医学细胞生物学课件：Signal Transduction II

SignalTransductionIIDiversity,Receptors,GTPaseswitchproteins,proteinkinases,adaptorproteins,proteinphosphatases

A.Cell-surfacereceptorpathways(receptorsthatareIonchannels,SignalingthroughG-protein-coupledreceptors,SignalingthroughEnzyme-linkedreceptors,

Signalingthatinvolvedinproteolysis)

B.Intracellularreceptorpathways(Nitricoxidepathway,Nuclearreceptorpathway)I.Signalingmolecules:II.SignalingPathways:B.Theadaptationoftargetingcells:Receptor

sequestration,down-regulation,

inactivation,Productionofinhibitoryprotein,InactivationofsignalingproteinIII.Theregulationofcellsignaling:A.Theinteractionbetweendifferentsignalingpathways(Convergence,Divergence,Crosstalk)Keycontentsforsignaltransduction:SignalMoleculeG-proteincoupledreceptorReceptortyrosinekinaseGproteinGproteinAdenylylcyclasecAMPPKAGeneregulatoryproteinsCaMKinasePKCMAPKPKBManytargetproteinsPLCIP3DAGCalmodulinCa2+

Grb2Ras-GEFRasMAPKKKMAPKKPI-3kinasePI(3,4,5)P3PDK13.SignalingthroughEnzyme-linkedreceptors3.1ReceptortyrosinekinasepathwayLigands:Insulin,epidermalgrowthfactor(GF),FibroblastGF,neurotrophinsandotherGFs.Receptors:Singletransmembraneα

helix;intrinsicproteintyrosinekinaseactivityincytosolicdomain;dimerizationandphosphorylationinducedbybindingtoligand.Signaltransduction:A.MAPkinasepathwayB.IP3/DAGpathway.

C.PI-3kinasepathwayRitaLevi-Montalcini(c.1986)b.1909StanleyCohen(c.1986)b.1922NobelPrizefordiscoveryofgrowthfactorsPeptidegrowthfactors

RitaLevi-Montalcini1950:StudyoftransplantablemousesarcomaGraftedthetumorondevelopingchickenembryoEmbryosensorygangliainnervatedthetransplantSensoryandsympatheticnervefibersinvadedthe

tumorandbranchedalongthetumorcellsbutdid

notestablishsynapticjunctionsUndertheseconditions,theembryoshowed

aberrantgrowthanddistributionofnervefibersThissuggestedthetumorreleasedahumoral

factorthatactsonsensoryandsympathetic

gangliaPurifiednervegrowthfactor(NGF)

StanleyCohen1956:Promotesoutgrowth@1-10ngIncreasesgangliabyenhancingdifferentiation

andpreventingcelldeathPromotesdifferentiationoftumorcellssuchas

neuroblastomacellsinvitroRemovesNGFdegenerationEpidermalGrowthFactor

StanleyCohenUsedmousesubmaxillaryglandas

sourceofNGFNotedanotheractivityFactorcausedprecociousincisor

eruption,earlyeyelidopeningand

lungmaturation-epithelialcellsPurificationwiththeseassaysThestructureofReceptortyrosinekinaseDiversityofreceptorstructureDifferentreceptorsinteractwithdownstream

moleculeswithuniqueSH2domainsActivationofPLCafteractivationofEGFreceptorPLCisactivatedbyEGF.EGFactivatesPI3kinase.EGFactivatesMAPkinase.ActivationofRasafteractivationofEGFreceptorGrb2isanadaptorproteinSosisaguaninenucleotideexchangefactorSosdisplacesGDPMAPkinasepathwayActivationoftheMAPkinasepathwayRafMekMAPkinaseInductionofgenetranscriptionbyactivatedMAPkinaseIP3/DAGpathwayisactivatedbyEGFPhosphatidylinositol3kinase(PI3K)pathwayIdentifiedbyitsassociationwithactivatedPDGFRConsistsof85kDaand110kDasubunitsPhosphorylatedinositolatthe3positionoftheinositolringActivationofphosphatidylinositolkinase1(PDK1)GenerationofIP3/DAGGenerationofPI3phosphatesPhosphatidylinositolpathwayRecruitmentandactivationofPKBbyphosphatidylinositolsThePHdomainofPKBdockstothe

