The Pennsylvania State University. The Graduate School. Eberly College of Science THE ROLE OF R RAS IN ARF6 ACTIVATION DOWNSTREAM OF THE HGF C MET

Transcription

1 ThePennsylvaniaStateUniversity TheGraduateSchool EberlyCollegeofScience THEROLEOFR RASINARF6ACTIVATIONDOWNSTREAMOFTHEHGF C MET SIGNALTRANSDUCTIONPATHWAY AThesisin Biochemistry,Microbiology,MolecularBiology by HeatherPursel 2011HeatherPursel SubmittedinPartialFulfillment oftherequirements forthedegreeof MasterofScience May2011

2 ThethesisofHeatherPurselwasreviewedandapproved*bythefollowing: LorraineSanty AssistantProfessorofBiochemistryandMolecularBiology ThesisAdvisor MelissaRolls AssistantProfessorofBiochemistryandMolecularBiology PamHankey ProfessorofImmunology ScottB.Selleck ProfessorandHead, DepartmentofBiochemistryandMolecularBiology *Signaturesonfileinthegraduateschool ii

3 ABSTRACT Epithelialcellsgrowassheetsthatfunctionasaselectivelypermeablebarrierthatis sealedtogetherwithanextensivenetworkofjunctions.undernormalcircumstances,this networkforcesthecellstobenon motile.however,itispossibleforthesecellstobecome motileduringembryonicmorphogenesisandthemetastasisofepithelialcancers.this movementispromptedbyavarietyofgrowthfactorssuchashgfthatcancauseepithelial celllinescatteringandmotilityinvitro.thebindingofhgftoitsreceptorc Methasthe abilitytostimulatetheactivationofpi3 kinaseandthesmallgtpaseras.rasandother subfamilymemberssuchasr rashavebeenknowntoplayaroleinproliferationandcell survivalaswellascellmigrationandmorphology.thesmallgtpasearf6,whichregulates actincytoskeletalorganizationandcanbeactivatedbyhgf,hasalsobeenassociatedwith, andisrequiredfor,epithelialcellmovement.whileitisknownthatr RasandArf6are bothrequiredforepithelialmotility,thesignalingpathwaylinkingthesecomponentstocell shapechangesremainsunclear.therefore,wehypothesizethatr rasandarf6are downstreamofthehgf cmetsignaltransductionpathwaytoregulateepithelialcell migration. TheinvolvementoftheRasrelatedprotein,R Ras,withinthispathwaywasinitially investigatedviaamorphologicalstudy.r rasactivitywasalteredusingeitherdominant negativeorconstitutivelyactivemutantsandcellmorphologyandmigrationwas monitored.areductioninr rasactivityinhibitedcellspreadingandscatteringafter stimulationwithhgfwhileconstantr rasactivityledtocellscatteringwithoutfurther additionofhgf,ashadbeenpreviouslyshown.themigrationpromotedbyactiver ras iii

4 wasinhibitedbytheadditionofsecinh3,acytohesininhibitor.theseobservationswere furthersupportedbywesternblotanalysisofarf6activationinthemutantexpressing cells.adecreaseinr rasactivityreducedarf6activationinthepresenceofhgf.the constantactivationofr rasalsocausedadecreaseinarf6activationinthepresenceof HGF.Together,thisdatashowsthatR RasisdownstreamoftheHGF c Metsignal transductionpathwayandhastheabilitytoaltercellshapeandmigration. iv

5 TABLEOFCONTENTS ListofFigures.vii ListofAbbreviations..ix Acknowledgments...x CHAPTER1:INTRODUCTION EpithelialCellGrowthandStructure 1 CellMigration..2 HGFandc MetReceptor...5 HGFandMDCKCells...6 TheSmallGTPaseRas 7 TheRasIsoforms..9 TheRasFamilyGTPaseRelated Ras(R ras)...10 TheSmallGTPaseArf6..11 TheArf6GEFS.12 ConnectingR rasandarf6..15 v

6 Hypothesis AimsofStudy..17 SpecificAim1.17 SpecificAim2.18 CHAPTER2:MATERIALSANDMETHODS CellCultureandTransfectionMethod 19 Antibodies,Plasmids,andReagents.19 Immunofluorescence.20 Arf6PulldownAssay..20 CHAPTER3:TheinvolvementofR RasintheHGF inducedarf6activationsignal transductionpathway...22 Experiment1:UseofT43NR rasmutant...23 Experiment2:UseoftheG38VR rasmutant...25 vi

