Ras Subcellular Localization Defines Extracellular Signal-Regulated Kinase 1 and 2 Substrate PDF
Preview Ras Subcellular Localization Defines Extracellular Signal-Regulated Kinase 1 and 2 Substrate
MOLECULARANDCELLULARBIOLOGY,Mar.2009,p.1338–1353 Vol.29,No.5 0270-7306/09/$08.00(cid:1)0 doi:10.1128/MCB.01359-08 Copyright©2009,AmericanSocietyforMicrobiology.AllRightsReserved. Ras Subcellular Localization Defines Extracellular Signal-Regulated Kinase 1 and 2 Substrate Specificity through Distinct (cid:1) Utilization of Scaffold Proteins Berta Casar,1 Imanol Arozarena,3 Victoria Sanz-Moreno,3 Ad´an Pinto,1 Lorena Agudo-Ib´an˜ez,1 Richard Marais,3 Robert E. Lewis,4 María T. Berciano,2 and Piero Crespo1* DepartamentodeBiologíaMolecular1andDepartamentodeAnatomíayBiologíaCelular,2FacultaddeMedicina,InstitutodeBiomedicinay BiotecnologíadeCantabria(IBBTEC),ConsejoSuperiordeInvestigacionesCientíficas-IDICAN-UniversidaddeCantabria,Santander,39011 Cantabria,Spain;InstituteofCancerResearch,CancerResearchUKCentreforCellandMolecularBiology,237FulhamRoad, LondonSW36JB,UnitedKingdom3;andEppleyInstituteforResearchinCancerandAlliedDiseases,Universityof NebraskaMedicalCenter,Omaha,Nebraska68198-76964 Received27August2008/Returnedformodification2October2008/Accepted17December2008 Subcellular localization influences the nature of Ras/extracellular signal-regulated kinase (ERK) signals by unknown mechanisms. Herein, we demonstrate that the microenvironment from which Ras signals emanate determines which substrates will be preferentially phosphorylated by the activated ERK1/2. We show that the phosphorylation of epidermal growth factor receptor (EGFr) and cytosolic phospholipase A (cPLA ) is most 2 2 prominentwhenERK1/2areactivatedfromlipidrafts,whereasRSK1ismainlyactivatedbyRassignalsfromthe disorderedmembrane.Wepresentevidenceindicatingthattheunderlyingmechanismofthissubstrateselectivity isgovernedbytheparticipationofdifferentscaffoldproteinsthatdistinctivelycoupleERK1/2,activatedatdefined microlocalizations,tospecificsubstrates.Assuch,weshowthatforcPLA activation,ERK1/2activatedatlipidrafts 2 interactwithKSR1,whereasERK1/2activatedattheendoplasmicreticulumutilizeSef-1.Tophosphorylatethe EGFr, ERK1/2 activated at lipid rafts require the participation of IQGAP1. Furthermore, we demonstrate that scaffold usage markedly influences the biological outcome of Ras site-specific signals. These results disclose an unprecedentedspatialregulationofERK1/2substratespecificity,dictatedbythemicrolocalizationfromwhichRas signalsoriginateandbytheselectionofspecificscaffoldproteins. RasGTPasesoperateaskeymolecularswitchesthatconvey Thepathwayleadingtotheactivationofextracellularsignal- extracellular signals from surface receptors to the interior of regulatedkinase1and2(ERK1/2)mitogen-activatedprotein the cell, thereby regulating essential processes, including pro- kinases is a key component of Ras signals (29). To date, evi- liferation, differentiation, and survival (10). It has been long dence is mounting indicating that the cellular microenviron- knownthatRasproteinsmustbeattachedtotheinnerleaflet ment can play an essential role in the regulation of ERK1/2 of the plasma membrane (PM) to be functional, but only re- functions. ERK1/2 can be regulated by processes strictly de- cently has it been established that Ras isoforms are distinc- pendent on subcellular localization (33), and ERK1/2 them- tivelysegregatedindifferentPMmicrodomains:H-Rascanbe selvesregulatesomebiochemicalprocessesinacompartment- found in the bulk membrane and in lipid rafts, both caveolar dependent fashion (14). Furthermore, the balance among andnoncaveolar.K-Rasisexclusivelylocatedinthebulkmem- differentcompartment-specificcomponentsofERK1/2signals brane(27,28),whileN-Rasismainlydetectedinnoncaveolar seemsdeterminantforthebiologicaloutcomesresultingfrom lipidrafts(20).Moreover,Rasproteinsarepresentandfunc- ERK1/2 activation (3, 30). ERK1/2 phosphorylate a great tional in endomembranes, such as endosomes, the endoplas- variety of substrates distributed in different subcellular local- micreticulum(ER),andtheGolgicomplex(9,26,32).How- izations and microenvironments (40). However, whether, de- ever, how these distinct microenvironments affect the signals pending on the activating stimuli, ERK1/2 display variable generated by Ras is just beginning to be unfolded. In this selectivity toward these different pools of substrates and the respect,recentfindingssupportthenotionthattheRassignals extent to which the subcellular atmosphere can regulate emanating from these different sublocalizations are quantita- ERK1/2 interactions with their cognate targets are largely tivelyandqualitativelydifferent,intheeffectorroutesthatthey unansweredissues. engage in and in the resulting transcriptomal and biological ERK1/2 signals are modulated by different types of regula- outcomes(1,9,15,21). tory proteins. Scaffold proteins serve such a purpose by con- nectingthedifferentcomponentsofthecascadeintoanenzy- maticmacrostructurebywhichtheamplitudeanddurationof *Correspondingauthor.Mailingaddress:InstitutodeBiomedicina ERK1/2 signals are fine-tuned, while providing signal fidelity yBiotecnologíadeCantabria(IBBTEC),ConsejoSuperiordeInves- by isolating the signaling complex from undesired, molecular