3-phosphatepartialactivation

InunstimulatedcellPKBisincytosolFormationofPI3,4-biphosphatePDK1alsobindsPI3,4-bisphosphatePDK1phosphorylationresultsin

activationofPKBPDKisphosphoinositoldependentkinasePKB,AktI,influencescellsurvivalPI3KinasesignalingTheinteractomemapwithincells3.SignalingthroughEnzyme-linkedreceptors3.2Cytokinereceptorspathway3.1receptortyrosinekinasepathway3.3TGFreceptorspathway3.2CytokinereceptorspathwayLigands:Interferons,erythropoitin(EPO),someinterleukins(IL-2,IL-4)andothers.Receptors:Singletransmembraneahelix;JAKkinaseassociatedwithintracellulardomain.Signaltransduction:DirectactivationofcytosolicSTAT(Signaltransducerandactivatoroftranscription)transcriptionfactors.B.PI3kinasepathwayC.MAPkinasepathwayD.IP3/DAGpathway.

Basicfunction:Proliferation,differentiationandmaturationofbloodcellsandimmunecells.ErythropoietinandredbloodcellsEpointeractionwithprecursorstemcellsCloningoferythropoietin

majorsuccessof

biotechnologyM.Socolovskyetal.,Blood98:3261Janus,RomantwofacedGodofGateJanusKinase:AtyrosinekinasetightlyboundtocytosolicdomainofEporeceptorJanusKinase/STATsignalingpathwaySTAT(Signaltransducerandactivatoroftranscription)Lodishetal.,ed,MolecularCellBiology5th,2004.TheJAK-STATsignalingpathwayActivatedbyerythropoietinSTATtranscriptionfactorTheSH2domainofSTATbindstophosphorylatedtyrosinePhosphorylatedSTATsdimerizemovetonucleusBindingtointerferonstimulatedresponseelement(ISRE)orinterferonγactivatedsequence(GAS).Lodishetal.,ed,MolecularCellBiology5th,2004.Erythropoietin(acytokine)mayactivateseveralpathwaysEPOEpoRJAK2?ExpressionoferythrocytesinmutantmiceTargeteddisruptionofEporeceptororJAK2ThesemicearewildtypeforJAK2+/+ThesemicearewildtypeforEpoR+/+Note:JAK2isdownstreamofEpoRErythrocytes(EpoR)Erythrocytes(JAK2)Wuetal.,1995Cell85:59Neubaueretal.,1998Cell93:307Fromhttp://www.N.GFP-knock-inmouseTissue-specificCre-transgenicmouseHarvardPh.D.’67,JimWatson"...theonethingthatIthinkisextremelyimportant,isthatanyonecandoit,ifgivenachance,ifgiventheopportunity."TelephoneinterviewwithMarioR.Capecchiimmediatelyfollowingtheannouncementofthe2007NobelPrizeinPhysiologyorMedicine,8October2007.TheinterviewerisAdamSmith,Editor-in-ChiefofN.KirkR.ThomasandMarioR.Capecchi

Site-directedmutagenesisbygenetargetinginmouseembryo-derivedstemcells

Cell1987,51:503-512.WelcometoBio-Lab!ThelegendsofNobelwinners@Harvard

HarvardBio-LabHarvardPh.D.’76,TheNobelPrizeinPhysiologyorMedicine2013JamesE.RothmanRandyW.SchekmanThomasC.SüdhofTheNobelPrizeinPhysiologyorMedicine2013wasawardedjointlytoJamesE.Rothman,RandyW.SchekmanandThomasC.Südhof

"fortheirdiscoveriesofmachineryregulatingvesicletraffic,amajortransportsysteminourcells".HarvardPh.D.’68,TheNobelPrizeinPhysiologyorMedicine2011BruceA.BeutlerJulesA.HoffmannRalphM.SteinmanOnehalfjointlytoBruceA.BeutlerandJulesA.Hoffmann"fortheirdiscoveriesconcerningtheactivationofinnateimmunity"andtheotherhalftoRalphM.Steinman"forhisdiscoveryofthedendriticcellanditsroleinadaptiveimmunity".

