmicproteinsthatcontainspecialdomains,calledSh2 domains. SH2 domains specifically recognize phosphorylated tyrosinesandtheadjacentaminoacids.OneproteinthatbindstophosphotyrosineresiduesintheEGFreceptorisanadaptorproteincalledGrb2.Grb2,inturn,recruitsaproteincalledSos.ThusbindingofEGFto

Figure 10.CdkinhibitorsbindtoandinactivateCdks.

Cdk2/4-cyclin D (G1-Cdk)Cdk2-cyclin E (G1/S-Cdk)

Cdk2-cyclin A (S-Cdk)

Cdk2-cyclin E (G1/S-Cdk)

p21 p27

p16


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theEGFreceptorrecruitsbothGrb2andSostotheintracellularportionofthereceptor.

Sos,inturn,actsonasmallGTPbindingprotein,Ras.TheRasproteinisboundtotheinnersurfaceoftheplasmamembrane.LiketheG-proteinsdiscussedbyDr.FultoninthePrologue,Rascanexistintwostates:aninactivestateinwhichGDPisboundandanactivestateinwhichGTPisbound.SosactivatesRasbypromotingthereleaseofitsGDPandbindingofGTP.Thus,SosisaguaninenucleotideexchangefactorforRas(Figure12).RecruitmentofSostotheplasmamembranewhereRasislocatedresultsintheactivationofRas.

Figure 11.ActivationoftheEpidermalGrowthFactor(EGF)receptortyrosinekinase.EGFbindstotheEGFreceptorthroughanextracellularligandbindingdomain,leadingtodimerizationofthereceptor.Dimerizationcausesonesubunittophosphorylatetheother(transphosphorylation)on specific tyrosine residues. TheSH2domainoftheGrb2adaptorproteinbindsthephosphorylatedtyrosines.Grb2inturn,bindsaanotherproteincalledSos.

EGFLigand

Binding Domain

KinaseDomain

P SH2 SH3

Grb2(adaptor protein)

Sos

Figure 12.SosactivatesRasandaGTPase-activatingproteininactivatesit.InitsGDPboundform,Rasisinactive.WhenSosisboundtoGrb2attheEGFreceptor,itcausesRastoreleaseitsGDPandbindaGTPinitsplace.AGTPase-activatingproteincauseshydrolysisofGTPtoGDP.

Sos

Ras-GDP Ras-GTP

GTP

GDP

inactive active

GAP

GTPase-activating protein

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RascanbereturnedtoitsinactiveformthroughthehydrolysisofGTPtoGDP.ThisstepoccurswhenaGTPase-activating proteinbindsRas andinducesthehydrolysisofitsGTP(seeFigure12).

InitsGTP-bound(active)state,Rasturnsonaproteinkinasecascade,inwhichproteinkinasessequentiallyactivateeachotherthroughphosphorylation(Figure13).ActiveRasbindstoandactivatesaproteinkinasecalledRaf.Inturn,RafphosphorylatesandactivatesanotherkinasecalledMEK.MEKinturnphosphorylatesandactivatesmitogen-activatedproteinkinase(MAP kinase).Thischainofphosphorylationeventsiscalled the MAP kinase cascade. MAP kinase phosphorylates gene-specific transcriptionfactorsinthecellnucleusthatbindtothepromotersofgenesandpromotecellproliferation.OneimportanttranscriptionfactorthatisupregulatedbytheMAPkinasecascadeisMYC,whichistheproductoftheC-MYCgene.

OneofthetargetsofthesetranscriptionfactorsisthecyclinDgene.Thus,amulti-tieredpathwayconnectsthepresenceofamitogen(EGF)outside

Figure 13.RasactivatestheRaf-MEK-MAPKcascade.RasbindsdirectlytoRaf,whichactivatesitskinaseactivity.RafphosphorylatesakinasecalledMEK(alsocalledMAPkinasekinase).AfterithasbeenphosphorylatedbyRaf,MEKphosphorylatesMAPkinase(mitogenactivatedproteinkinase,MAPK).ActiveMAPKthenphosphorylatesitstargetproteins,leadingtotheactivationofcelldivision.

Ras-Raf

MEK MEK-P

MAPK MAPK-P

Target Target-P

inactive

inactive

inactive

active

active

active

Figure 14.ActivationofMAPKleadstothetranscriptionofcyclinD.MAPKphosphorylatestranscriptionfactors.Thisinturnleadstothetranscriptionofthemycgene,whichitselfencodesatranscriptionfactorforthecyclinDgene.