tigaciones Científicas-IDICAN-Universidad de Cantabria, Departa- interferences(12,17,23).Furthermore,scaffoldscanalsoserve mentodeBiologíaMolecular,FacultaddeMedicina,Santander,39011 a role in the spatial control of ERK1/2 signals by regulating Cantabria,Spain.Phone:34-942-200959.Fax:34-942-201945.E-mail: [email protected]. their activity in a sublocalization-specific fashion. In this re- (cid:1)Publishedaheadofprinton29December2008. spect, KSR1 acts preferentially on ERK1/2 signals emanating 1338 VOL.29,2009 SPATIAL REGULATION OF ERK1/2 SUBSTRATE SPECIFICITY 1339 from PM cholesterol-rich domains (22). MP-1 regulates FocusformationandsenescenceassaysinMEFs.Thefocusformationand ERK1/2atendosomes(35),SefregulatesERK1/2attheGolgi senescenceassayswereperformedexactlyasdescribedpreviously(18). Luciferaseassays.Luciferaseassayswereperformedasdescribedpreviously complex(37),andpaxillinregulatesERK1/2atfocaladhesions (33),usingthereporterpGE51-lucandavectorencodingthetransactivation (16). But the precise mechanisms whereby scaffolds can domain of Elk-1 (amino acids 307 to 428) fused to the GAL4 DNA-binding achieve spatial specificity for ERK1/2 signals are largely un- domain.Theluciferaseactivitiesweredeterminedusingacommercialkit(Pro- known. mega,Madison,WI)andnormalizedbythe(cid:5)-galactosidaseactivity. Herein, we have taken a look at these questions. We dem- Immunofluorescence.Cellsweregrownonglassslides,washedtwiceinPBS, fixed with 3.7% formaldehyde in PBS for 10 min at room temperature, and onstratethatthesignalingplatformfromwhichtheactivating permeabilized,whennecessary,with0.5%TritonX-100–PBSfor20min.The RassignalsspringdictatesERK1/2selectivitytowarddifferent preparationsweresequentiallyincubatedwith0.5%TritonX-100–PBSfor15 substrates.Inthisprocess,weunveiltheparticipationofscaf- min and 0.1 M glycine-PBS for 30 min and blocked with 1% bovine serum foldproteinsthatdistinctivelycoupleERK1/2activatedatde- albumin,0.01%Tween20inPBSfor5min.Thepreparationswerethenrinsed inPBS–0.05%Tween20,incubatedfor1hwiththeprimaryantibody,washed, finedmicrolocalizationstospecificsubstrates.Ourresultspro- andincubatedfor45minwiththeappropriatesecondaryantibodies.Coverslips vide the first evidence pointing both to the activating signal weremountedinVectaShieldandsealedwithnailpolish.Confocalmicroscopy microenvironment and to a differential scaffold usage, as reg- wasperformedwithaBio-RadMRC-1024microscope,usingexcitationwave- ulatorsofERK1/2substratespecificity. lengthsof488nm. RESULTS MATERIALSANDMETHODS Plasmids.Theexpressionvectorsencodingthelocalization-targetedH-RasV12 ERK1/2 cytoplasmic substrates are differentially activated andthewildtype(5,21)havebeenpreviouslydescribed.Myc-Sefwasagiftfrom depending on Ras localization. To take an initial glance into Z. Chang (38). UO126, SB203580, and epidermal growth factor (EGF) were howRassublocalizationaffectedERK1/2functions,welooked fromCalbiochem.Lysophosphatidicacid(LPA)andtetradecanoylphorbolac- attheactivationofseveralbonafideERK1/2substrates,which etate(TPA)werefromSigma. Cell culture and transfection. HEK293T cells and wild-type, H-Ras(cid:2)/(cid:2) areknowntobepresentindifferentsubcellularcompartments. N-Ras(cid:2)/(cid:2)(H/N-Ras(cid:2)/(cid:2))andKsr(cid:2)/(cid:2)mouseembryonicfibroblasts(MEFs)were To this end, we utilized the previously described H-RasV12 growninDulbecco’smodifiedEagle’smedium-10%fetalcalfserum,andNIH constructsselectivelytetheredtodefinedmicrolocalizationsby 3T3cellsweregrownin10%calfserum.Subconfluentcellsweretransfectedby specificlocalizationsignals:theER(avianinfectiousbronchitis Lipofectamine.SmallinterferingRNAs(siRNAs)werefromDharmacon;apool virusM1protein),lipidrafts(LCKmyristoylationsignal),the offourdifferentsiRNAswasusedineachparticularcase.Sequencesareavail- able upon request. siRNAs were transfected by following the manufacturer’s disordered membrane (CD8(cid:6) transmembrane domain), and guidelines. the Golgi complex (KDEL receptor N193D) (21), transiently Immunoblotting and immunoprecipitations. Immunoblotting and immuno- expressedin293Tcells.WiththeexceptionoftheGolgisignal, precipitationswereperformedexactlyasdescribedpreviously.Immunoprecipi- whichevokedlittleERKactivation,endogenousERK1/2were tationswereperformedforlysatesnormalizedbytotalproteincontents(33). Mouse monoclonal antihemagglutinin (anti-HA), -Myc, and -AU5 were from activated to similar levels independently of Ras localization Covance.Themousemonoclonalantibodiesanti-ERK1/2and-phospho-ERK; andofRasexpressionlevels(Fig.1A).Theexpressionlevelsof therabbitpolyclonalsanti-KSR1,-RSK1,-caveolin,and-phospho-RSK1;and the different site-specific Ras constructs were determined by the goat polyclonal anti-cytosolic phospholipase A (anti-cPLA) were from 2 2 theamountofRasthatcanfitintoeachofthesublocalizations, Santa Cruz. The mouse monoclonal antibodies anti-Elk1 and -phospho-Elk1 rather than on the intrinsic expression capacity of the con- werefromCellSignaling.Themousemonoclonalantibodiesanti-IQGAP1and -transferrinreceptorwerefromBDTransduction.Therabbitpolyclonalanti- structs themselves, as extensively discussed before (21). This EGFreceptor(anti-EGFr)wasagiftfromAtanasioPandiella(Salamanca). pattern of activation was almost identical to that previously