HarvardPh.D.66,Harvard’72Harvard’69,Ph.D.’77,WoodyHastingHarvardPh.D.’67,JimWatson"fortheirdiscoveryofRNAinterference-genesilencingbydouble-strandedRNATheNobelPrizeinPhysiologyorMedicine2006AndrewZ.Fire

CraigC.Mello

1/2oftheprize1/2oftheprizeSchoolofMedicineStanfordUniversity

USAMedicalSchoolUniversityofMassachusetts

Worcester,MA,USAb.1960b.1959Ph.D,MITPh.D,’90HarvardDanStinchcombV617FmutationsofJAK2inchronicmyeloproliferativedisorders

OverexpressionofmiR-375inhibitstheproliferationofgastriccancercellsDownregulationofmiR-375in90%ofgastriccancerTheroleofJAK2ingastriccarcinogenesisJAK2isamiR-375targetgeneingastriccancercellsmiR-375JAK2ProliferationDingetal.,CellResearch2010ThepotentialroleofJAK2ingastriccancer美国哈佛大学校园（2003年1月）3.SignalingthroughEnzyme-linkedreceptors3.2Cytokinereceptorspathway3.1receptortyrosinekinasepathway3.3TGFreceptorspathway3.3TGFreceptorspathwayLigands:Transforminggrowthfactorsuperfamily(TGF),bonemorphogeneticprotein,activin,inhibins,Nodal(mammals)andDpp(Drosophila),etc.Receptors:Intrinsicserine/threoninekinaseproteinactivityincytosolicdoman(typeIandII),TypeIIIisglycanwithoutkinaseactivity.Signaltransduction:DirectactivationofcytosolicSmad(mammalianhomologuesofC.eleganSmaandDrosophilaMad)transcriptionfactors.Basicfunction:Negativeregulationofcellproliferation,lossofTGFsignalingcontributesabnormalcellproliferationandmalignancy.SmadsfamilyR-Smads(receptorregulatedSmads):Smad2andSmad3,etc.Co-Smads(commonSamds):Smad4I-Smads(InhibitorySmads):Smad6andSmad7.TGFbindstypeIII/typeIIreceptorTypeII(kinase)dimerizeswithtypeIandphosphorylatesitPhosphorylationoftypeIresultsinactivationofkinaseactivityPhosphorylationofSmadleadstodimerizationandtranslocationtothenucleusTGFbindinginvolvesserinephosphorylationLodishetal.,ed,MolecularCellBiology5th,2004.TGFsignalingpathwayTFE3:TranscriptionfactorE3Ran:smallGproteinSmadscomplexinducesexpressionofplasminogenactivatorinhibitor(PAI)Lodishetal.,ed,MolecularCellBiology5th,2004.TGFsignalingpathway(2009)TGFpathwayinphysiologyandpathologyHumMolGenet.16SpecNo1:R14-20.(2007)Implicationsforawiderspectrumofdisorders,includingcancer,fibrosis,healingandinflammatoryandcardiovasculardiseases.NatureGenetics

36,790-792(2004)HHT:hereditaryhemorrhagictelangiectitasiaJPS:juvenilepolyposissyndromeMFS:MarfansyndromeENG:TypeIIIreceptorACVRL1:TypeIreceptorTGFsignalingandCancerTheRoleofTGFβinCancer.Innormalandpremalignantcells,TGFβenforceshomeostasisandsuppressestumorprogressiondirectlythroughcell-autonomoustumor-suppressiveeffects(cytostasis,differentiation,apoptosis)orindirectlythrougheffectsonthestroma(suppressionofinflammationandstroma-derivedmitogens).However,whencancercellsloseTGFβtumor-suppressiveresponses,theycanuseTGFβtotheiradvantagetoinitiateimmuneevasion,growthfactorproduction,differentiationintoaninvasivephenotype,andmetastaticdisseminationortoestablishandexpandmetastaticcolonies.

