Signal Transduction II .ppt

1、Signal Transduction IIBICH202Signal transduction through the MAPK pathways Protein-protein interactions Protein kinase cascades.OverviewThe MAPK signalling pathways Cells in an organism receive a variety of extracellular stimuli for cell proliferation.The best characterized mitogenic pathway is the

2、mitogen-activated protein kinase(MAPK)cascades.Many growth stimulation converges on the kinase cascade that activates the MAPK(also called ERK1 and ERK2=extracellular signal-regulated kinase 1 and 2).The MAPK signalling pathways The RAS-activated MAPK pathway:RASRAFMEKMAPKrepresents the first exampl

3、e where all the steps in a complete signalling cascade from the cell surface receptor PTK,to the nuclear transcription is known.Upon activation,MAPK then translocate into the nucleus and induce the transcription of the immediate early genes(e.g.MYC).Immediate early genes:genes that are expressed ear

4、ly during stimulation(in contrast to late response genes).They are usually transcription factorsThe MAPK signalling pathways It should be noted that the RAS-RAF-MEK-MAPK pathway is only one example of so called“MAPK pathways”.Two other mammalian MAPK pathways involving JNK1 and p38,are involved in s

5、tress responses(they are also“MAPK pathways).The MAPK signalling pathwaysRAS-RAF-MEK-MAPK Ligand binds receptor PTK Ligand binds receptor PTK Autophosphorylation on tyrosine PPPPRAS-RAF-MEK-MAPK Ligand binds receptor PTK Autophosphorylation on tyrosine GRB2(a SH2-and SH3-containing protein)binds to

6、the receptor phosphotyrosine motif Y-V/L-N-X via its SH2 domainPPPPRAS-RAF-MEK-MAPKSH2SH3GRB2SOS Ligand binds receptor PTK Autophosphorylation on tyrosine GRB2(a SH2-and SH3-containing protein)binds to the receptor phosphotyrosine motif Y-V/L-N-X via its SH2 domain The SH3 of GRB2 binds constitutive

7、ly to the proline-rich sequence in the C-terminus of SOS(a guanine nucleotide exchange factor for RAS).PPPPSH2SH3GRB2SOSRAS-RAF-MEK-MAPK Recruitment of SOS to the close proximity of RAS in the membranePPPPSH2SH3GRB2SOSRASGDPRAS-RAF-MEK-MAPK RAS becomes activated by exchanging GDP for GTPPPPPSH2SH3GR

8、B2SOSGDPGTPRASRAS-RAF-MEK-MAPKGTP The RAS-GTP interacts with the N-terminal regulatory region of the RAF(serine/threonine protein kinase),hence recruiting RAF to the membrane and changing its conformationPPPPSH2SH3GRB2SOSRASRAFRAS-RAF-MEK-MAPKGTP14-3-3 Activation of RAF(localization to membrane and

9、conformation changes induced by RAS-GTP:cause phosphorylation of RAF and binding to the scaffold protein 14-3-3)PPPPSH2SH3GRB2SOSRASRAFRAS-RAF-MEK-MAPKGTP14-3-3 Activation of RAF(localization to membrane and conformation changes induced by RAS-GTP:cause phosphorylation of RAF and binding to the scaf

10、fold protein 14-3-3)PPPPSH2SH3GRB2SOSRASRAFRAS-RAF-MEK-MAPKPGTP14-3-3 Activated RAF in turn activates MEK(also called MAPK kinase;a dual specificity kinase)by phosphorylation on two conserved serine residues in MEK.PPPPSH2SH3GRB2SOSRASRAFMEKP PRAS-RAF-MEK-MAPKPGTP14-3-3 Activated RAF in turn activat

11、es MEK(also called MAPK kinase;a dual specificity kinase)by phosphorylation on two conserved serine residues in MEK.PPPPSH2SH3GRB2SOSRASRAFMEKP PRAS-RAF-MEK-MAPKPGTP14-3-3 Activated MEK activates MAPK(a serine/threonine protein kinase)by phosphorylation of conserved threonine and tyrosine residues.P

12、PPPSH2SH3GRB2SOSRASRAFMEKP PMAPKP PRAS-RAF-MEK-MAPKPGTP14-3-3 Activated MEK activates MAPK(a serine/threonine protein kinase)by phosphorylation of conserved threonine and tyrosine residues.PPPPSH2SH3GRB2SOSRASRAFMEKP PMAPKP PRAS-RAF-MEK-MAPKPGTP14-3-3 Activated MAPK phosphorylates a number of substr

