1Generic Signal Transduction PathwaysProliferationSurvivalDifferentiationApoptosisCytoskeletonLIGANDSRECEPTORSADAPTORSEFFECTORSOUTPUTSDownward, J. 2001Nature N&VLnRnGnCHANNELSAC cAMPcAKinasePDE cGMPcGKinaseCHANNELSPLAPLCArachidonicacidProstaglandinsEicosanoidsPIP2IP3Ca++CaMKinaseDAGKinase CPI derivativesKinase B/AktPI3K2RECEPTORSTRANSDUCERS EFFECTORS (proximal)“SECOND MESSENGERS”SERPENTINE (7TM)HETEROTRIMERICG PROTEINSADENYLATE CYCLASEPHOSPHODI- ESTERASEPHOSPHO-LIPASE C!PI3 KINASECHANNELScAMPcGMPDERIVATIVESDERIVATIVES Ca++PHOSPHATIDYLINOSITOLPHOSPHATESIONS+DIACYLGLYCEROLIP3GsGiGtGoGq !"Gt!"various incl.!"ADENYLATE CYCLASEPHOSPHO-DIESTERASEGs GiGtcAMPcGMPNote:There are alsoGuanylate KinaseReceptors3SECOND MESSENGER EFFECTORScAMPcGMPCa++DAGPKC Protein Kinases CPKA Protein Kinase ACalmodulin (CaM)CaM KinasesOther Ca++-activated kinases eg MLCKOther effectors - cAMP phosphodiesterase- Calcineurin, a Ca++-activated phosphatase- cytoskeleton Some but not all Ca++- dependentActivated by phorbol estersChannels/PumpscGMP-dependent kinasesPM-NUCLEUSSee alsoBrivanlou & DarnellScience 295:813-818(2002)4RTK-P RTK PhosphataseLigandSignalingGGTPGGDPLigandSignalingGEFsGAPsRGSsGDIsReceptor Protein Tyrosine KinasesIgFn3HGF/SFNeurotrophinsEphrins Gas65Dimerization Trans-phosphorylationRecruitmentActivationHGH/HGHR1 monomeric ligand2 receptorsR-R interactionsAsymmetricAlso Epo/EpoRKosiakoff, De Vosand Wells6VEGF/VEGFR1 dimeric ligand2 receptorsR-R interactionsSymmetricalso PDGF, NGF, TGF/BMPWiesman et al, Cell 91:695-704 1997EGF/EGFR2 monomeric ligands2 receptorsR-R interactionsSymmetric7FGF/FGFR2 monomeric ligands2 receptors2 heparin chainsR-R interactionsOverall symmetric but L-R binding istwo-site, asymmetricSchlessinger et al, Mol. Cell 6: 743-750 2000Mohammadi et al, Curr Opin Struc Biol. 15: 506-516, 2005Receptor Autoinhibition• EGFR extracellular domain undergoes conf. change on EGF binding• BUT EGFR extracellular domain does not dimerize on EGF binding• Intact EGFR DOES dimerize on EGF binding• MODEL - ECD conformation blocks dimerization (autoinhibition)and that is relieved by ligand• sEGFR is mostly monomeric and low affinity (400-550 nM)• there is a small population (~5%) with high affinity (2-20 nM)• deletion of domain IV converts it to high affinity (13-21 nM)8Autoinhibition of EGFRsIVCho and LeahyScience (2002)EGFEGFEGFAutoinhibition of FGFRsFGFRFGFFGFD3D2D3D2D1D1D19PDGF-ReceptorRecruitment of signal transducersto tyrosine phosphatesProtein Modules Recruiting Signaling Proteins to Receptors and to the Membrane10Insulin Receptor Substrate (IRS)etcAn alternative way to recruitsignal transducers-Y-PP-Y--Y-PY-PP-YIRS11Different Mechanisms for Activating Signaling Proteins Recruitmentplus Activation& PhosphorylationRecruitment with ConformationalChange -> ActivationRecruitment plus Phosphorylation -> ActivationReceptor -> MAPKinase pathwayMAPKMAPKKMAPKKKMAPKK and MAPKactivations involveBOTH Y and S/Tphosphorylations12Evolutionary Conservation ofMAPKinase PathwaysMAPKMAPKKMAPKKKEffectorsMAP Kinase Pathways/CascadesWhy cascades? How does the cell keep modules separate?13switchesFerrell, TIBS 21: 460-466 (1996)Cascade as a switch mechanismMos -> ->ErkIs there amplification? Model Results0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 500.10.20.30.40.50.60.70.80.91Fig. 1: Replica of Fig. 2A - Predicted responses on a linear plotInput stimulus (E1tot in multiples of the EC50)Predicted Steady-State Kinase ActivityMAPKKKMAPKKMAPKMAPKKKMAPK10-610-510-410-310-210-100.10.20.30.40.50.60.70.80.91Fig. 2: Replica of Fig. 2B - Predicted responses on a semi-logarithmic plotInput stimulus (E1tot)Predicted Steady-State Kinase ActivityMAPKKKMAPKKMAPKMAPKKKMAPKnH = 1.0nH = 4.9Huang C.F. & Ferrell J.E. (1996) PNAS 93, 10078-8314Experimental ValidationHuang C.F. & Ferrell J.E. (1996) PNAS 93, 10078-83Scaffolds for MAPK modulesSte11pSte7pFus3pMAPKKK MAPKK MAPKSte5pMLKMKK7JNKJIP1MLKMKK3p38JIP2kinesin15More Scaffolds for MAPK modulesMEK1ERK1MAPKKK MAPKK MAPKMP1late endosomesp14FocalcontactsSH2-SH3-SH3CrkIIp130CaspaxillinC3GSOSDOCK180(via PPxLPxK)GEFsalso in JNKMutation blocks JNK activation by EGF/rac but not by UV!-arrestinsGPCR/arr/raf/erkcomplexes onendocytosisMorrison & DavisARCDB 19:91-118(2003)RTK-P RTK PhosphataseLigandSignalingKinase*KinaseRLigandSignalingPhosphataseReceptor-intrinsickinasesReceptor-associatedkinases16ITAM - YxxLx6-8YxxLSrc family kinaseITAM-binding(2xSH2) kinaseMHC/peptideCD3 complexTCRT Cell Receptor SignalingP PCytokine ReceptorsShared signaling subunits - different ligand-binding subunits17JAK/StatIRS etcCytokine ReceptorsSignaling PathwaysTGF! Receptor Family - S/T KinasesIIIIII18TGF! Receptor Family - SMAD SignalingTGF! Receptor Family -Regulators and Feedback Loops19Protein Tyrosine PhosphatasesProtein Tyrosine Phosphatases20Structure of PTPase domainD1 from receptor-likeProtein phosphatase-#Bilwes et al. Nature 382:555-559(1996)wedgeactive siteT Cell Receptor SignalingSrc family kinaseITAM-binding(2xSH2) kinaseMHC/peptideCD3 complexTCRCD4521Negative Feedback
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