Massage.Cell,2008OverviewofEnzyme-linkedreceptors4.Signalingthatinvolvedinproteolysis4.1NF-B(nuclearfactorB)pathway4.2Notch/Deltasignalingpathway4.3Hedgehog(Hh)pathway4.4Wntpathway4.1NF-B(nuclearfactorB)pathwayLigands:Tumornecrosisfactoralpha(TNF-),Interleukin1,Spatzle(Drosophila).Receptors:TNFreceptor,IL1receptor,Toll-likereceptor.Signaltransduction:Phosphorylation-dependentdegradationofinhibitorproteinwithreleaseofactiveNF-Btranscriptionfactors.Basicfunction:immunityandinflammationNF-kBsignalingpathwayLodishetal.,ed,MolecularCellBiology5th,2004.I-kB:inhibitorofNF-kB

TAK1:TGFactivatedkinase1TNF-alphainhibitorsavailablebyprescriptionAdalimumab(Humira):rheumatoidarthritispsoriaticarthritis.Etanercept(Enbrel):RheumatoidarthritisJuvenilerheumatoidarthritisPsoriaticarthritisPsoriasisInfliximab(Remicade):ModeratetosevereCrohn'sdiseaseRheumatoidarthritisPsoriaticarthritisAnkylosingspondylitisUlcerativecolitisVarfolomeevetal.,IAPAntagonistsInduceAutoubiquitinationofc-IAPs,NF-κBActivation,andTNFα-DependentApoptosis.Cell131,669-681,16November2007.Vinceetal.,IAPAntagonistsTargetcIAP1toInduceTNFα-DependentApoptosis.Cell131,682-693,16November2007.

TNF-alphaandNF-κBsignalingandCancer4.Signalingthatinvolvedinproteolysis4.1NF-B(nuclearfactorB)pathway4.2Notch/Deltasignalingpathway4.3Hedgehog(Hh)pathway4.4WntpathwayCellrecognition--communicationbyplasmamembrane-boundproteinsDeltaproteinDeltareceptor4.2Notch/DeltasignalingpathwayLigands:Membrane-boundDeltaproteinorSerrateprotein.Receptors:ExtracelluarsubunitofDeltareceptor(Notch)noncovalentlyassociatedwithtransmembranesubunit.Signaltransduction:Intramembraneproteolyticcleavageofreceptortransmembranedomainwithreleaseofcytosolicsegmentthatfunctionasco-activatoroftranscriptionfactors.Basicfunction:Regulationofdevelopmentanddeterminationofcellfate.Notch/DeltasignalingpathwayTACE:TNFαconvertingenzyme(αsecretase)ICN:intracellulardomainofNotchCSL:CBF1/Suppressorofhairless/Lag-1,aco-repressor.ICN(γSecretase)CSLLodishetal.,ed,MolecularCellBiology5th,2004.ProteolyticcleavageofAPP,aneuronalplasmaproteinAPP:amyloidprecursorprotein-secretase,

APPcleavingenzyme1(BACE1)Amyloidhypothesis:Anaberrantformationofamyloid-peptides(A42)isamajorcauseofAlzheimer'sdisease.Lodishetal.,ed,MolecularCellBiology5th,2004.4.Signalingthatinvolvedinproteolysis4.1NF-B(nuclearfactorB)pathway4.2Notch/Deltasignalingpathway4.3Hedgehog(Hh)pathway4.4Wntpathway4.3Hedgehog(Hh)pathwayLigands:HedgehogReceptors:BindingofHhtoPatched(Ptc),whichhas12transmembranehelices;activationofsignalingfromSmoothened(Smo),with7transmembranehelices.Signaltransduction:Proteolyticreleaseofatranscriptionalactivatorfromaproteincomplexinthecytosol.Basicfunction:amasterregulatorofembryonicdevelopmentandpostembryonictissuehemeostasis.DiseasesHedgehogPathwayanddiseasesScience318,66(5October2007)4.Signalingthatinvolvedinproteolysis4.1NF-B(nuclearfactorB)pathway4.2Notch/Deltasignalingpathway4.3Hedgehog(Hh)pathway4.4Wntpathway4.4WntpathwayLigands:SecretedWnt(mammals),Wg(Drosophila).Receptors:Frizzled(Fz)withseventransmembranehelices;associatedwithmemberane–boundLDLreceptor-relatedprotein(LRP)requiredforreceptoractivitySignaltransduction:Assemblyofmultiproteincomplexatmembranethatinhibitstheproteasome-mediatedproteolysisofcytosolic–catenintranscriptionfactor,resultinginitsaccumulationBasicfunction:celldifferentiation,cellproliferationanddeterminationofcellfateWntsignalingpathwayLigand:WntReceptor:LRP+FrizzledAPC,adenomatouspolyposiscoli;-cat,-catenin;CBP,CREB-bindingprotein;CK,caseinkinase;DKK,Dickkopf;DSH,Dishevelled;GBP,GSK3-bindingprotein;GSK,glycogensynthasekinase;LRP,LDL-receptor-relatedprotein;P,phosphorylation;sFRP,secretedFrizzled-relatedprotein;TCF,T-cellfactor.NatRevGenetics2004,5,689WntpathwayanddiseasesNatRevGenetics2004,5,689Overviewofcellsurfacereceptors5.Intracellularreceptor--NitricoxidepathwayLigands:Nitricoxide.Receptors:NOreceptorSignaltransduction:GenerationofcGMPBasicfunction:VasodilationandneurotransmitterRegulationofcontractilityofarterialsmoothmusclebyNOandcGMPNOpathwayinphysiologyandpathologyCardiovasculardiseasesNeurologicaldiseases:BrainischemiaandchronicdegenerativediseasesofthenervoussystemErectiledysfunction(impotence):Sildenafil,popularlyknownbythetradenameViagra,stimulateserectionsprimarilybyenhancingsignalingthroughthenitricoxidepathwayinthepenis.TheNobelPrizeinPhysiologyorMedicine1998RobertF.Furchgott

