線蟲發(fā)育及基因功能研究韓珉

線蟲發(fā)育及基因功能研究韓珉第1頁/共45頁P(yáng)athwayinearly1992RASEGFR第2頁/共45頁R7R8WTsevenlessR7receptorisrequiredforUVresponsesevBoss第3頁/共45頁Strategy:movingfromRTKdown:discoveringSOSRASGDPRASGTP第4頁/共45頁SonofSevenless:hyperactivemutationRogerandBenejee,Cell1990ThestoryaboutRonRoger-+Sev/RTK(lf) SevenlessSev(lf)+SOS(gf) R7fineSevSOSR7fateLeadstoModel:A:yesB:maynot.第5頁/共45頁Drosophila:F1screenvsF2screenX**X**F3homozygotesmutagenXTMTM**F1F2screenmutagenX**F1F1screenTMAconversationwithRubin第6頁/共45頁MikeSimon:landmarkmodifyscreenSimonetal.1991CellSevRAS

SOSR7Rougheye,noR7Mostlywt,small%defectsRougheyeRougheyeHighTLowTLowTLowTTsalleleX**F1F1screenTM+/-+/-第7頁/共45頁SOSencodesanexchangefactorforGTPaseSimonetal.1991CellRASGDPRASGTP第8頁/共45頁第9頁/共45頁Movingfromreceptordown:biochemistryRASGDPRASGTP第10頁/共45頁DiscoveryofGRB2EGFRYEGFRLigandactivationY-PScreenlGT11proteinexpressionlibrary9GRBproteinsWhichoneisonemediatedRasactivation?Y-P第11頁/共45頁DiscoveryofGRB2:complementaryworksbybiochemistsandgeneticistsAcDNAcloneencodinganovel,widelyexpressedprotein(calledgrowthfactorreceptor-boundprotein2orGRB2)containingonesrchomology2(SH2)domainandtwoSH3domainswasisolated.ImmunoblottingexperimentsindicatethatGRB2associateswithtyrosine-phosphorylatedepidermalgrowthfactorreceptors(EGFRs)andplatelet-derivedgrowthfactorreceptors(PDGFRs)viaitsSH2domain.Interestingly,GRB2exhibitsstrikingstructuralandfunctionalhomologytotheC.elegansproteinsem-5.Ithasbeenshownthatsem-5andtwoothergenescalledlet-23(EGFRlike)andlet-60(raslike)liealongthesamesignaltransductionpathwaycontrollingC.elegansvulvalinduction.ToexaminewhetherGRB2isalsoacomponentofrassignalinginmammaliancells,microinjectionstudieswereperformed.WhileinjectionofGRB2orH-rasproteinsaloneintoquiescentratfibroblastsdidnothavemitogeniceffect,microinjectionofGRB2togetherwithH-rasproteinstimulatedDNAsynthesis.TheseresultssuggestthatGRB2/sem-5playsacrucialroleinahighlyconservedmechanismforgrowthfactorcontrolofrassignaling.Lowensteinetal.Cell.Aug.1992第12頁/共45頁Thesem-5story-early1992SEM-5wasidentifiedasbyasuppressormutantscreen Clarketal.NatureMar1992lin-15(-)

Multivulvasem-5(-) Vulvalesslin-15(-);sem-5(-) VulvalessRasSEM-5LIN-15EGFRLIN-15RasVulvalinductionKnownSince1990SEM-5ishomologoustoGRB-2.Combinethedatafromthetwofield:RasGRB2/SEM-5EGFRras(gf)

Mutivulvasem-5(-) Vulvaless

ras(gf);sem-5(-) Multivulva第13頁/共45頁Therestofthestoryisthereinarushandclash-mid1993OlivierJP,RaabeT,HenkemeyerM,DicksonB,MbamaluG,MargolisB,SchlessingerJ,HafenE,Pawson.ADrosophilaSH2-SH3adaptorproteinimplicatedincouplingthesevenlesstyrosinekinasetoanactivatorofRasguaninenucleotideexchange,Sos.Cell.1993Apr9;73:179-91.EganSE,GiddingsBW,BrooksMW,BudayL,SizelandAM,WeinbergRA.AssociationofSosRasexchangeproteinwithGrb2isimplicatedintyrosinekinasesignaltransductionandtransformation.Nature.1993May6;363:45-51.Rozakis-AdcockM,FernleyR,WadeJ,PawsonT,BowtellD.TheSH2andSH3domainsofmammalianGrb2coupletheEGFreceptortotheRasactivatormSos1.Nature.1993May6;363:83-5.BudayL,DownwardJ.Epidermalgrowthfactorregulatesp21rasthroughtheformationofacomplexofreceptor,Grb2adapterprotein,andSosnucleotideexchangefactor.Cell.1993May7;73:611-20.ChardinP,CamonisJH,GaleNW,vanAelstL,SchlessingerJ,WiglerMH,Bar-SagiD.HumanSos1:aguaninenucleotideexchangefactorforRasthatbindstoGRB2.Science.1993May28;260:1338-43.SkolnikEY,BatzerA,LiN,LeeCH,LowensteinE,MohammadiM,MargolisB,SchlessingerJ.ThefunctionofGRB2inlinkingtheinsulinreceptortoRassignalingpathways.Science.1993Jun25;260:1953-5.BaltenspergerK,KozmaLM,CherniackAD,KlarlundJK,ChawlaA,BanerjeeU,CzechMP.BindingoftheRasactivatorsonofsevenlesstoinsulinreceptorsubstrate-1signalingcomplexes.Science.1993Jun25;260:1950-2.SimonMA,DodsonGS,RubinGM.