Nucleocytoplasmicfractionations.Nucleocytoplasmicfractionationswereper- described for NIH 3T3 cells stably expressing these targeted formedasdescribedpreviously(3). RasV12constructs(21). S100/P100separations.S100/P100separationswereperformedasdescribed previously(4). First, we explored whether the platform in which Ras-acti- Sucrosegradients.Cellswereresuspendedin25mMTris(pH7.4),150mM vatingsignalsweregeneratedcoulddetermineiftheactivated NaCl,5mMEDTA,and0.25%TritonX-100withrockingat4°Cfor1h.The ERK1/2 preferentially selected substrates localized at distinct lysatesweresettoasucroseconcentrationof41%.Layersof8.5mlof35% sites. We assayed the phosphorylation of the EGFr, an ERK sucrose(in10mMTris[pH7.4])andof2.5mlof16%sucroseweresequentially substratelocalizedatthePM(24).The293Tcellsweretrans- overlaidandcentrifugedinaBeckmanSW41rotorfor18hat35,000rpm.Ten to 12 1-ml fractions were collected, precipitated in 6.5% trichloroacetic acid, fectedwiththetargetedRasV12constructsandmetabolically resuspendedinloadingbuffer,andfractionatedon12%sodiumdodecylsulfate- labeledwithorthophosphateinordertodisclosethephosphor- polyacrylamidegelelectrophoresisgels. ylationofEGFr.ItwasfoundthatEGFrwasintensivelyphos- cPLA activation assays. The cPLA activation assays were performed as 2 2 phorylated in cells expressing RasV12 at lipid rafts and, to a describedpreviously(33).Briefly,cellswerelabeledwith[3H]arachidonicacid(1 (cid:3)Ciperwellina24-wellplate)for18h.Thecellswerethenwashedtwicewith lesserextent,atthedisorderedmembrane.Ontheotherhand, phosphate-bufferedsaline(PBS),andnewmedium(Dulbecco’smodifiedEagle’s RasV12 at endomembranes, whether ER or Golgi, was not medium–1%fattyacid-freebovineserumalbumin)wasadded.Whereindicated, capableofinducingasubstantialphosphorylationoftheEGFr thecellswerestimulatedwithEGF(100ng/ml)for30min.Themediumwas (Fig. 1B, top panels). To validate that the RasV12 signals removedandcentrifugedat2,000(cid:4)g,andfree,labeledarachidonicacidre- inducing the phosphorylation of EGFr were mediated by leasedintothemediumwasmeasuredbyliquidscintillationcounting. EGFrphosphorylationassays.32Pmetaboliclabelingwasperformedasde- ERK1/2,weusedtheMEKinhibitorUO126,whichwasshown scribedpreviously(33).After18hofstarvationandupontreatmentwithmito- tocompletelyinhibitthephosphorylationofEGFrinducedby gensorinhibitorswhereappropriate,thecellswerelysedandthelysateswere Ras,irrespectiveofitslocalization(Fig.1B,bottompanels).In immunoprecipitated with anti-EGFr antibody. The immunoprecipitates were NIH 3T3 cells expressing ectopic EGFr, LCK-RasV12 could resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and evokethephosphorylationofaproteinofthesamesizeasthe EGFr phosphorylation was made evident by prolonged exposition of the driedgel. onedetectedin293Tcells,whereasinparentalNIH3T3cells 1340 CASAR ET AL. MOL.CELL.BIOL. FIG. 1. Ras sublocalization defines ERK1/2 substrate specificity. (A) Phosphorylation of ERK1/2 resulting from compartmentalized Ras activation.293TcellsweretransfectedwithH-RasV12(0.5(cid:3)g),untargeted(V12)orspecificallytetheredtotheER(M1),lipidrafts(LCK),the disordered membrane (CD8), or the Golgi complex (KDELr). Phosphorylated and total ERK1/2 levels were determined in total lysates by immunoblotting.Anti-HAWesternblotting(WB)revealstheexpressionlevelsoftheRasconstructs.C,control.(B)PhosphorylationofEGFr resulting from compartmentalized Ras activity. Top panel, EGFr phosphorylation was determined by immunoprecipitation (IP) in 32P-labeled 293TcellstransfectedwiththeRasconstructs.Bottompanel,EGFrphosphorylationisERKdependent.293Tcellstransfectedwiththetargeted Rasconstructsandincubatedwith2(cid:3)MU0126for2h((cid:1))wereanalyzedforEGFrphosphorylation.(C)PhosphorylationofRSK1inducedby compartmentalizedRasactivation.TotalandphosphorylatedRSK1levelsweredeterminedinstarvedNIH3T3and293TcellsexpressingtheRas constructs.Rightpanel,RSK1phosphorylationisERKdependent.293TcellstransfectedwithH-RasV12andincubatedwithU0126for2h((cid:1)) wereanalyzedforRSK1phosphorylation.(D)RegulationofcPLA activationbyRassublocalization.3H-labeledarachidonicacidreleasewas 2 measuredinNIH3T3cells.(E)ERK1/2substratespecificityisunaffectedbytargetedRasexpressionlevels.Cellsweretransfectedwithincreasing concentrations(0.5to5(cid:3)g)ofLCK-orCD8-RasV12andRSK1phosphorylationandcPLA activationwereexamined.(DandE)Shownarethe 2 averages(cid:7)standarderrorsofthemeansoftheresultsfromthreeindependentexperimentsrelativetothelevelsinthecontrolcells.(cid:6),anti. thatdonotexpressEGFr,thisbandwasnotapparent(datanot WethendeterminedifERK1/2cytoplasmicsubstrateswere shown),therebyvalidatingEGFridentity.Sinceproteinkinase also subject to differential regulation depending on the Ras D can phosphorylate EGFr (6), we investigated the possible activation site. To do so, we monitored RSK1, a substrate participationofthiskinasebysuppressingitsexpressionusing phosphorylated by ERK1/2 primarily at the cytoplasm (13). siRNA interference. It was found that the downregulation of BothinNIH3T3andin293Tcells,RSK1phosphorylationwas protein kinase D had no effects on EGFr phosphorylation mostprominentwheninducedbyRassignalsoriginatedatthe inducedbyRasV12(datanotshown). disordered membrane. UO126 completely abrogated RSK1 VOL.29,2009 SPATIAL REGULATION OF ERK1/2 SUBSTRATE SPECIFICITY 1341 FIG. 2. EffectsofRassublocalizationontheactivationofERK1/2nuclearsubstrates.