细胞信号转导与肿瘤细胞信号转导与肿瘤中山大学中山大学2005级分子医学博士生课程第四讲级分子医学博士生课程第四讲2005年年9月月27日日黎孟枫医学博士中山大学基础医学院微生物学教研室mengligzsums.edu.cn引言：细胞信号转导与生命过程引言：细胞信号转导与生命过程问题的问题的提出和理论的产生提出和理论的产生细胞信号转导理论概述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细胞信号转导经典文献举例引言引言信号转导与生命过程信号转导与生命过程问题的提出和理论的产生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信号转导理论建立以前的细胞生物学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)组织生长需要组织生长需要细胞分裂增殖细胞分裂增殖细胞细胞生长因子生长因子细胞周期细胞周期蛋白表达蛋白表达病原体侵入病原体侵入抗感染状态抗感染状态细胞细胞抗原抗原细胞因子细胞因子表达分泌表达分泌细胞过度生长细胞过度生长细胞死亡细胞死亡细胞细胞死亡因子死亡因子胞内致死胞内致死分子表达分子表达细胞骨架蛋细胞骨架蛋白表达、激活白表达、激活牵动细胞移动牵动细胞移动(Cellmovement)趋化因子趋化因子细胞粘附细胞粘附细胞存活细胞存活(Survival)抗凋亡因子抗凋亡因子表达、激活表达、激活胞外信号胞外信号信号作用信号作用于细胞于细胞基因表基因表达改变达改变细胞表细胞表型改变型改变细胞信号转导理论概述细胞信号转导理论概述胞外信号分子胞外信号分子(可溶性分子、细胞表面分子、组织基质分子可溶性分子、细胞表面分子、组织基质分子)靶细胞跨膜分子靶细胞跨膜分子(狭义受体如狭义受体如EGFR或广义受体如或广义受体如Integrin)靶细胞受体（胞内段）化学变化（如磷酸化、二聚体形成）靶细胞受体（胞内段）化学变化（如磷酸化、二聚体形成）靶细胞内信号转导分子化学变化与激活靶细胞内信号转导分子化学变化与激活（如磷酸化、去磷酸化、聚体形成）（如磷酸化、去磷酸化、聚体形成）激活的信号转导分子进入胞核激活的信号转导分子进入胞核进入胞核的转导分子作用于基因转录调控区进入胞核的转导分子作用于基因转录调控区基因表达改变基因表达改变ExtracellularSignalMolecules1.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.StressSignal Transducing Receptors1.Transmembranereceptorsthathaveintrinsicenzymaticactivity.AutophosphorylationPhosphorylationofothersubstratesA)Tyrosinekinases:PDGF-R,insulin-R,EGFRandFGF-RB)Tyrosinephosphatases:e.g.CD45C)Guanylatecyclases:e.g.natriureticpeptidereceptors)D)Serine/Threoninekinases:activinandTGF- receptors2.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信号转导过程中的生物化学磷酸化反应（酪氨酸激酶、丝磷酸化反应（酪氨酸激酶、丝/苏氨苏氨酸激酶）酸激酶）蛋白质构象改变蛋白质构象改变去磷酸化反应（磷酸酶）去磷酸化反应（磷酸酶）受体或其他信号转导分子的聚体化受体或其他信号转导分子的聚体化SignalTransducersReceptor Tyrosine Kinases (RTKs) contains:An extracellular ligand binding domain. An intracellular tyrosine kinase domain. An intracellular regulatory domain. A transmembrane domain.TyrosinephosphorylationInteractwithandphosphorylateSrchomologydomain2(SH2)-containingproteins(e.g.,PLC- ,Ras,PI-3K,etc)Phosphorylateotherkinasesphosphorylateproteins,whichuponphosphorylated,canenterthenuclearandbindDNAregulatoryregions.ClassExamplesStructural Features of ClassIEGF receptor, NEU/HER2, HER3cysteine-rich sequencesIIinsulin receptor, IGF-1 receptorcysteine-rich sequences; characterized by disulfide-linked heterotetramersIIIPDGF receptors, c-Kitcontain 5 immunoglobulin-like domains; contain the kinase insertIVFGF receptorscontain 3 immunoglobulin-like domains as well as the kinase insert; acidic domainVvascular endothelial growth factor (VEGF) receptorcontain 7 immunoglobulin-like domains as well as the kinase insert domainVIhepatocyte