7 CHAPTER4:ThemorphologicaleffectsofR Rasoncellularmotility...28 MorphologicalStudyPart1 EffectofT43NR ras...29 MorphologicalStudyPart2 EffectofG38VR ras...34 InhibitionofG38V inducedmigrationbysecinh CHAPTER5:DISCUSSION...38 REFERENCES...46 vii

8 ListofFigures Chapter1:Introduction 1.1:EpithelialCellMigrationona2 Dsubstrate 1.2:IntroductiontoGTPases 1.3:Arf6GuanineNucleotideExchangeFactors(GEFs) Chapter3:TheinvolvementofR RasintheHGF inducedarf6activationsignal transductionpathway 3.1:TheproposedsignalingpathwayleadingfromHGFtoactiveArf6 3.2A:DominantnegativeR Ras(T43N)decreasesArf6activation 3.2B:DominantnegativeR Ras(T43N)decreasesArf6activation 3.3A:ConstitutivelyactiveR ras(g38v)increasesarf6activationinthepresenceof HGF 3.3B:ConstitutivelyactiveR ras(g38v)increasesarf6activationinthepresenceof HGF Chapter4:ThemorphologicaleffectsofR Rasoncellularmotility 4.1A:DominantnegativeR Ras(T43N)inhibitscellspreadingandscatteringin MDCKcells(0hours) 4.1B:DominantnegativeR Ras(T43N)inhibitscellspreadingandscatteringin MDCKcells(4hours) viii

9 4.1C:DominantnegativeR Ras(T43N)inhibitscellspreadingandscatteringin MDCKcells(18hours) 4.2:DominantnegativeR ras(t43n)inhibitstypicalcellmigrationinthepresence ofhgf 4.3:DominantnegativeR ras(t43n)inhibitscellscatteringafter18hoursofhgf incubation 4.4:ConstitutivelyactiveR ras(g38v)caninducecellspreadingandscatteringin theabsenceofhgf 4.5:Thecytohesininhibitor,SecinH3,reducescellmigrationinducedby constitutivelyactiver ras. Chapter5:Dicussion 5.1:NewlyproposedpathwayleadingfromHGFtoArf6activation ix

10 ListofAbbreviations EMT EpithelialtoMesenchymaltransition GTPase GuanosineTriphosphatase HGF/SF HeptaocyteGrowthFactor/ScatterFactor PI3 K Phosphotidylinositide3 kinase MDCK MadinDarbyCanineKidneycells GTP Guanosinetriphosphate GDP Guanosinediphosphate GEF Guaninenucleotideexchangefactor GAP GTPaseactivatingprotein Arf6 ADP ribsoylationfactor6 ARNO ARFnucleotidebindingsiteopener x

11 ACKNOWLEDGEMENTS IwouldfirstandforemostliketothankDr.LorraineSantyforallofherinvaluable guidanceandsupport.withoutherhelp,iwouldneverhavebeenabletofinishthisdegree andstillhavemysanity.iwouldalsoliketogiveabigthank youtopastandpresentsanty labmembers:joe,myriam,seungja,dave,andmike.thesefivepeopleweregreatfriends andlabmates.tomymom,kathy,mydad,rickandtherestofmyfamily,thankyoufor yourencouragementeventhoughyoureallyhavenoideawhatidoatworkeveryday.to mybrother,sean,youmademylifemucheasierinstatecollege.andtomyboyfriend, Randy,youcouldalwaysmakemesmile,evenfrom200milesaway.Thankyouforyour unendingloveandsupport. xi