(A)PhosphorylationofElk1bycompartmentalizedRas signals. Total and phosphorylated Elk1 levels were determined by immunoblotting in NIH 3T3 and 293T cells expressing the targeted Ras constructs.C,control.(B)EffectsofcompartmentalizedRassignalsonERK1/2nucleocytoplasmicdistribution.TotalandphosphorylatedERK1/2 levelswereexaminedinnuclear(N)andcytoplasmic(C)fractionsfrom293TcellsexpressingthetargetedRasconstructs.Bottompanel,thepurity ofthefractionswasascertainedbyblottingagainstRho-GDPdissociationinhibitor((cid:6)GDI)andElk1.(C)Elk1activationdependenceonRas compartmentalized signals. Left panel, Elk1 transactivation was assayed in NIH 3T3 cells transfected with GAL4-Elk1 and the targeted Ras constructsplussiRNAsagainstERK1and-2orJNK1and-2(10ng),asshowninthekey.C,control.Shownaretheaverages(cid:7)standarderrors ofthemeansoftheresultsfromthreeindependentexperimentsrelativetothelevelsinthecontrolcells.Rightpanel,diminishedexpressionof the endogenous mitogen-activated protein kinases resulting from siRNA treatments. V12, untargeted H-RasV12; M1, H-RasV12 specifically tetheredtotheER;LCK,H-RasV12specificallytetheredtolipidrafts;CD8,H-RasV12specificallytetheredtothedisorderedmembrane;KDELr, H-RasV12specificallytetheredtotheGolgicomplex. phosphorylationinducedbyRasV12(Fig.1C),demonstrating RasV12, was remarkably higher than that generated by the that ERK1/2 were the sole mediators in this event. It was site-specific RasV12 constructs, among which there were no importanttoknowwhetherotherERKcytoplasmicsubstrates majordifferences.OnlyRassignalsemanatingfromtheGolgi responded similarly or if this behavior was unique for RSK1. complex were slightly less effective. Similar results were ob- As such, we analyzed a second cytoplasmic substrate, cPLA served in 293T cells (Fig. 2A). To further substantiate this 2 (19). Upon examination of radiolabeled arachidonic acid re- observation, we looked at how Ras sublocalization affected lease in cells expressing the tethered Ras constructs, it was phosphorylated ERK1/2 nuclear influx. To this end, nuclear found that cPLA was activated mostly by Ras at lipid rafts and cytoplasmic fractions were purified from 293T cells ex- 2 and, to a lesser extent, by signals elicited from the ER (Fig. pressing the targeted Ras constructs. It was found that Ras 1D). These results demonstrated that ERK1/2 activated from sublocalization was not a factor for determining ERK1/2 nu- different Ras signaling platforms selectively targeted distinct cleocytoplasmic distribution, again except with Golgi Ras sig- cytoplasmic substrates. To ascertain that ERK1/2 substrate nals, which were less effective for eliciting ERK1/2 nuclear selectivity was not altered by the levels of the targeted Ras entry(Fig.2B). proteins,weanalyzedcPLA andRSK1activationinresponse SinceElk1isalsoasubstrateforotherMAPKs,suchasp38 2 to increasing concentrations of LCK- or CD8-RasV12. As and JNK, we investigated the contribution of each of these shown in Fig. 1E, even the highest levels of active Ras at the MAPKstoitsphosphorylation,inresponsetocompartmental- disordered membrane failed to affect cPLA activation. Like- izedRassignals.Forthis,weutilizedaGAL4-Elk1-dependent 2 wise,Rastargetedtolipidraftscouldnotelicitanysignificant luciferasereporter.Emulatingtheresultsobtainedbyassaying RSK1phosphorylation,irrespectiveofitsexpressionlevels. Elk1 phosphorylation, untargeted RasV12 induced the great- RaslocalizationdoesnotaffectERK1/2nuclearsubstrates. est transactivation of the Elk1 reporter, whereas few differ- Next,wefocusedonERK1/2nuclearsubstrates.Noticeably,in enceswerefoundamongthesite-specificRasconstructs(Fig. NIH 3T3 cells expressing the Ras constructs, the phosphory- 2C, left panel). The depletion of ERK1/2 levels by siRNA lation of Elk1 transcription factor, induced by untargeted interference (Fig. 2C, right panel) resulted in pronounced 1342 CASAR ET AL. MOL.CELL.BIOL. FIG. 3. cPLA butnotRSK1activationisimpairedinMEFsdevoidofRasatlipidrafts.(A)ERK1/2activationinH/N-Ras(cid:2)/(cid:2)MEFs.Total andphosphorylat2edERK1/2levelswereanalyzedinwildtype(wt)andH/N-Ras(cid:2)/(cid:2)MEFsafterstarvation((cid:2))andstimulationwith10ng/mlEGF for5min((cid:1)).FiguresshowERK1/2phosphorylationlevelsrelativetothosefoundinunstimulated,wild-typeMEFs.(B)cPLA activationis impairedinH/N-Ras(cid:2)/(cid:2)MEFs.3H-labeledarachidonicacidreleasewasmeasuredinwild-typeandH/N-Ras(cid:2)/(cid:2)MEFsafterstimula2tion((cid:1))with EGF.Shownaretheaverages(cid:7)standarderrorsofthemeansoftheresultsfromthreeexperimentsrelativetothelevelsinunstimulatedcells.***, P(cid:8)0.001,witha95%confidenceinterval.(C)EGFrphosphorylationisimpairedinH/N-Ras(cid:2)/(cid:2)MEFs.EGFrphosphorylationwasassayedin wild-type and H/N-Ras(cid:2)/(cid:2) MEFs after stimulation with LPA (5 (cid:3)M) for 5 min ((cid:1)). (D) Phosphorylation of RSK1 and Elk1 is unaffected in H/N-Ras(cid:2)/(cid:2)MEFs.TotalandphosphorylatedRSK1/Elk1levelsweredeterminedbyimmunoblottinginwild-typeandH/N-Ras(cid:2)/(cid:2)MEFsafter stimulationwithEGF.