growth factor (HGF) and scatter factor (SC) receptorsheterodimeric like the class II receptors except that one of the two protein subunits is completely extracellular. The HGF receptor is a proto-oncogene that was originally identified as the Met oncogeneVIIneurotrophin receptor family (trkA, trkB, trkC) and NGF receptorcontain no or few cysteine-rich domains; NGFR has leucine rich domainCharacteristics of the Common Classes of RTKs Non-ReceptorProteinTyrosineKinases(PTKs)Twonon-receptorPTKfamilies:1)ThearchetypaplPTKfamilty:Src-relatedproteins2)Januskinase(Jak)familyMostnon-receptorPTKscoupletocellularreceptorsthatlackenzymaticactivitythemselves(e.g.,CD4,CD8,TCRandallcytokinereceptorssuchasIL-2RReceptorSerine/ThreonineKinases(RSTKs)Typicalexample:ReceptorsfortheTGF- superfamilyofligandsTheTGF- superfamilyinclude30multifunctionalproteins,e.g.,activins,inhibinsandthebonemorphogeneticproteins(BMPs).17RSTKsisolatedarein2subfamilies:typeIandtypeIIreceptors.NuclearproteinsrespondingtoTGF- activationincludec-MycandSmadLigandsbindtothetypeIIreceptorsComplexedwithtypeIreceptorsTypeIIRphosphorylatestypeIreceptorInitiationofsignalingcascadeNon-ReceptorSerine/ThreonineKinases1)cAMP-dependentproteinkinase(PKA)2)ProteinkinaseC(PKC)3)Mitogenactivatedproteinkinases(MAPKorERK)(requiringphosphorylationofbothtyrosineandthreonine)G-ProteinCoupledReceptors1.1000GPCRs,mostofwhichareorphanreceptors)2.ThreedifferentclassesofGPCR:1)GPCRsthatmodulateadenylatecyclaseactivityandproducecAMP2)GPCRsthatactivatePLC-gleadingtohydrolysisofpolyphosphoinositides:angiotensin,bradykininandvasopressinreceptors.3)PhotoreceptorIntracellularHormoneReceptors1.Residingwithinthecytoplasm.2.Thesteroid/thyroidhormonereceptorsuperfamily(e.g.glucocorticoid,vitaminD,retinoicacidandthyroidhormonereceptors):bindsteroid/thyroidhormone,translocatetonuclearandbindspecificDNAsequenceshormoneresponseelements(HREs).*PhosphatasesinSignalTransduction1.TransmembranePTPs:e.g.,CD45.2.IntracellularPTPs.胞外信号分子胞外信号分子(可溶性分子、细胞表面分子、组织基质分子可溶性分子、细胞表面分子、组织基质分子)靶细胞跨膜分子靶细胞跨膜分子(狭义受体如狭义受体如EGFR或广义受体如或广义受体如Integrin)靶细胞受体（胞内段）化学变化（如磷酸化、二聚体形成）靶细胞受体（胞内段）化学变化（如磷酸化、二聚体形成）靶细胞内信号转导分子化学变化与激活靶细胞内信号转导分子化学变化与激活（如磷酸化、去磷酸化、聚体形成）（如磷酸化、去磷酸化、聚体形成）激活的信号转导分子进入胞核激活的信号转导分子进入胞核进入胞核的转导分子作用于基因转录调控区进入胞核的转导分子作用于基因转录调控区基因表达改变基因表达改变信号转导研究中的重大信号转导研究中的重大理论问题及热点领域理论问题及热点领域信号转导通路的调控信号转导通路的调控磷酸化磷酸化去磷酸化调控去磷酸化调控信号转导分子消长的调控（分子半衰期）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不同通路之间的效应调控胞内内源性抑制物的调控功能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?