13、ates in the plasma membrane and the cytoplasm;PPPPSH2SH3GRB2SOSRASRAFMEKP PMAPKP PSubstratesSubstratesPPRAS-RAF-MEK-MAPKPGTP14-3-3 Activated MAPK phosphorylates a number of substrates in the plasma membrane and the cytoplasm;it also translocated into the nucleus(within minutes)where it phosphorylate

14、s nuclear transcription factors.PPPPSH2SH3GRB2SOSRASRAFMEKP PMAPKP PSubstratesRAS-RAF-MEK-MAPKMAPKP PPGTP14-3-3 Activated MAPK phosphorylates a number of substrates in the plasma membrane and the cytoplasm;it also translocated into the nucleus(within minutes)where it phosphorylates nuclear transcrip

15、tion factors.PPPPSH2SH3GRB2SOSRASRAFMEKP PSubstratesRAS-RAF-MEK-MAPKMAPKP PMAPKP PPPGTP14-3-3 Activated MAPK phosphorylates a number of substrates in the plasma membrane and the cytoplasm;it also translocated into the nucleus(within minutes)where it phosphorylates nuclear transcription factors.Trans

16、cription of genes important for cell proliferation.PPPPSH2SH3GRB2SOSRASRAFMEKP PRAS-RAF-MEK-MAPKSubstratesMAPKP PMAPKP PPP14-3-3 More detail for each componentsPPPPSH2SH3GRB2SOSRASGTPRAFMEKP PSubstratesPMAPKP PRAS-RAF-MEK-MAPKP14-3-3SH2SH3GRB2SOSRASGTPRAFMEKP PMAPKP PGRB2 24 kDa adaptor molecule.Onl

17、y contains an SH2 domain between two SH3 domains(only one is shown in the diagram).PPPPRAS-RAF-MEK-MAPKPGRB2 The SH2 of GRB2 binds to phosphotyrosine in a number of receptor PTK:for EGF,PDGF,colony-stimulating factor-1(CSF-1),stem cell factor(SCF);all lead to the activation of RAS pathway.i.e.GRB2 f

18、unctions as a point of convergence for Ras-activated signaling pathways.RAS-RAF-MEK-MAPK14-3-3SH2SH3GRB2SOSRASGTPRAFMEKP PMAPKP PPPPPP14-3-3SH2SH3GRB2SOSRASGTPRAFMEKP PMAPKP PSOS 180 kDa guanine nucleotide exchange factor for RAS;stimulate the formation of active GTP-bound RAS.PPPPRAS-RAF-MEK-MAPKP1

19、4-3-3SH2SH3GRB2SOSRASGTPRAFMEKP PMAPKP PPPPPRAS-RAF-MEK-MAPKSOS SOS belongs to the DBL family of oncogenes(14 members).Most of these activate the RHO family of GTP-binding proteins involved in cytoskeletal reorganization.SOS is one of the few known member of the family for RAS.P14-3-3SH2SH3GRB2SOSRA

20、SGTPRAFMEKP PMAPKP PRAS 21 kDa GTPase protein.A special case of G protein monomer GTP-bound form:active GDP-bound form:inactive.originally identified as oncogenes in several retroviruses(rat sacroma),and are activated in a variety of human tumors.Amino acid substitution in one of two positions impai

21、rs its GTPase actvity and the activity cannot be shut off.PPPPRAS-RAF-MEK-MAPKP14-3-3SH2SH3GRB2SOSRASGTPRAFMEKP PMAPKP PPPPPRAS Oncogenic forms of RAS often have point mutations that lock RAS in the active GTP-bound form.Evidence:3T3 cells were induced to proliferate by+PDGF and EGF.Microinjection o

22、f anti-RAS antibodies into the cells blocked the cell proliferation.Microinjection of a constitutively active mutant of RAS caused cells to proliferate in the absence of PDGF and EGF.RAS-RAF-MEK-MAPKP14-3-3SH2SH3GRB2RASGTPRAFMEKP PMAPKP PRAF RAF is a serine/threonine protein kinase RAF is a MAPK kin

23、ase kinase(MAPKKK).Can transform cells when constitutively active or when overexpressed.PPPPSOSRAS-RAF-MEK-MAPKP14-3-3SH2SH3GRB2RASGTPRAFMEKP PMAPKP PPPPPSOSRAF The 14-3-3 family of scaffold proteins interacts constitutively with RAF via the phosphorylated Ser621 in RAF-both 14-3-3 and RAS are requi

24、red for activation of RAF.RAS-RAF-MEK-MAPKP14-3-3SH2SH3GRB2RASGTPRAFMEKP PMAPKP PPPPPSOSRAF RAS recruits RAF to the membrane.Membrane targeting of RAF is necessary to fully activate RAF.(Expression of mutant RAF that cannot bind RAS no stimulation of cell proliferation by a constitutively active RAS