LouisJ.Ignarro

FeridMurad

fortheirdiscoveriesconcerningnitricoxideasasignalingmoleculeinthecardiovascularsystem

6.Intracellularreceptor--NuclearreceptorpathwayLigands:steroidhormones,thyroxine,retinoidacidsandfattyacidetc.Receptors:HighlyconservedDNA-bindingdomain,somewhatconservedhormone-bindingdomain,andavariabledomain;locatedwithincytosolornucleus.Signaltransduction:activationofreceptors’transcriptionfactoractivitybyligandbinding.Basicfunction:Biologicalhomeostasisandsexregulation,etc.Intracellularreceptors（Steroidhormonereceptors）III.ThenetworkofcellsignalingThemajorsignalingpathwaysrelevanttocellgrowthinhumancells.III.TheregulationofcellsignalingTheinteractionbetweendifferentsignalingpathwaysTheadaptationoftargetingcells:Desensitization

ConvergenceDivergenceCrosstalkReceptorsequestrationReceptordown-regulationReceptorinactivationInactivationofsignalingproteinProductionofinhibitoryproteinThemajorsignalingpathwaysactivatedbyGPCRsandRTKsSignalMoleculeG-proteincoupledreceptorReceptortyrosinekinaseGproteinGproteinAdenylylcyclasecAMPPKAGeneregulatoryproteinsCaMKinasePKCMAPKPKBManytargetproteinsPLCIP3DAGCalmodulinCa2+

Grb2Ras-GEFRasMAPKKKMAPKKPI-3kinasePI(3,4,5)P3PDK1ThemajorsignalingpathwaysactivatedbyGPCRsandRTKsSignalMoleculeG-proteincoupledreceptorReceptortyrosinekinaseGproteinGproteinAdenylylcyclasecAMPPKAGeneregulatoryproteinsCaMKinasePKCMAPKPKBManytargetproteinsPLCIP3DAGCalmodulinCa2+

Grb2Ras-GEFRasMAPKKKMAPKKPI-3kinasePI(3,4,5)P3PDK1ConvergenceDivergenceCrosstalkErythropoietin(acytokine)mayactivateseveralpathwaysEPOEpoRJAK2TheadaptationoftargetingcellsFivewaysinwhichtargetcellscanbecomedesensitizedtoasignalmolecule.Theinactivationmechanismsshownhereforboththereceptorandtheintracellularsignalingproteinofteninvolvephosphorylationoftheproteinthatisinactivated,althoughothertypesofmodificationarealsoknowntooccur.Inbacterialchemotaxis,desensitizationdependsonmethylationofthereceptorprotein.GPCRDesensitizationDependsonReceptorPhosphorylationReceptorinactivation:TheycanbecomealteredsothattheycannolongerinteractwithGproteins.Receptorsequestration:Theycanbetemporarilymovedtotheinteriorofthecell(internalized)sothattheynolongerhaveaccesstotheirligand.Receptordown-regulation:TheycanbedestroyedinlysosomesafterinternalizationTherolesofG-protein-linkedreceptorkinases(GRKs)andarres