(E)ThepresenceofRasatlipidraftsinH/N-Ras(cid:2)/(cid:2)MEFsrestoresPLA activation.3H-labeledarachidonicacidrelease wasmeasuredinH/N-Ras(cid:2)/(cid:2)MEFstransfectedwithwild-typeH-Rastargetedtoitsdifferentmicro2localizations(1(cid:3)g)afterstimulationwithEGF. Shownaretheaverages(cid:7)standarderrorsofthemeansoftheresultsfromtwoexperimentsrelativetothelevelsdetectedinunstimulatedcells transfectedwiththerespectivesite-specific,wild-typeH-Rasconstructs.*,P(cid:8)0.05,witha95%confidenceinterval.(F)TherestorationofRas atlipidraftsinH/N-Ras(cid:2)/(cid:2)MEFsdoesnotaffectRSK1activation.TotalandphosphorylatedRSK1levelsweredeterminedforH/N-Ras(cid:2)/(cid:2)MEFs transfectedwithLCK-orCD8-tetheredwild-typeH-Ras(1(cid:3)g)afterstimulationwithEGF((cid:1)).TargetedH-Rasproteinexpressionlevelswere determinedbyanti-HAimmunoblotting.(cid:6),anti;c,control. drops in Elk1 transactivation in all cases. Noticeably, Elk1 EGFstimulationwassignificantlyreducedcomparedtothatin activationelicitedbyGolgiRassignalswastheleastresponsive wild-type MEFs (Fig. 3A). When we evaluated the activation to ERK1/2 downregulation but was the most affected by of cPLA in H/N-Ras(cid:2)/(cid:2) MEFs, the response to EGF was 2 JNK1/2 decline. On the other hand, p38 inhibition by treat- reducedbynearly70%incomparisontothatofthewild-type ment with SB203580 was ineffective in all cases (data not MEFs (Fig. 3B). The same situation occurred when we ana- shown). lyzedEGFrphosphorylationinducedbyLPAstimulation(Fig. It was essential to validate the above findings in a physio- 3C). Conversely, EGF-induced RSK1 and Elk1 phosphoryla- logically relevant context. For this purpose, we resorted to tions were similar to those encountered in wild-type MEFs fibroblasts obtained from H-Ras/N-Ras knockout mice, in (Fig. 3D), thereby corroborating our hypothesis. To further which lipid rafts are completely devoid of Ras (21), since K- ascertain our case, we restored Ras at the lipid rafts of H/N- Ras is exclusively located in disordered membrane (27). We Ras(cid:2)/(cid:2)MEFsbytransfectingthemwiththeLCK–H-Raswild reasonedthatinthesecells,thephosphorylationofERKsub- type. The renewed presence of Ras in lipid rafts markedly strates regulated by Ras signals from lipid rafts, but not of rescued cPLA activation upon EGF stimulation (Fig. 3E), 2 those regulated from disordered membrane, should be com- whereasincellstransfectedwithH-RastetheredtotheER,the promised. In these MEFs, ERK1/2 activation resulting from rescueofcPLA activationwasonlymodestandinsignificantin 2 VOL.29,2009 SPATIAL REGULATION OF ERK1/2 SUBSTRATE SPECIFICITY 1343 FIG. 4. SublocalizationdictatesERK1/2substrateselectivityunderphysiologicalstimulation.(A)SubcellularfractionationoftargetedERK2 proteins.293TcellsexpressingAU5-taggedERK2specificallytetheredtolipidrafts(LCK)ortothedisorderedmembrane(CD8)(1(cid:3)g)were solubilizedin0.25%TritonX-100andpartitionedinasucrosegradient.ThepresenceofthetargetedERK2proteinsinthedifferentfractionswas analyzedbyanti-AU5immunoblotting.Anti-caveolin-1((cid:6)Cav)identifiesthelipidraftfractions,andanti-transferrinreceptor((cid:6)Tfr)identifiesthe disordered membrane fractions. (B) Targeted ERK2 proteins recognize signals in site-specific fashion. Cells were transfected with LCK- or CD8-AU5-taggedERK2inadditiontoLCK-RasV12orCD8-RasV12(1(cid:3)geach)asshown.TotalandphosphorylatedERKlevelswereexamined in anti-AU5 immunoprecipitates. (C) Time-dependent activation profiles of the targeted ERK2 proteins. 293T cells were transfected with the tetheredERK2constructsandtheirkineticsofactivationwerecomparedwhenstimulatedwithEGF(10ng/ml),LPA(5(cid:3)M),orTPA(100nM) fortheindicatedtimes(inminutes)bymonitoringtotalandphosphorylatedERKlevelsinanti-AU5immunoprecipitates.Forcomparison,the phosphorylationkineticsofendogenousERK1/2areshown(toptwopanels).(D)EGFrphosphorylationcorrelateswiththeactivationofERK2 atlipidrafts.32P-labeled293Tcellswerestimulated,afterstarvation,withLPAfortheindicatedtimesandphosphorylationoftheEGFrwas analyzed.NoticethatthekineticsofEGFrphosphorylationmatchthoseofLCK-ERK2stimulatedwithLPA.(E)RSK1phosphorylationcorrelates withtheactivationofERK2atthedisorderedmembraneandvariesaccordinglydependingonthestimulus.293Tcellswerestimulated,after starvation,withEGForTPAfortheindicatedtimes,andthephosphorylationofRSK1wasanalyzed.NoticethatthekineticsofRSK1activation matchthoseofCD8-ERK2stimulatedwithEGForwithTPA. thosecellsharboringdisorderedmembrane-orGolgi-tethered activation in the immediate proximity of these microenviron- Ras.Conversely,therestorationofH-Rasatlipidraftshadno ments, upon the receipt of external stimuli. To verify the re- effectsinthephosphorylationofRSK1inducedbyEGF,which sponsespecificityoftheseconstructstosignalsemanatingfrom was slightly augmented in those cells transfected with disor- lipid rafts or disordered membrane, they were cotransfected dered membrane-tethered Ras (Fig. 3F). These results dem- with CD8- and LCK-RasV12. As shown in Fig. 4B, CD8- onstrated that, both in physiological contexts and in ectopic RasV12 only induced the phosphorylation of the disordered expression systems, the Ras signaling platform regulated membrane-tethered ERK2, whereas LCK-RasV12 just af- ERK1/2selectivitytowarddifferentsubstrates. fectedlipidraft-boundERK2,therebycorroboratingtheirspa- SublocalizationdictatesERK1/2substrateselectivityunder tialspecificity. physiologicalstimulation.Subsequently,weaskedwhetherfor These