12 CHATPTER1 INTRODUCTION EpithelialCellGrowthandStructure Epithelialcellsgrowassheetsconnectedthroughasystemofcell to cellandcell tomatrixjunctions.thesesheetsactasaselectivelypermeablebarrier,separatingthe compartmentsofthebody.thishighlyorganizedbarrierallowsforthepolarizationofthe cellandthereforetransportbetweencompartments(balkovetz1998).occludingand adheringjunctionsaretwomaintypesofadhesiveelementsresponsibleforthistissue architecture.occludingjunctions,suchastightjunctions,formcell to cellcontactsand createtheselectivelypermeablebarrier.adheringjunctions,suchascadherinsand integrins,formcontactsbetweenadjacentcellsandtheextracellularmatrix,whichis crucialinmaintainingtissueintegrity(gumbiner1996). Thistypeofstructureandorganizationusuallyleavestheepithelialcellsimmobile. However,duringembryonicmorphogenesis,tissuerepairandregenerationcellscan becomemotile.embryonicdevelopmentiscoordinatedbycellmigrationwherecollectively migratingcellsformtheembryoandsubsequenttissuesandorgans(ridley,schwartzetal. 2003).Formatureepitheliatobecomemotile,thesheetofcellsmustbetriggeredtomove byachemotaticfactorsuchashepatocytegrowthfactor.thesefactorsbindthroughcell surfacereceptorsandtranducesignals,whichinduceactincytoskeletonrearrangements, activatevesiculartransportandpromotere polarization. 1

13 2DCellMigration Theearlieststagesofepithelialcellmigrationcanbeseeninadevelopingembryo duringcompactionoftheblastula.fortheformationofmorecomplexbodystructures, epithelialcellsmusttransitionfromtheirrestrainedphenotypetoonethathasan enhancedmigratorycapacityandincreasedinvasivenesssuchasthatofthemesenchymal cell.thesetwocellphenotypesareahallmarkofnormaldevelopmentandallowfora highlydynamiccellularenvironment.theconversionbetweenepithelialandmesenchymal cellsisaprocesscalledepithelialtomesenchymaltransition(emt)(kalluriandweinberg 2009).EMTstartswiththedissolutionoftight,adherens,andgapjunctions.Thisallowsfor themixingofapicalandbasolateralmembranecomponentsandthedegradationofthe basementmembrane.next,cellsurfaceproteinssuchase cadherinandepithelial specific integrinsarereplacedbyn cadherinandamoretransienttypeofintegrinspecificto mesenchymalmigration.thisdissolvesconnectionswithneighboringcellsandthe basementmembrane.eventually,thecellwilltakeonaspindlelikeshapeandforegoits cuboidal,epithelialshape.theupregulationofn cadherinandextracellularcomponent bindingintegrinsdrivesthecelltomigrateinthedirectionofextracellularcuesandaway fromtheepitheliallayerinwhichitwasderived(kalluriandweinberg2009;micalizzi, Farabaughetal.2010). Thefirststepinsinglecellmigration,whichoccursafterthecompletionoffullEMT, istheformationofamembraneprotrusionattheleadingedge(rorth2009).thisfingerlike protrusionispushedforwardbypolymerizedactin,whichhasbeenrearrangedby activatedsmallgtpasesracandcdc42.theprotrusioncanbealarge,broadlamellipodia 2

14 oraspike likefilopodia.next,theprotrusionwilladheretotheextracellularmatrixfor stability.thenewlyformedadhesionsserveasfootingforthecellasitmovesforwardand eventuallymatureintofocaladhesions.anothersmallgtpase,rho,thenregulatesthe translocationofthecell.acto myosinfilamentsareassembledandcontract,propellingthe cellbodyinthedirectionofmovement.finally,thetrailingendofthecellwillretractby disassemblingcell substrateadhesivestructuresandthemigrationcyclecancontinue (Ridley,Schwartzetal.2003;Yamazaki,Kurisuetal.2005).Thisprocesscanbeseenin Figure1.1. 3

15 Figure1.1:EpithelialCellMigrationona2 Dsubstrate.Migrationconsistsoffour consecutiveprocesses:protrusion,adhesion,translocation,andretraction.allfourare requiredforprogressivecellmovement(adaptedfromyamazaki,kurisuetal.2005). EMTprovidestheflexibilitytotransitionbetweencelltypes,whichisrequiredfor singlecellmigration.however,itisnotalwaysnecessarytoundergoafullepithelialto mesenchymaltransitiontomigrate.epithelialplasticityrequiressomedisruptionofthe epithelialtissuesfixedpositionandtheadoptionofsomemesenchymalproperties.an exampleofaprocessthatdoesnotrequireafullepithelialtomesenchymaltransitionis woundhealing(micalizzi,farabaughetal.2010).thisisatypeofcollectivecellmigration 4