替代通路替代通路（AlternativePathways)信号转导的研究方法信号转导的研究方法与工具与工具一、蛋白质磷酸化状态的检测一、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四、信号转导分子的表达或活性抑制1、Anti-sense2、RNAi3、Geneknock-out4、Dominantnegativemutants(1)Ligand-bindingsite(2)Phosphorylationsite(3)Dockingsite(4)Protein-proteinbindingsite(5)DNAbindingsite5、Small-moleculeinhibitors：e.g.,tyrosinekinaseinhibitor(TKi)6、Inhibitoryoligopeptides信号转导在肿瘤发生信号转导在肿瘤发生发展中的意义发展中的意义Signalingmoleculesinvolvedincancerdevelopment/progressionReceptors1)Growthfactorreceptors:EGFR2)Hormonereceptor:ER,AR3)Angiogenicreceptros:VEGF,PDGF,IGF4)Deathreceptors5)TheIntegrinsystemTransducers1)Ras2)Raf3)Rhofamily4)PI-3K/Akt5)Deathtransducers6)STAT-37)Transcriptionfactors1)c-Myc2)c-Junandc-fos3)STAT-34)BiologicalEffectsofSignalingRelatedtoCancerDevelopment/ProgressionCellimmobilizationAbrogationofapoptosisActivationofcellcycleandremovalofcellcyclecheckpointsAngiogenesisCellinvasionMetastasisDrugresistancePhosphorylationtargetsofPI-3KAktForkhead-relatedtranscriptionfactor1(FKHR-L1)14-3-3bindingFKHR-L1retainingincytosolabrogationofgeneactivationbyFKHR-L1AktBad14-3-3bindingReleaseofBcl-2andBcl-XCellsurvivalAktGSK3GSK3catalyticactivityturnedoffPermittingactivationofc-MycandcyclinDPDK1phosphorylationofotherkinases(p70S6-kinasse,CISK,PKC)Cellgrowthandsurvival信号转导与肿瘤临床信号转导与肿瘤临床诊断、预防与治疗诊断、预防与治疗Expressionlevel,mutationsandantibodiesofsignalingmoleculesincancerdiagnosis1)EGFR:lung,H&N,glioma2)TGF-receptor(typeII):lung,H&N,3)Ras4)Androgenreceptoranddownstreammolecules5)Estrogenreceptoranddownstreammolecules6)Proteinphosphorylationandcancerdiagnosis1)Determinationofsinglephosphorylatedsignalingmolecules:EGFR2)Phospho-proteinprofiling:proteomics3)Phospho-proteinbasedimagingtechnologySignalinginhibitorandcancerprevention/therapy1)EGFR-selectiveTKi:Iressa2)EGFRantibodies3)FarnesylationorGeranylgeranylationinhibitorstargetingRasandRho4)VEGFRantibody(andTKi):Avastin经典文献举例经典文献举例Stat3asanOncogeneJacquelineF.Bromberg1,MelissaH.Wrzeszczynska1,GeetaDevgan1,YanxiangZhao2,RichardG.Pestell3,ChrisAlbanese3andJamesE.Darnell,Jr.,1,1LaboratoryofMolecularCellBiology,TheRockefellerUniversity,NewYork,NewYork10021-6399,USA2LaboratoryofMolecularBiophysics,TheRockefellerUniversity,NewYork,NewYork10021-6399,USA3TheAlbertEinsteinCancerCenter,DepartmentofDevelopmentalandMolecularBiology,andDepartmentofMedicine,AlbertEinsteinCollegeofMedicine,Bronx,NewYork10461,USAReceived30March1999;Revised23June1999.Availableonline27September2000Cell Volume 98, Issue 3 , 295-303 信号转导理论在各生命科学领信号转导理论在各生命科学领域中的普遍意义域中的普遍意义以本课程中各讲为例：以本课程中各讲为例：干细胞蛋白质组学细胞凋亡肿瘤转移血管增生