信号通路和肿瘤.ppt

细胞信号转导与肿瘤中山大学2005级分子医学博士生课程第四讲2005年9月27日,黎孟枫医学博士中山大学基础医学院微生物学教研室mengli,引言：细胞信号转导与生命过程问题的提出和理论的产生细胞信号转导理论概述信号转导研究中的重大理论问题及热点领域信号转导的研究方法与工具信号转导理论研究及应用举例：在肿瘤发生发展中的信号转导的意义信号转导与肿瘤临床：诊断和治疗细胞信号转导经典文献举例,引言,信号转导与生命过程问题的提出和理论的产生,细胞信号转导理论建立以前的细胞生物学,细胞的显微结构（胞膜、胞浆、胞核）细胞的生理功能（生存、“活性”、分裂增殖、胞间连接、吞饮、分泌、迁移、死亡)细胞组分的生物化学（脂、糖、核酸、蛋白）细胞的超微结构和亚细胞结构（脂质双层膜结构、细胞器),细胞信号转导理论概述,ExtracellularSignalMolecules,GrowthFactorsPDGF(Platelet-DerivedGrowthFactor),EGF(EpidermalGrowthFactor),TGF-(TransformingGrowthFactor-),EPO(Erythropoietin),NGF(NerveGrowthFactor),IGF(Insulin-likeGrowthFactor),TPO(Thrombopoietin)2.CytokinesIFN-(Interferon-),IFN-(Interferon-),TNF(TumorNecrosisFactor),Interleukins(1,2,3,4)3.DeathmoleculesFas4.AdhesionmoleculesCadherins,Adhesin5.HormoneInsulin6.Stress,SignalTransducingReceptors,Transmembranereceptorsthathaveintrinsicenzymaticactivity.AutophosphorylationPhosphorylationofothersubstratesA)Tyrosinekinases:PDGF-R,insulin-R,EGFRandFGF-RB)Tyrosinephosphatases:e.g.CD45C)Guanylatecyclases:e.g.natriureticpeptidereceptors)D)Serine/Threoninekinases:activinandTGF-breceptors,2.Receptorsthatarecoupled,insidethecell,toGTP-bindingandhydrolyzingproteins(G-proteins).e.g.,adrenergicreceptors,odorantreceptors,andcertainhormonereceptors(e.g.glucagon,angiotensin,vasopressinandbradykinin).3.Receptorsthatarefoundintracellularlyanduponligandbindingmigratetothenucleuswheretheligand-receptorcomplexdirectlyaffectsgenetranscriptione.g.,STAT1,3,4,5,6(Signaltransducerandactivatoroftranscription)4.Simplereceptors:e.g.,ion-channelsthatleadtochangesinmembraneelectricpotential,信号转导过程中的生物化学,磷酸化反应（酪氨酸激酶、丝/苏氨酸激酶）蛋白质构象改变去磷酸化反应（磷酸酶）受体或其他信号转导分子的聚体化,SignalTransducers,ReceptorTyrosineKinases(RTKs)contains:Anextracellularligandbindingdomain.Anintracellulartyrosinekinasedomain.Anintracellularregulatorydomain.Atransmembranedomain.,CharacteristicsoftheCommonClassesofRTKs,Non-ReceptorProteinTyrosineKinases(PTKs)Twonon-receptorPTKfamilies:1)ThearchetypaplPTKfamilty:Src-relatedproteins2)Januskinase(Jak)family,Mostnon-receptorPTKscoupletocellularreceptorsthatlackenzymaticactivitythemselves(e.g.,CD4,CD8,TCRandallcytokinereceptorssuchasIL-2R,ReceptorSerine/ThreonineKinases(RSTKs),Typicalexample:ReceptorsfortheTGF-superfamilyofligandsTheTGF-superfamilyinclude30multifunctionalproteins,e.g.,activins,inhibinsandthebonemorphogeneticproteins(BMPs).17RSTKsisolatedarein2subfamilies:typeIandtypeIIreceptors.NuclearproteinsrespondingtoTGF-activationincludec-MycandSmad,Non-ReceptorSerine/ThreonineKinases1)cAMP-dependentproteinkinase(PKA)2)ProteinkinaseC(