constructs were transfected in 293T cells that were externalstimuliactingupstreamofRas,ERK1/2alsoexhibited then treated with different agonists. Interestingly, the activa- a localization-dependent substrate selectivity. To this end, we tionofERK2atthesemicrodomainsfolloweddifferentkinet- engineeredAU5-taggedERK2constructsspecificallytethered ics depending on the stimulus. At lipid rafts, upon treatment to disordered membrane and to lipid rafts (Fig. 4A), from withEGF,thephosphorylationofERK2reachedarapidmax- which the activations of RSK1 and EGFr were, respectively, imum after just 2 min and faded progressively afterwards. regulated.SuchconstructswouldenableustomonitorERK2 Conversely, at the disordered membrane, ERK2 exhibited a 1344 VOL.29,2009 SPATIAL REGULATION OF ERK1/2 SUBSTRATE SPECIFICITY 1345 slower phosphorylation course, with a late peak 10 min after stimulation that was still apparent after 30 min (Fig. 4C). Stimulation with LPA evoked a similar pattern of activation: the activation of ERK2 at the disordered membrane lagged behindthatonetakingplaceatlipidrafts.Interestingly,when cellsweretreatedwiththephorbolesterTPA,ERK2followed similarpatternsofactivationatbothlocalizations,withrobust peaks5minafterstimulationthatweresustainedafter30min. WethenexaminedifthekineticsofERK2activationatlipid raftsanddisorderedmembranecorrelatedwiththeactivation patterns exhibited by those substrates specifically regulated fromeachoftheselocalizations,namely,EGFrandRSK1.In a physiological setting, such as parental 293T cells stimulated withLPA,thephosphorylationofEGFrdisplayedamaximum after 5 min, decreasing thereafter (Fig. 4D). Noticeably, this activationcoursewassimilartotheoneexhibitedbyERK2at FIG. 6. RSK1 and cPLA occupy distinct cytoplasmic microdo- lipid rafts upon LPA stimulation (Fig. 4C). In the same fash- 2 mains.ImmunogoldelectronmicroscopywasperformedonNIH3T3 ion,thephosphorylationofRSK1inducedbyEGFdisplayeda cells.ThesecondaryantibodiesdisclosingRSK1(asterisks)andcPLA 2 late onset (Fig. 4D). Remarkably, RSK1 activation paralleled (arrows) were labeled with 15-nm and 10-nm gold particles, respec- EGF-induced ERK2 activation at the disordered membrane. tively.Scalebar(cid:9)100nm. More interestingly, the RSK1 activation pattern completely changed when the activating stimulus was TPA (Fig. 4D). In this case, its kinetics resembled that of TPA-induced ERK2 activation at disordered membrane, with an early peak and cPLA andRSK1werelabeledwithgoldparticlesofdifferent 2 sustained activation. These results added further support to diameters. Interestingly, instead of appearing intermixed and the notion that, regardless of the activating stimulus, the mi- randomlyscatteredthroughoutthecytoplasm,bothcPLA and 2 croenvironment in which ERK1/2 are activated dictates their RSK1 formed clusters at specific sites, clearly separated from substrateselectivity. eachother(Fig.6andTable1).Thiswasnotageneralfeature cPLA and RSK1 occupy distinct subcytoplasmic domains. forallcytoplasmresidents,sincebothcPLA andRSK1were 2 2 Wewishedtounderstandhowtwosubstratesthat,supposedly, intermixedwithanothercytoplasmicproteinsuchasubiquitin shared the same subcellular compartment were distinctively (data not shown). These results suggested that depending on selected by ERK1/2 activated at different localizations. We the membrane localization from which the Ras signal ema- hypothesizedthateventhoughRSK1andcPLA bothresideat nated,activatedERK1/2weredirectedtodefinedsubcytoplas- 2 the cytoplasm, they could be present at different microdo- miccompartmentswheredifferentsubstratesresided. mains.Totestthispossibility,weexploredthelocalizationsof ERK1/2site-specificsubstrateselectivityentailsdistinctus- RSK1andcPLA byimmunofluorescence.Interestingly,even age of scaffold proteins. It was important to understand the 2 thoughthesetwoproteinsexhibitedclearcytoplasmicstaining, molecular mechanisms underlying ERK1/2 site-dependent hardly any colocalization was apparent, suggesting that they substrate specificity. Since some scaffold proteins have been could be occupying different sites within the cytoplasm (Fig. shown to regulate ERK1/2 signals in a localization-regulated 5A, profile A). Next, we examined the colocalization of these fashion (17), we investigated their implication in substrate two substrates with activated ERK1/2 in cells in which the specificity. To this end, we first analyzed the activation of activating Ras signal originated at different sites. In cells ex- severalERKsubstrateswhentheexpressionofKSR1,ascaf- pressingRasV12atlipidrafts,phosphorylatedERK1/2mark- foldinvolvedinERK1/2signalingorchestratedfromlipidrafts edlycolocalizedatthecytoplasmwithcPLA ,inparticularin (22),wasdownregulatedbysiRNAinterferencein293Tcells. 2 the perinuclear region and at the cellular periphery (Fig. 5B, Whereas the reduction of KSR1 levels forestalled EGF-in- profileB),butverylittlecolocalizationwithRSK1wasevident ducedcPLA activationbymorethan75%,RSK1phosphory- 2 (Fig.5C).Conversely,incellsexpressingRasV12atthedisor- lationwascompletelyunaltered(Fig.7A).Furthermore,grad- dered membrane, a profuse colocalization of phosphorylated ually increasing the amount of KSR1 that was transfected ERK1/2withRSK1butnotwithcPLA wasapparent(Fig.5D resultedinabiphasiccPLA activationpatterninresponseto 2 2 andE).Westudiedthemicrolocalizationofthesesubstratesin EGF stimulation, typical of scaffold proteins, whereas RSK1 further detail by immunogold electron microscopy, in which activation was, again, unaffected (Fig. 7B). These results sug- FIG. 5. RSKandcPLA arelocatedindistinctsubcytoplasmicmicrolocalizations.