SH3-SH2-SH3proteinisrequiredforp21Ras1activationandbindstosevenlessandSosproteinsinvitro.Cell.1993Apr9;73:169-77.第14頁/共45頁ManySmallGTPaseshaveaconservedC-terminus第15頁/共45頁Rasislipidmodifiedforitsmembranebinding第16頁/共45頁RASGDPSH2SH3SH3SOSSH3SH3SH2GRB2EGFRYRASGDPSH2SH3SH3SOSEGFRY-PRASGTP第17頁/共45頁HalfwaydoneRASGDPRASGTP第18頁/共45頁Summaryoftheoriginalscreenslin-15(-),Muvulvalin-15(-);suppressor;

Vulvalessmutagenwormbiochembindingtophosphorylatedtyosineofgrowthfactor:Grb2DiscoveryofGAP.RTKGRB2SOSRaswormLET-23SEM-5SOS-1LET-60flySevenlessDRKSOSRas1sev(lf),sev(lf);suppressor/enhancer;

flyR7fatereduced

mutagenR7fatebetterorworseGAPGAP-1Gap1第19頁/共45頁Theholygrail:whatistheeffectorofRas?第20頁/共45頁TheRaseffector1.MustinteractwiththeRaseffectordomain2.TheinteractionmustberequiredforRasfunction3.MustberequiredtointeractwithactivatedRas(oncoproteins).A:Yes,B:noRASGDPRASGTP第21頁/共45頁TheRaseffectordomainisdefinedby1.MutationsintheregionaffectRasbiologicalfunction2.Themutationshavenoeffectonotherbiochemproperties3.Thedomainisproposedtointeractwiththeeffector/target1259116effector185164第22頁/共45頁GAP=RaseffectorNature.April1988第23頁/共45頁GAP=RaseffectorScience.April1988第24頁/共45頁KeyargumentA.ThebiochemicalandgeneticcriteriaforaRaseffector:1.ItshouldinteractwithGTP-boundnotGDP-boundRas.2.ItshouldnotinteractwithaninactiveRasB.

FactorsinterferingwithRasfunctionblocktheRas/GAPinteractionKeydataPositionofRasmutationsGAPactivation(hydrolysis)InteractionWithGAPRasfunction12,59,61abolished++++35,36,38insensitive--39Sensitive++1259116effectorGAPbindingMembranebinding185164GAPistheeffector.A:Convincing.B.Somewhatconvincing.C.Notconvincing.第25頁/共45頁ThefieldwasconvincedCellJune15,1990RASGDPRASGTP第26頁/共45頁ThefieldwasconvincedAnnuRevCellBiol.1991;7:601-32.第27頁/共45頁ThefieldwasconvincedAnnuRevCellBiol.1991;7:601-32.第28頁/共45頁ThefieldwasconvincedAnnRevCellBiol1991;7:601-32.第29頁/共45頁FunctionsGAPalsoactsastheeffectorRASGDPRASGTP第30頁/共45頁GeneticsGaul,MardonandRubin.,:AputativeRasGTPaseactivatingproteinactsasanegativeregulatorofsignalingbytheSevenlessreceptortyrosinekinase.CellMarch1992Buchberg,Cleveland,Jenkins,Copeland:Sequencehomologysharedbyneurofibromatosistype-1geneandIRA-1andIRA-2negativeregulatorsoftheRAScyclicAMPpathway.Nature.1990SepStanaka,….,Matsumoto,Kaziro,Toh-e:S.cerevisiaegenesIRA1andIRA2encodeproteinsthatmaybefunctionallyequivalenttomammalianrasGTPaseactivatingprotein.Cell.March1990Xu,….,Tamanoi:Thecatalyticdomainoftheneurofibromatosistype1geneproductstimulatesrasGTPaseandcomplementsiramutantsofS.cerevisiae.Cell.1990NovTanaka,Matumoto,andToh-E:IRA1,aninhibitoryregulatoroftheRAS-cyclicAMPpathwayinS.cerevisiae.MolCellBiol.1989FebHajnal….Kim:InhibitionofC.elegansvulvalinductionbygap-1andbylet-23receptortyrosinekinase.GenesDev.Oct.1997