PKC)3)Mitogenactivatedproteinkinases(MAPKorERK)(requiringphosphorylationofbothtyrosineandthreonine)G-ProteinCoupledReceptors1.1000GPCRs,mostofwhichareorphanreceptors)2.ThreedifferentclassesofGPCR:GPCRsthatmodulateadenylatecyclaseactivityandproducecAMPGPCRsthatactivatePLC-gleadingtohydrolysisofpolyphosphoinositides:angiotensin,bradykininandvasopressinreceptors.Photoreceptor,IntracellularHormoneReceptors1.Residingwithinthecytoplasm.,2.Thesteroid/thyroidhormonereceptorsuperfamily(e.g.glucocorticoid,vitaminD,retinoicacidandthyroidhormonereceptors):bindsteroid/thyroidhormone,translocatetonuclearandbindspecificDNAsequenceshormoneresponseelements(HREs).,*PhosphatasesinSignalTransduction1.TransmembranePTPs:e.g.,CD45.2.IntracellularPTPs.,信号转导研究中的重大理论问题及热点领域,信号转导通路的调控,磷酸化去磷酸化调控信号转导分子消长的调控（分子半衰期）不同通路之间的效应调控胞内内源性抑制物的调控功能,Cross-Talk,信号转导效应的特异性WhenandWhere?,Cooperationwithothersignalingpathways?Pre-existingtranscriptionco-factorsdifferentiallyexpressedandactivatedindifferentcelltypes?Pre-existingco-activatorsoftargetproteins?Subcellularlocalizationoftransducers?Optimallevel(orathreshold)ofphosphorylation/dephosphorylation?,替代通路（AlternativePathways),信号转导的研究方法与工具,一、蛋白质磷酸化状态的检测1、免疫印迹(phospho-proteinspecificantibodies)2、免疫沉淀(protein-specificantibody+phospho-AAantibody3、流式细胞仪分析4、Luminex分析二、信号转导分子过度表达或过度激活1、Overexpressionbygenetransduction2、Constitutivelyactivatedmutants三、基因转录活性测定1、Electrophoreticmobilityshiftanalysis(EMSA)、Reportergeneexpressiondetection,四、信号转导分子的表达或活性抑制1、Anti-sense2、RNAi3、Geneknock-out4、DominantnegativemutantsLigand-bindingsitePhosphorylationsiteDockingsiteProtein-proteinbindingsiteDNAbindingsite5、Small-moleculeinhibitors：e.g.,tyrosinekinaseinhibitor(TKi)6、Inhibitoryoligopeptides,信号转导在肿瘤发生发展中的意义,Signalingmoleculesinvolvedincancerdevelopment/progression,ReceptorsGrowthfactorreceptors:EGFRHormonereceptor:ER,ARAngiogenicreceptros:VEGF,PDGF,IGFDeathreceptorsTheIntegrinsystem,TransducersRasRafRhofamilyPI-3K/AktDeathtransducersSTAT-3Transcriptionfactorsc-Mycc-Junandc-fosSTAT-3,BiologicalEffectsofSignalingRelatedtoCancerDevelopment/Progression,CellimmobilizationAbrogationofapoptosisActivationofcellcycleandremovalofcellcyclecheckpointsAngiogenesisCellinvasionMetastasisDrugresistance,PhosphorylationtargetsofPI-3K,AktForkhead-relatedtranscriptionfactor1(FKHR-L1)14-3-3bindingFKHR-L1retainingincytosolabrogationofgeneactivationbyFKHR-L1AktBad14-3-3bindingReleaseofBcl-2andBcl-XCellsurvivalAktGSK3GSK3catalyticactivityturnedoffPermittingactivationofc-MycandcyclinDPDK1phosphorylationofotherkinases(p70S6-kinass