16 inwhichthecellsoneitheredgeofaninjurymigratetowardseachotherinordertoclose theopenwound.collectivecellmigrationisadvantageousinthissituationbecauseit allowsthetissuetostaycontinuousduringtheremodelingprocessandhelpsretainthe tissueintegrity(rorth2009).becausethecellsaremigratingasacollectivegroup,they maintaincell cellcontactsanddonotexperienceafullemt.however,theleadingedge cellsdisplaymesenchymalpropertiessuchasdevelopingacellprotrusion,whichexhibits directionalcellmovement.theleadingedgecellsalsoloseapical basalpolaritybutmanage tomaintainthebasicepithelialphenotypeandcompletelyretainthisoncethewoundis closed(micalizzi,farabaughetal.2010). ThoughtheprocessofcellmigrationandEMTarecriticalduringdevelopmentand wouldhealing,propertiesofeachhavebeenconnectedtocancermetastasis.whilethe processofmetastaticmigrationisnotaswell defined,manyhumancancershavethebasic featuresofemtandinappropriatelyexpressregulatorsofthiscellulartransitionandcell motility.cancerouscellshavetheabilitytoreactivateepithelialdevelopmentalprograms andmanipulateenvironmentalcuestoincreaseinvasionandmigration,whichwillleadto metastaticspread(micalizzi,farabaughetal.2010). HGFandc Metreceptor Aknownregulatorofepithelialplasticityandparticularlypotentepithelialmotogen ishepatocytegrowthfactororscatteringfactor(hgf/sf)(birchmeier,birchmeieretal. 2003).Thispolypeptidegrowthfactorcannotonlyinducecellmotilitybutcanstimulate 5

17 mitogenesisandthedevelopmentoftissues(balkovetz,pollacketal.1997).synthesizedas asinglechain,whichislargelyinactive,theproteinislaterconvertedproteolyticallyintoa 2 chain,activeheterodimer.theamino terminalfragmentofthismolecule,nk1,isthe mainreceptor bindingsiteandcausesreceptordimerization.c Met,theonlyreceptorfor HGF,isareceptortyrosinekinasethatfunctionsasaheterodimer(Birchmeier,Birchmeier etal.2003).thistransmembranereceptoriscomprisedofanαchainthatisexposedatthe cellsurfaceandβchainthatspanstheplasmamembrane.bothchainsarerequiredforhgf bindingwhichinturncausesdimerizationandtheautophosphorylationofthereceptoron tyrosines1349and1356(stellaandcomoglio1999).oncephosphorylated,bothsitesare abletobindsubstratessuchasthegrb2adaptorforsos,thegab1multiadaptorprotein, phosphotidylinositide3 kinase(pi3 K)andothers.TheseproteintransducerscontainSrchomology2domainbindingsitesandcanmediateinteractionbetweendownstream effectors.therearetwomajorcascadesactivatedfollowingc Metactivation:thePI3 Kand Ras/MAPkinasepathways.Couplingitsp85subunittothec Metreceptoractivatesthe PI3 KpathwayandtheGrb 2adaptorproteinlinksc MettoSOStoactivatetheRas/MAP kinasepathway(zhangandvandewoude2003). HGFandMDCKcells c MetactivationstimulatedbyHGFbindingcaninduceseveraldifferentmotogenic andmorphogenicevents,particularlyinthemadin Darbycaninekidney(MDCK)epithelial cellline.whentreatedwithhgf,cellsinculturewillexperienceadisruptionin 6

18 intracellularjunctions,takeonafibroblastmorphology,andscatterawayfromcolonies hencethename scatterfactor (Stoker1989).MDCKcellsgrownwithinacollagenmatrix willalsoundergocellshapechangeswhentreatedwithhgf.notonlywillmdckcells becomemoreinvasiveinthepresenceofhgfbuttheywillalsoformbranchingtubules (Weidner,Behrensetal.1990;Montesano,Matsumotoetal.1991).Researchhasalso shownthatbothpi3 KandMAPkinasemustbeactivatedforHGFtoinducescatteringand branchingtubulogenesisinmdckcells.(khawaja,lehmannetal.1998;potempaand Ridley1998).TheirresponsivenesstoHGFinadditiontotheabilitytoformacontinuous, polarizedmonolayerinculturearecharacteristicsthatmakemdckcellsaconvenient modelinthestudyofepithelialmotility. TheSmallGTPaseRas AkeycomponentintheactivationoftheMAPkinaseandPI3 Ksignalingpathways isras.rasisasmallguanosinetriphosphatase(gtpase),whichactsasamolecularswitch, cyclingbetweenanactiveandinactiveform(figure1.2).whileintheactiveform,rasis boundtogtpandwaspromptedintothisstatebyregulatoryproteinscalledguaninenucleotideexchangefactors(gefs).thisproteinfacilitatestheexchangebetweengdpand GTP.ItisintheactiveformthatRascaninteractwithdownstreameffectors(Wennerberg, Rossmanetal.2005).Forcyclingtocontinue,aGTPaseactivatingprotein(GAP)mustact onthegtpaseandhelptoincreasetherateofgtphydrolysis.thiswillallowthe hydrolysisofgtptogdpandpromotetheformationoftheinactiveform.another 7