25、).RAS-RAF-MEK-MAPKP Interaction between RAS and RAF(or between any two proteins in general)can be demonstrated by:(1)Co-immunoprecipitationABCMethods Interaction between RAS and RAF(or between any two proteins in general)can be demonstrated by:(1)Co-immunoprecipitationABCMethodsAntibodies against B

26、on a solid matrix(e.g.beads coated with Staphylococcal Protein A-which binds IgG)Interaction between RAS and RAF(or between any two proteins in general)can be demonstrated by:(1)Co-immunoprecipitationABCMethodsWash out unbound proteinsThe presence of protein A(or absence of C)in the immunoprecipitat

27、es can then be detected by immunoblotting.Immunoblotting(Western blot)SDS-PAGE-+View from the sideMethodsImmunoblotting(Western blot)SDS-PAGE-+View from sideMethodsImmunoblotting(Western blot)SDS-PAGE-+View from sideMethodsImmunoblotting(Western blot)-View from side+Transfer to membraneMethodsImmuno

28、blotting(Western blot)-View from side+Transfer to membraneMethodsImmunoblotting(Western blot)MethodsImmunoblotting(Western blot)Probe with primary antibodiesMethodsImmunoblotting(Western blot)Wash away unbound antibodiesMethodsImmunoblotting(Western blot)Probe with secondary antibodies(HRP-conjugate

29、d)MethodsImmunoblotting(Western blot)Wash away unbound secondary antibodiesMethodsImmunoblotting(Western blot)Add substrate for HRP,expose to filmMethodsImmunoblotting(Western blot)Add substrate for HRP,expose to filmMethodsImmunoblotting(Western blot)Develop filmMethods(view from the front)-+?AB(im

30、munoprecipitation)(Immunoblotting)What is this?(2)yeast two-hybrid systemTranscription factorDNA-BindingDomainActivatingdomainReporter geneMethodsBActivatingdomainDNA-BindingDomainAMethods(2)yeast two-hybrid systemTranscription factorDNA-BindingDomainActivatingdomainReporter geneDNA-BindingDomainACA

31、ctivatingdomainNo interactionInteractionMethods(2)yeast two-hybrid systemReporter geneBActivatingdomainDNA-BindingDomainAReporter gene?What controls are needed for designing immunoprecipitation experiments?What are the disadvantages of yeast two-hybrid?14-3-3RASGTPRAFMEKP PMEK Also called MAPK kinas

32、e(MAPKK).Phosphorylated on Ser218 and Ser222 by activated RAF.Mutation of MEK that leads to constitutive activity(by replacing the two Ser with glutamic acids or aspartic acids-by mimic phosphorylation)MAPK activation,mitogenicity,and cellular transformation.PPPSH2SH3GRB2PSOSMAPKP PRAS-RAF-MEK-MAPKP

33、14-3-3RASGTPRAFMEKP PPPPSH2SH3GRB2PSOSMAPKP PMEK The first true dual-specificity kinase to be discovered.RAS-RAF-MEK-MAPKP14-3-3RASGTPRAFMEKP PMAPK Five isoforms of ERK have been identified,but ERK1 and ERK2 have been most studied.Activated by phosphorylation on the 183T-E-Y185-located similarly to

34、the conserved Ser in MEK.PPPSH2SH3GRB2PSOSMAPKP PRAS-RAF-MEK-MAPKP14-3-3RASGTPRAFMEKP PPPPSH2SH3GRB2PSOSMAPKP PMAPK MAPK activation is biphasic:a transient peak within 5-10 min,and a sustained peak lasting several hours.The different activation kinetics different cellular response(e.g.in PC12 cells,

35、EGF induces only the transient response and stimulate cell growth;but NGF(nerve growth factor)induces the sustained response and stimulate differentiation).RAS-RAF-MEK-MAPKP14-3-3RASGTPRAFMEKP PPPPSH2SH3GRB2PSOSMAPKP PMAPK Inactivation of MAPK is achieved by several phosphatases:the serine/threonine

36、 phosphatase PP2A(may be for short term inactivation of MAPK);the dual-specificity phosphatase MKP1(and the related PAC)and PTP1(a tyrosine phosphatase,may be for long term negative feedback control of MAPK).RAS-RAF-MEK-MAPKMKP1:MAPK phosphatase 1PMAPK phosphorylates the consensus motif:P-X-S/T-P.In

37、 the cytoplasm,MAPK phosphorylates its upstream components in a negative feedback loop-MAPK phosphorylates SOS,RAF,MEK inhibition of MAP kinase pathway.Substrates of MAPK:In the nucleus,MAPK phosphorylates a number of transcription factors(e.g.Elk1)increase transcription(e.g.of c-Fos mRNA).Many othe