TranscriptionFactorsMAPK cyclin D


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thecelltoincreasedexpressionofakeycomponentofthecellcyclecontrolmachinery(thecyclinDgene)inthenucleus.IncreasedexpressionofthecyclinDgeneleadstotheactivationofG1-Cdk(Figure14).

B. Wnt signalingWnt proteinsaregrowthfactorsanalogoustoEGF.Theyfunctioninasignalingpathwaythatactivatescellproliferation(Figure15).NotonlyistheWntsignalingpathwayimportantforthedevelopmentoftheorganism,italsoservesimportantfunctionsinadults.Forexample,Wntsignalingisnecessaryfortheproliferationofstemcellsintheproliferativezonesinthegutepithelium(thecryptsthatliebetweenthemicrovillioftheepithelium)(Figure16).Hyperactivationofthispathwayisassociatedwithcoloncancer.

Figure 15. TheWntsignalingpathway.

GSK-3

Wnt

Frizzled

β-catenin β-cateninAPC APC

P P

degradation

APCTCF

β-catenin

TCF

cyclin D

microvillus microvillus

crypt(proliferatingstem cells)

Expressionof APC

low

high

Figure 16.Shownisaschematicofamicrovillusinthegutepitheliumshowingthezoneofproliferation(crypts)andthegradientofexpressionoftheAPCgene,whoseproteinproductinhibitsWntsignaling.

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WntproteinsbindtoacellsurfacereceptorcalledFrizzled.Frizzledcontrolsthestabilityofaproteincalled ß-catenin,whichfunctionstogetherwithaproteincalledTCFtoformatranscriptionfactorthatactivatesthecyclinDpromoter.

OnceboundtoWnt,FrizzledturnsoffaproteinkinasecalledGSK-3.GSK-3normallyfunctionstopromotethedegradationß-catenin,thuspreventingitfromactivatingthecyclinDpromoter.Phosphorylationofß-cateninbytheproteinkinaseGSK-3resultsinitsdegradation.However,GSK-3canonlyphosphorylateß-cateninwhenß-cateninisboundtoaproteincalledAPC (adenomatous polyposis coli).Thus,APCisnecessarytoholdß-cateninincheck.

OnceGSK-3isinhibitedbyFrizzled,ß-cateninisnolongerdegraded,allowingittoassociatewithTCFandactivatethecyclinDpromoterandpromotecellproliferation.Thus,Wntsignalingpromotescellproliferationthroughtheactionofb-cateninoncyclinD.

WhileWntproteinsaretheextracellulargrowthfactorsthatactivatethispathway,cellsalsocontrolthepathwayfromwithinthecellbyvaryingthetranscriptionoftheAPCgene,whoseproteinproductinhibitstheWntsignalingpathway.Forexample,intheepitheliumofthecolon,thereexistsagradientofAPCexpressionwhichishighestintheterminallydifferentiatednondividingcellsinthemicrovilliandlowestintheproliferatingstemcellsin

Figure 17.HowTGFbßarrestscelldivision.TGFßbindstotheTGFßreceptor.BindingofTGFßactivatesthereceptor’sintracellularproteinkinasedomain,leadingtophosphorylationofSmadproteins.PhosphorylatedSmadsenterthenucleusandbindtopromotersofgenestocontroltranscription.Akeytargetisthep21gene.

plasmamembrane

nucleus

TGFβ

kinasedomain

Smad

Smad-P

p27 gene

promoter

Smad-P

TGFβ receptor

p21

Cdk4-cyclin DCdk2-cyclin ECdk2-cyclin A


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thecrypts(Figure16).(MoreabouttheroleofAPCincoloncancertocomeinlecturesonColonCancerandFamilialandHereditaryCancerSyndromes.)

C. An anti-mitogenic pathway: the TGFß-Smad pathwayLikeEGF,TGFßisanextracellularproteinthatbindsacellsurfacereceptor.However,insteadofcausingcellproliferation,thismoleculecausescellstoarresttheircellcycleandenterG0.Howdoesthisoccur?TheTGFßreceptorisatransmembraneserine-threoninekinase.UponbindingtoTGFß,thereceptorphosphorylatesproteinsinthecytoplasmcalledSmads (Figure17).Oncephosphorylated,Smadproteinsthenenterthenucleusandfunctionastranscription factors to turn on specific target genes. A key gene turned on byTGFßistheG1/S-Cdkinhibitorp21discussedabove.Theactivationofp21leadstothearrestofthecellcycle.Thus,TGFßarrestscelldivisionbyactivatingthegeneforaCdkinhibitor.