(A)RSKandcPLA donotcolocalize.GrowingNIH3T3cells 2 2 werecostainedwithanti-RSKandanti-cPLA antibodiesandvisualizedbyimmunofluorescenceandconfocalmicroscopy.(BandC)cPLA butnot 2 2 RSK1colocalizeswithphospho-ERK(p-ERK)inNIH3T3cellsstablyexpressingLCK-RasV12.Inset,detailofthesublocalizationatthecytoplasm (thick arrow). (D and E) RSK1 but not cPLA colocalizes with phospho-ERK in NIH 3T3 cells stably expressing CD8-RasV12. To highlight the 2 cytoplasmicsignals,thecellswerenotpermeabilized(exceptinpanelB),andthenuclearsignalsarequenched.Pseudocolorswereredandgreenand asindicated;yellowindicatesamerge.Scalebar(cid:9)10(cid:3)M.ProfileA,pseudocolorintensityprofile,acrossthesectionshowninpanelA.Noticethatthe colocalizationofpeaksisminimal.ProfileB,pseudocolorintensityprofileataPMlocalization(thinarrow)showninpanelB. 1346 CASAR ET AL. MOL.CELL.BIOL. TABLE 1. RSKandcPLA arelocatedindistinct tents, we then investigated whether KSR1 would couple 2 subcytoplasmicmicrolocalizationsa ERK1/2 to the activation of cPLA , irrespective of the site 2 where the activating Ras signal was generated. For this, we Distancebetweengold Particles Pvalue particles(nm) evaluated cPLA activation in 293T cells transfected with 2 cPLA-cPLA 8.21(cid:7)1.21 (cid:10)0.5 RasV12, tethered to lipid rafts, the ER, and the disordered RSK12-RSK12 8.02(cid:7)1.88 (cid:10)0.5 membrane,plusasiRNAagainstKSR1.Itwasfoundthatthe cPLA-RSK1 160.82(cid:7)13.08 (cid:8)0.005 downregulationofKSR1expressionaffectedonlycPLA acti- 2 2 vationelicitedfromlipidraftsandnotthatresultingfromRas aDistancebetweenRSK1andcPLA particles.Immunogoldelectronmicros- copywasperformedwithNIH3T3ce2lls.Thesecondaryantibodiesdisclosing activation at the ER or at the disordered membrane (Fig. RtivSeKly1. aRnedsuclPtsLAsh2owwetrheeladbiestlaendcewsithbe1tw5-enemn agnodld1p0a-nrtmiclgeosldasptahreticalevse,rargeespse(cid:7)c- 10A), thereby demonstrating that KSR1 exhibited both sub- strateandlocalizationspecificity. standard errors of the means of six sections. Student’s t test highlights the significanceofthedistancebetweenRSK1andcPLA. Itwasofinteresttoidentifythescaffoldproteinthatcoupled 2 ERK to cPLA activation when the Ras signal was coming 2 from the ER. To do so, we analyzed the activation of cPLA 2 gested that KSR1 was the scaffold utilized by ERK1/2 for under conditions where the availability of different scaffold activatingcPLA butnotRSK1. proteins was reduced. The 293T cells were transfected with 2 Following this argument, we examined whether KSR1 cou- ER-targetedRasV12andsiRNAsagainstasetofwell-known pledERKandcPLA activationsinasite-dependentfashion. ERK1/2scaffolds,namely,dystroglycan,IQGAP1,KSR1,(cid:5)-ar- 2 Todoso,weanalyzedthecompositionoftheKSR1complex restin, MORG, MP-1, paxillin, and Sef. These siRNAs effi- uponthereceiptofRassignalsoriginatedatdifferentlocaliza- ciently downregulated the expression of all the scaffold pro- tions.Itwasfoundthatthelevelsoftotalandphosphorylated teinstested(8),butonlythedropinSefexpressionlevelshad ERK1/2 that assembled to endogenous KSR1 markedly in- a negative impact on cPLA activation induced by ER Ras 2 creasedincellsstimulatedwithEGFandinthosetransfected (Fig.10B).Moreover,increasingconcentrationsofectopically with lipid raft-associated RasV12 but not in those expressing expressed Sef brought about a biphasic response for cPLA 2 RasV12tetheredtothedisorderedmembrane(Fig.7C).These activation(Fig.10C),pointingtoSefhavingascaffoldfunction results were ascertained in the entirely physiological back- in this process. We next asked if Sef would also intervene in groundofferedbytheH/N-RasnullMEFsandunderectopic cPLA activation as regulated from other sublocalizations. In 2 stimulation. Whereas in wild-type MEFs EGF induced an in- this respect, cPLA activation induced by lipid raft-targeted 2 crease in the total and phosphorylated ERK1/2 bound to en- RasV12wascompletelyunaffectedbythesiRNA-induceddrop dogenousKSR1,inH/N-Ras(cid:2)/(cid:2)MEFs,devoidofRasatlipid inSefexpression(Fig.10D).Furthermore,whenweanalyzed rafts,thelevelsoftotalandactivatedERK1/2whichassociated the composition of Sef complexes upon the receipt of Ras withKSR1uponEGFstimulationwhereunchanged(Fig.7D). signals originated at different localizations, cPLA associated 2 Most interestingly, endogenous cPLA was completely absent withSefinanactivatedERK-dependentfashionincellstrans- 2 from KSR1 immunoprecipitates under resting conditions, but fectedwithER-boundRasV12butnotinthoseexpressinglipid significantproportionsofcPLA werefoundtoassociatewith raft-associatedRasV12(Fig.10E).Inconclusion,theseresults 2 KSR1 in cells stimulated with EGF or transfected with lipid indicatedthatSefwasthescaffoldproteinspecificallyregulat- raft-associated RasV12. As we had previously demonstrated ing cPLA activation by ERK1/2 responding to Ras signals 2 (8),theinteractionbetweencPLA andKSR1wascompletely comingfromtheER. 