38、r substrates of MAPK probably unknown-identification is difficult as in the case of CDKs-see Cell cycle lectures).Substrates of MAPK:JNK pathwayGenericpathwayMAPKKKMAPKKMAPKERK/MAPkinase pathwayRAF1MEK1,2ERK1,2RASGRB2/SOSReceptor PTKJNK/SAPKpathwayRAC/CDC42MEKK1-3MEK4JNK/SAPKTAKMEK3,6p38p38pathwayPA

39、KStress responsesProliferation/differentiationJNK pathway In response to extracellular stresses like cycloheximide treatment,UV irradiation,heat shock,or TNF-a treatment,a family of MAPK relatives known as JNKs(also called stress-activated protein kinase(SAPKs)become activated.JNK pathway RAC1 and C

40、DC42 are two members of the RHO family of GTP-binding proteins.RAC1/CDC42GTP RAC1 can also be activated by RAS,explaining why receptor PTK can sometimes contribute to JNK activation.However,this is a minor pathway.RAC1 and CDC42 are mainly activated by stress response independent of RAS.STRESSJNK pa

41、thway GTP-bound form of RAC1 and CDC42 bind and activate the serine/threonine protein kinase PAK,PKN,and PtdIns kinases.RAC1/CDC42GTPSTRESSPAKPAK:p21-activated protein kinasePKN:protein kinase NPtdIns kinase:phosphatidylinositol kinaseJNK pathway GTP-bound form of RAC1 and CDC42 bind and activate th

42、e serine/threonine protein kinase PAK,PKN,and PtdIns kinases.STRESSPAKRAC1/CDC42GTPJNK pathway these kinases phosphorylate and activate MEKK1-3STRESSPAKMEKK1-3P PRAC1/CDC42GTPMEKK1-3JNK pathwaySTRESSPAKMEKK1-3P PMEK4P PRAC1/CDC42GTP MEKK1-3 phosphorylate and activate MEK4(also called JNKK)(MEK in MA

43、PK pathway;45%identical in sequence with MEK)MEK4JNK pathway MEK4 phosphorylates JNK at two similar sites as in ERK(but T-P-Y in JNK instead of T-E-Y in MAPK)i.e.conservation between the ERK and JNK pathways at the level of proteins and mode of regulation!STRESSPAKMEKK1-3P PMEK4P PJNKP PRAC1/CDC42GT

44、PJNKJNK pathway JNK translocation into the nucleusphosphorylation of the transcription factor c-JUN at the N-terminal residues(Ser63 and Ser73)activation of transcription by c-JUN STRESSPAKMEKK1-3P PMEK4P PJNKP Pc-JUNPRAC1/CDC42GTPc-JUNRAC1/CDC42GTPSTRESSPAKMEKK1-3P P14-3-3PPPPSH2SH3GRB2SOSRASGTPRAF

45、MEK4P PMEKP PJNKP PMAPKP PSpecificity of MAP kinase pathways When cells are treated with mitogenic agents(e.g.growth factors),ERK(MAPK)become strongly activated but JNK is poorly activated.Conversely,when cells are challenged with stress,JNK is activated but ERK(MAPK)is only weakly activated.i.e.it

46、seems JNK and ERK pathways are biologically distinct.However,they are both protein kinases with similar substrate specificity-most in vitro substrates are the same for both.Yet these pathways must result in unique transcriptional activity-because stress and mitogen must elicit different responses.Si

47、milarly,there are at least five parallel MAP kinase pathways in mammalian cells.How is specificity achieved?One way is by scaffold proteins.Specificity of MAP kinase pathwaysMAPKKKMAPKKMAPKScaffold protein AMAPKKKMAPKMAPKKScaffold protein BSpecificity of MAP kinase pathwaysMAP kinase pathways in yea

48、st Most,perhaps all,eukaryotic cells contain kinase cascades that activate MAP kinase.In multicellular organisms,MAP kinase pathway is activated by ligand binding to receptor PTK.But there is no receptor PTK in yeast.How is MAP kinase pathways activated in yeast?At least five independent MAPK-relate

49、d cascades exist in yeast.Various functions:Mating(related to proliferation)Adaptation to nitrogen starvation(related to stress)Response to external osmolarity(related to stress)Polarized growth(related to proliferation)Sporulation(related to proliferation)MAP kinase pathways in yeastMating In S.cer

50、evisiae,a MAP kinase pathway is linked to the receptors for the mating factors a and a a.Mating factors control mating between haploid yeast cells of the opposite mating type:an a haploid cell secretes the a mating factor and has cell surface receptors for the a a factor;an a a haploid cell secretes