V. APOPTOSISThenumberofcellsinatissueisnotonlycontrolledbycellproliferationbutalsobyprogrammedcelldeath,orapoptosis.Foratissuetostaythesamesize,cellproliferationandcelldeathmustbeperfectlybalanced.Apoptosisplaysimportantrolesbothduringdevelopmentandinmaturetissues(Figure18).Duringdevelopmentofalimb,tissuepresentbetweenthedigitsmustberemoved.Thisoccursthroughlocalizedapoptosis.Intheimmunesystem,onemechanismbywhichTcellskilltargetcellsistoproduceaproteincalledFasligand.Fasligandbindsitsreceptor,Fas,ontargetcells.BindingofFasligandtoFasinducesapoptosis.Therealsoexistextracellularproteins,calledsurvival factors,whichpreventapoptosisbybindingtotheirreceptorsonthesurfaceofthecell.

AsdescribedinthePrologueblock,activationofapoptosisfromoutsidethecelliscausedbytheassociationofprocaspase-8withthecytoplasmicdomainof“deathreceptors”suchasFas(Figure19a).Thisassociationismediatedbyadaptor

Figure 18.Programmedcelldeath.Duringdevelopmentoflimb,tissuepresentbetweenthedigitsisremovedbyapoptosis.KillerTcellstriggerapoptosisbyproducingFasligand.

Apoptosis

Killer T-cell Target Cell

Fas ligandFas

Apoptosis

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Figure 19.Inductionofapoptosisbyeitherextracellularorintracellularsignals.(A)Extracellularactivation.AdaptorproteinsbindtheintracellularregionofaggregatedFasproteins,causingtheaggregationofprocaspase-8molecules.Thesethencleaveoneanothertoinitiatethecaspasecascade.(B)Intracellularactivation.Mitochondriareleasecytochromec,whichbindsandcausestheaggregationoftheadaptorproteinApaf-1.Apaf-1bindsandaggregatesprocaspase-9molecuels,whicharetherebyactivated(withoutcleavage)totriggeracaspasecascade.FromAlbertsetal.,MolecularBiologyoftheCell,2002.

proteinsthatbindbothFasandprocaspase-8.Theaggregatedprocaspase-8moleculescleaveeachother,initiatingthecaspase cascadethatleadstoapoptosis.

Whencellsarestressed(e.g.,hypoxia)ordamaged(e.g.,unrepairedDNAdamage)theycanalsoactivateapoptosisfrominsidethecellbytriggeringprocaspaseaggregationandactivation.Inresponsetostressordamagen,pro-apoptoticsignalsinducemitrochondriatoreleatecytochrome cintothecytosol,whereitbindsandactivatesanadaptorproteincalledApaf-1.Thiscomplexactivatesacaspasecalledprocaspase-9,whichleadstotriggeringofthecaspacecascade(Figure19-b).

ThereleaseofcytochromecfromthemitochondriaistightlycontrolledbymembersoftheBcl-2 familyofproteins.Withinthisfamilyofproteins,therearetwosub-classes:proteinsthatpromoteapoptosis(“proapoptotic”)suchasBad,BaxandPUMA,andproteinsthatantagonizeapoptosis(“antiapoptotic”)suchasBcl-2andBcl-xL.Theantiapoptoticfamilymemberscandirectlybindto


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Figure 20.HowproteinsoftheBcl-2familycontrolapoptosis.ProapoptoticmembersoftheBcl-2familysuchasBadformchannelsinthemitochondrialmembranewhichpermitthereleaseofcytochromecintothecytoplasm.CytochromecbindstoApaf-1whichinturnactivatesprocaspase-9.AntiapoptoticmembersoftheBcl-2familypreventtheBadchannelfromforming.

Bad channel

cytochrome C apoptosis

Bad Bcl-2family

member

theproapoptoticfamilymembersandinhibitthem(Figure20).Intheabsenceoftheantiapoptoticfamilymembers,theproapoptoticfamilymembers,suchasBadandBax,formchannelsinthemitochondrialmembranethatresultsinthereleaseofcytochromecintothecytoplasmwhereitcanactivateprocaspase-9viaApaf-1.PUMAfunctionsinadifferentmannerasdescrbedbelow.

VI. P53, ThE CELL CYCLE, AND APOPTOSISThecellcycleiscontrolledatcertainstagesbycheckpoints.Thesearebiochemicalmechanismsthatstopthecellcycleifcertainconditionsarenotmet.