2 dependent on ERK1/2 activation, since preincubation with Our results had demonstrated that Ras signals from lipid UO126 completely inhibited cPLA binding to KSR1 (Fig. rafts regulated the phosphorylation of two ERK substrates, 2 7E).Ontheotherhand,RSK1wasfoundnottoassociatewith cPLA and EGFr, and that KSR1 was the ERK scaffold me- 2 KSR1,eitherincellstransfectedwithH-RasV12orinresponse diating in cPLA activation. As such, we questioned whether 2 toEGFstimulation(Fig.7E).Inasimilarfashion,inwild-type KSR1 also participated in ERK1/2 signals directed to EGFr, MEFs,EGFinducedamarkedincorporationofcPLA tothe thus behaving as a “general” lipid raft scaffold. Remarkably, 2 KSR1 complex, whereas in H/N-Ras(cid:2)/(cid:2) MEFs, the levels of EGFr phosphorylation resulting from Ras activation at lipid cPLA which associated with KSR1 upon EGF stimulation rafts was unaffected by the downregulation of KSR1 levels 2 whereminimal(Fig.7F). (Fig.11A),speakingagainstKSR1havingascaffoldroleinthe Theseresultswerefurthersubstantiatedbyimmunofluores- activation of this substrate. Accordingly, we looked for the cencestudies,inwhich,inwild-typeMEFs,cPLA wasshown scaffold mediating in this event by assaying the phosphoryla- 2 to profusely colocalize with KSR1 at PM localizations in re- tionofEGFr,asinducedbylipidraft-tetheredRasV12,under sponsetoEGFstimulation(Fig.8AandB,arrows),whilesuch limitinglevelsofscaffoldproteins,broughtaboutbytheutili- interactionwaslostinH/N-Ras(cid:2)/(cid:2)MEFs(Fig.8CandD).An zationofthesiRNAbatterymentionedbefore.Inthiscase,it identical situation was observed in biochemical studies evalu- wasfoundthatadropinIQGAP1expressionevokedamarked atingEGF-inducedKSR1andcPLA associationwiththePM, reduction in EGFr phosphorylation (Fig. 11B). Further, in 2 extractedfromthesetwotypesofMEFs(Fig.9).Overall,these cellstransfectedwithlipidraft-tetheredRasV12,IQGAP1was results supported the notion that KSR1 coupled activated foundinassociationwiththeEGFr(Fig.11C),therebypoint- ERK1/2selectivelytoitssubstrate,cPLA ,inresponsetosig- ing to IQGAP1 as the scaffold participating in the lipid raft- 2 nalsgeneratedatlipidrafts. inducedERKsignal,directedtothisparticularsubstrate.Since In light of our data demonstrating that cPLA could be Rassignalsderivedfromlipidraftsalsoregulatedtheactivationof 2 activated from distinct Ras platforms, though to different ex- cPLA ,wetestedwhetherIQGAP1couldalsointerveneinthis 2 VOL.29,2009 SPATIAL REGULATION OF ERK1/2 SUBSTRATE SPECIFICITY 1347 FIG. 7. KSR1couplesRassignalsfromlipidraftstocPLA activation.(A)DepletionofKSR1preventscPLA butnotRSK1activation.293Tcells 2 2 weretransfectedwith((cid:1))orwithout(-)asiRNA(10nM)forKSR1andstimulatedwithEGF(10ng/ml,5min)afterstarvation.EndogenouscPLA 2 activation was determined by [3H]arachidonic acid release. Bottom panels show total and phosphorylated RSK1 (p-RSK1), and KSR1 levels were determined by immunoblotting. (B) Biphasic activation of cPLA resulting from increasing KSR1 levels. Cells were transfected with increasing 2 concentrationsofFLAG-KSR1andstimulatedwithEGFafterstarvation.BottompanelsshowtotalandphosphorylatedRSK1,andKSR1(FLAG)levels were determined by immunoblotting. (A and B) Shown are the averages (cid:7) standard errors of the means of the results from three independent experimentsrelativetothelevelsinstarved(C)cells.(C)ERK1/2-KSR1associationisenhancedbyRassignalsfromlipidraftsbutnotthedisordered membrane.EndogenousERK1/2,totalandphosphorylated,presentinnativeKSR1immunoprecipitateswasdeterminedincellstransfectedwithRasV12 (0.5(cid:3)g)tetheredtolipidrafts(LCK)ortothedisorderedmembrane(CD8)orstimulatedwithEGF.(D)ERK1/2-KSR1associationisenhancedin wild-typebutnotinH/N-Ras(cid:2)/(cid:2)MEFs.ERK1/2,totalandphosphorylated,presentinKSR1immunoprecipitateswereanalyzedinMEFs,starved(C)or EGFstimulated.(E)TheassociationofKSR1andendogenouscPLA isenhancedbyEGFstimulationandbyRassignalsfromlipidraftsandisinhibited 2 byUO126(2(cid:3)M,25minpriortostimulation).Bottompanels,RSK1doesnotassociatewithKSR1inresponsetoRassignalscomingfromlipidrafts ortoEGFstimulation.(F)TheassociationofKSR1andendogenouscPLA inresponsetoEGFisdiminishedinH/N-Ras(cid:2)/(cid:2)MEFscomparedto 2 wild-typefibroblasts.TL,totallysate;IP,immunoprecipitationsperformedwithaspecificantibody;PI,immunoprecipitationsperformedwithpreimmune serum;(cid:6),anti. process.ItwasfoundthatthesiRNA-mediateddepletionofIQ- tion. Interestingly, the levels of RSK1 phosphorylation re- GAP1 levels did not affect whatsoever cPLA activation as in- mainedunalteredinallcases(Fig.11E),suggestingthatnone 2 ducedbyRasV12actingatlipidrafts(Fig.11D). of the tested scaffold proteins participated in the disordered Subsequently, we set out to find the scaffold protein medi- membrane-evokedERKsignaltargetingRSK1.Overall,these atingthephosphorylationofRSK1,asinducedbyRassignals results supported the notion that Ras localization within dif- flowing from the disordered membrane. With this aim, once ferent membrane microdomains could dictate ERK1/2 sub- again, we utilized the set of siRNAs previously described in stratespecificityandthatsuchaneffectwaspossibleduetothe cells expressing RasV12 tethered to this specific sublocaliza- selectiveparticipationofdefinedscaffoldproteins.
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