19 importantbiochemicalfeatureofrasisitslipidmodification.thec terminalendofrasis terminatedwithacaaxmotif,whichisasequencethatdeterminesthetypeofinteractions indifferentmembranecompartments(brownandsacks2009). Figure1.2:IntroductiontoGTPases.SmallGTPases,suchasRasandArf6,areproteins thatcyclebetweenanactiveandinactivestate.duringtheiractivestate,theproteinis boundtogtp.duringtheirinactivestate,theproteinisboundtogdp.onceintheactive state,thegtpboundproteinisfreetointeractwithandactivatedownstreameffectors. ThebestcharacterizedandmoststudiedRassignalingpathwayistheMEK/ERK cascade.ras GTPbindstoandrecruitsRafkinasetotheplasmamembrane.Rafkinase becomesfullyactiveandphosphorylatesmek1/2proteinkinase.thiskinasethen 8

20 phosphorylatesandactivateserk1/2protein,whichistranslocatedtothenucleus. ActivatedERKwillphosphorylatetranscriptionfactorsandconsequentlypromote proliferationanddifferentiation(wennerberg,rossmanetal.2005). InadditiontoRas traditionallyobservedroles,italsoplaysapartincellmigration. Onceactivated,Rascanregulateadhesionmoleculesandcytoskeletalrearrangementsthat controlmigrationandinvasion.ithasbeenshownthatinhibitionofrascanpreventhgfinducedcellscatteringandadditionofactivatedrascaninducecellspreadingandactin reorganization(ridley,comoglioetal.1995).thisdatademonstratesthatrasisessential inepithelialmotilityandmorerecentstudieshaveimplicateddistinctrasisoforms responsiblefortheseeffects. RasIsoforms TherearethreedifferentRasisoforms:H Ras,K Ras,andN Ras.Thesemolecules havealmostcompletesequenceidentityinthen terminalregion,whichcontainsthe effectorinteractionsitesandnucleotidebindingregions.thehypervariableregion(hvr), locatedatthec terminalend,istheonlyareaofsequencedivergence(omerovic,laudeet al.2007).thoughtheseisoformsuseacommonsetofeffectorsandactivators,they generatedistinctoutputsandhaveindividualrolesinthecell.initialisoformknockout studiesrevealedthattheisoformsareexpressedinspecifictissuesandhavedifferentroles duringdevelopment.ithasalsobeenfoundthattheisoformscanbelocalizedtodifferent 9

21 microdomainsintheplasmamembranebecauseofadditional,distincthvrmodifications (Rajalingam,Schrecketal.2007). Thesedifferencesallowtheisoformstodisplayfunctionaldiversityduringcell migration.eachisoformhasaneffectoncellularmotility;forexample,ithasbeenshown thath Rascanpromoteproliferationanddownregulateintegrinaffinity(Zhang,Vuorietal. 1996)(Kwong,Wozniaketal.2003).K rashastheabilitytoregulatethepi3 K/AKT signalingpathwayswhilen rascanregulatethemek/erk1cascaderesponsesandneither cansubstitutetheother(liao,planchonetal.2006). TheRasFamilyGTPaseRelated Ras(R ras) ThereisalsoanothermemberoftheRassuperfamily,R ras,thathasaroleincell motility.r rasis55%identicaltoandsharesmanycommondownstreameffectorssuchas Raf1andPI3 KinasewiththeRasisoforms;however,itactsinoppositiontoallthree. UnlikeH,K,andN ras,r rascaninhibitcellproliferation,promotecellspreadingand adhesion,inducecellscatteringandfocaladhesionformation,andupregulateintegrin affinity(zhang,vuorietal.1996;khawaja,lehmannetal.1998;kwong,wozniaketal. 2003;Goldfinger,Ptaketal.2006).Inordertoperformthesetasks,R rasistargetedtothe plasmamembranewhereitcanco localizewitharf6,asmallgtpaseinvolvedin membranetrafficking,andβ1integrins.r rascanthenhelptolocalizeandgroupintegrins tospecificareasoftheplasmamembraneviaitseffectorbindingloopandcontroltheir endocytosis(oertli,hanetal.2000).thisallowsr rastoregulatetherecyclingofintegrins 10