OnecheckpointistheG1DNAdamagecheckpoint.IfcellscontainunrepaireddamagetotheirDNA,thecellcycleisarrestedinG1.Thisarrestrequiresakeytranscriptionfactor,p53,whichisactivatedbyDNAdamage.Therearethreecomponentstothesystem:1)aDNAdamagesensor,2)theMdm2proteinwhichnormallycausesp53tobedegraded,3)andp53itself(Figure21).DNAdamagecausesphosphorylationofp53andblocksthebindingofMdm2.Thisleadstothestabilizationandaccumulationofp53.p53canthenbindtothepromoterofthep21CDKinhibitordescribedearlierandactivateitstranscription.InhibitionofCdksleadstocellcyclearrest.

Ifp53activationcontinuesforaprolongedperiodoftime,apoptosisensues(Figure22).ThisprocesskillscellswithdamagedDNAthatremainunrepaired,andservestoremovecellsfromtissuesthatmayotherwiseaccumulatemutationsthatwouldbepassedontotheirdaughtercells.p53probablyactivatesapoptosisbyincreasingthetranscriptionofseveralgenes.OnetargetgeneistheproapoptoticBcl-2familymemberBax,whosegeneisdirectlyactivatedbyp53.

Recentresearchindicatesthatp53ismorethanatranscriptionfactor.Unexpectedly,somep53associateswithmitochondriatodirectlypromotethereleaseofcytochromeCfrommitochondria.Thiscytoplasmicpopulationofp53

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Figure 21.HowDNAdamageactivatesp53andcausescell-cyclearrest.DNAdamageactivatesaproteinkinasewhichphosphorylatesp53.

Figure 22.DNAdamagecanleadtoapoptosis.Prolongedactivationofp53inresponsetoDNAdamageresultsinapoptosis.p53activatesthetranscriptionofseveralgenesinvolvedinapoptosisincludingthatfortheproapoptoticBcl-2familymemberBaxshownhere.

BAX gene

promoter

p53

Prolonged p53

Activation

Bax channel

cytochrome C apoptosis


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isinhibitedbytheanti-apoptoticBcl-2familymemberBcl-xL,whichdirecltybindstop53.Oneofthetranscriptiontargetsofp53,PUMA(theproapoptoticBcl2familymemberdescribedabove),releasesp53fromBcl-xLThisinturnallowsp53tobindtoBaxwhichactivatescytochromeCrelease,andapoptosisensues.

Inlightoftheimportantrolep53playsinpreventingunrepairedDNAdamagetobepassedontodaughtercells,itisnotsurprisingthatp53isfoundtopayaroleincancerdevelopment.Infact,p53ismutatedinmanytypesofcancer,thusallowingdamagedDNAtoremainincellsastheyproliferate(moreinthelectureontumorsuppressorgenesandoncogenes).

VII. ThE SPINDLE ASSEMBLY ChECKPOINTInadditiontomonitoringthestateofDNA,cellsalsomonitorthestateofthemitoticspindleatthespindle assembly checkpoint(Figure24).Thespindleassemblycheckpointensuresthatmitosisdoesnotproceedunlessthespindleisproperlyassembled.ThischeckpointmonitorstheattachmentofspindlemicrotubulestoeachkinetochorethoughtheactionoftheMad2 protein.Therearetwokeyfeaturesofthecheckpoint:1)Mad2associateswithkinetochoresonlywhentheyarenotattachedtomicrotubules.and2)Mad2isactiveinarrestingmitosisonlywhenitisboundtokinetochores.Thuswhenthespindleisproperlyassembledandallofthekinetochoresareboundtomicrotubules,Mad2isinactiveallowingmitosistoproceed.Ifthereisaproblemwithspindleassembly,Mad2isactivatedandarreststhecellcycle.Notsurprisingly,cellsdefectiveinthespindleassemblycheckpointshowhighratesofaneuploidybecauseoferrorsinchromosomesegregationduringmitosis.Defectsinthespindle-assemblycheckpoint, and specifically in Mad2, have been associated with tumorigenesis.

p53 stabilization

accumulation innucleus

activation of Bax andPUMA transcription

accumulation incytoplasm

Bcl-XL sequestersp53

PUMA liberates p53

p53 activates BaxApoptosis

Figure 23. p53functionsbothinthenucleusandthecytoplasmtopromoteapoptosis

Cell Proliferation

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SisterChromatids

microtubules

kinetochore

centrosome Mad2

cell cycle arrest

Mad2

inactive

Figure 24.Spindleassemblycheckpoint.ThisisaccomplishedthroughtheactionoftheMad2protein,whichbindstokinetochoresthathavenotattachedtomicrotubules.Whenboundtokinetochores,Mad2triggerscellcyclearrest.Oncemicrotubulesareattachedtoallofthekinetichores,Mad2isnolongeractiveandthecellcycleproceeds.

cell proliferation and its regulationbiochemistry.ucsf.edu/programs/ptf/m3 links/cell proliferation...

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