第31頁/共45頁WhatdidgeneticssayRas(-) SignalingeliminatedGAP(-) SignalingincreasedQ:CanGAPbethemajoreffectorofRas?A:YesB:NoC:notsure第32頁/共45頁Theendof1992:GAPwasnolongerconsideredaRaseffector第33頁/共45頁TheStoryofRafCell1989Aug:DeborahK.Morrison,DavidR.Kaplan,JaimeA.Escobedo,UlfR.Rapp,ThomasM.RobertsandLewisT.Williams:Directactivationoftheserine/threoninekinaseactivityofraf-1throughtyrosinephosphorylationbythePDGFreceptor

Wehaveexaminedtheinteractionbetweentheserine/threoninekinaseproto-oncogeneproductRaf-1andthetyrosinekinasePDGFbeta-receptor.Raf-1tyrosinephosphorylationandkinaseactivitywereincreasedbyPDGFtreatmentof3T3cellsorCHOcellsexpressingwild-typePDGFreceptorsbutnotmutantreceptorsdefectiveintransmittingmitogenicsignals,suggestingthattheincreaseinRaf-1kinaseactivityisasignificanteventinPDGF-inducedmitogenesis.Concurrentwiththeseincreases,Raf-1associatedwiththeligand-activatedPDGFreceptor.Furthermore,bothmammalianRaf-1andRaf-1expressedusingarecombinantbaculoviralvector,associatedinvitrowithbaculoviral-expressedPDGFreceptor.Thisassociationwasmarkedlydecreasedbypriorphosphatasetreatmentofthereceptor.Followingincubationofpartiallypurifiedbaculoviral-expressedPDGFreceptorwithpartiallypurifiedRaf-1,Raf-1becamephosphorylatedontyrosineanditsserine/threoninekinaseactivityincreased4-to6-fold.Thisisthefirstdemonstrationofthedirectmodulationofaproteinactivitybyagrowthfactorreceptortyrosinekinase.第34頁/共45頁TheStoryofRafCell1989Aug:DeborahK.Morrison,DavidR.Kaplan,JaimeA.Escobedo,UlfR.Rapp,ThomasM.RobertsandLewisT.Williams:Directactivationoftheserine/threoninekinaseactivityofraf-1throughtyrosinephosphorylationbythePDGFreceptor

PDGFRY-PRaf-1Raf-1P-Isthismodelconvincing?A:Thereisnoconvincingdatatosupportit.B:Thedataisgood,theproposalisreasonable.第35頁/共45頁TheStoryofRaf:summer1993MoodieSA,WillumsenBM,WeberMJ,WolfmanA.

ComplexesofRas.GTPwithRaf-1andmitogen-activatedproteinkinasekinase.Science.1993Jun

WarnePH,VicianaPR,DownwardJ.

DirectinteractionofRasandtheamino-terminalregionofRaf-1invitro.Nature.1993Jul

ZhangXF,……,RappUR,AvruchJ.Normalandoncogenicp21rasproteinsbindtotheamino-terminalregulatorydomainofc-Raf-1.Nature.1993JulVojtekAB,HollenbergSM,CooperJA.MammalianRasinteractsdirectlywiththeserine/threoninekinaseRaf.Cell.1993Jul*****HughesDA,AshworthA,MarshallCJ.Complementationofbyr1infissionyeastbymammalianMAPkinasekinaserequirescoexpressionofRafkinase.Nature.1993Jul.VanAelstL,BarrM,MarcusS,PolverinoA,WiglerM.

ComplexformationbetweenRASandRAFandotherproteinkinases.PNAS.1993JulRafhadbeenaroundforalongtime,whydideveryoneallofasuddenthinkitwastheRaseffector?第36頁/共45頁Maindatainthesesixpapers1.RafdirectlybindstoRaseffectordomain2.OncogenicRasstillinteractswithRafforthefunction3.RaseffectordomainmutationsdisruptbindingtoRaf4.Raf’sabilitytobindRascorrelatestoitsfunctionHowever,allabovearealsotrueforGAP.Why?Whatwasmissing?Vote:A:Convincing,B:Notconvincing第37頁/共45頁Late1992andearly1993HanM,GoldenA,HanY,SternbergPW.C.eleganslin-45rafgeneparticipatesinlet-60ras-stimulatedvulvaldifferentiation.Nature.1993MayDicksonB,SprengerF,MorrisonD,HafenE.RaffunctionsdownstreamofRas1intheSevenlesssignaltransductionpathway.Nature.1992DecRas(gf) Signalingincreases(constitutive)Raf(lf) SignalingeliminatesRas(lf) SignalingeliminatesRas(gf);Raf(lf) SignalingeliminatesRasRaf第38頁/共45頁CompareRafwithGAPRas(lf) SignaleliminatedRaf(lf) SignaleliminatedGAP(lf) SignalincreasesRaf(lf)suppressactivatedRasRas(lf)suppressGAP(lf)RASGDPGAPRASGTPSOSGRB2EG