22 andmodulatecelladhesionandspreading(furuhjelmandperanen2003;conklin,ada Nguemaetal.2010). R rascanaccomplishitsuniquecellularfunctionswiththehelpofanr rasspecific effectornamedrlip76.thisproteinwasfoundusingaproteomicscreen,whichwasused toisolateras bindingproteins(goldfinger,ptaketal.2007).thestudyrevealsthatrlip76 bindstoonlyactivatedr rasbutnotadominant negativeversionofr ras,demonstrating thatrlip76bindstor rasinagtp dependentmanner.rlip76isalsorequiredforr ras inducedcellspreadingandappearstobeadownstreameffectorofr ras(goldfinger,ptak etal.2006). Ithasbeenfoundthatcellspreadingandmigrationcanbestimulatedbyactivated R ras,whichinturncanactivatetherhogtpase,rac1(holly,larsonetal.2005).through itslocalizationtotheplasmamembrane,rac1hasthecapacitytoaltertheactin cytoskeletonandproducechangestothecellsleadingedgesuchaslamellipodiaformation (HeasmanandRidley2008).Thoughthismaybecelltypespecific,ithasbeenfoundthatRrasanditseffectorRLIP76arerequiredforadhesion inducedracactivationand directionalcellmigration(wozniak,kwongetal.2005;goldfinger,ptaketal.2006). ThesmallGTPaseArf6 AnotherimportantregulatorofRacactivityisthesmallGTPaseArf6.TheADPribosylationfactorproteinsarealsomembersoftheRassuperfamilyandaredividedinto threeclasses.classiarfsincludearf1,arf2,andarf3.thisclassismorethan96% 11

23 identicalandregulatestheassemblyofcoatcomplexesonvesiclesinvolvedinthe secretorypathway.classiiarfsincludearf4andarf5,whichhavearoleinearlygolgi transport(d'souza SchoreyandChavrier2006).Arf6isthesolememberofclassIIIand caninfluencemembranetraffickingandrecyclingandactincytoskeletalchangesatthe plasmamembrane.earlyresearchshowedthatarf6isrequiredfortheconversiontoa motilecell(palacios,priceetal.2001).itisnowknownthatarf6canalsoregulatecell cell adhesionbyinitiatingadownregulationofrac1activityandpromotingendocytosisofecadherintodisassembleadherensjunctionsduringtheearlystagesofmigration.asthe migrationprocessmovesforward,arf6isabletomediateendosomalrecyclingand recruitmentofrac1totheplasmamembrane,whichinducesmodificationsoftheactin cytoskeletonandinfluencestheformationofleadingedgeprojectionssuchaslamellipodia (Schweitzer,Sedgwicketal.2010).Goldfingeretal.haveshownthatRaclocalizationand activationmediatedbyarf6iscontrolledbyrlip76inthataknockdownofrlip76blocks adhesion inducedarf6activation. TheArf6GEFS ThecellularfunctionsattributedtoArf6areobviouslycomplexandinvolvemultiple sitesofactionsuggestingthattheproteinisactivatedandinactivatedinseveralareas placesattheplasmamembrane(donaldson2003;schweitzer,sedgwicketal.2010).in orderforarf6tobecomeactive,itrequiresaguaninenucleotideexchangefactor(gef), whichstimulatesgtploading.eukaryoticarfgefscanbedividedinfivedifferentfamilies 12

24 basedondomainsimilaritiesandorganization.thesefivefamiliesincludegbf/big, cytohesins,efa6,brags,andfbox(figure1.3).eachfamilyofgefscontainthesec7 domainwhichisthecentralcatalyticdomain200aminoacidslongthatisresponsiblefor nucleotideexchange.ofthefivefamilies,arf6isknowntointeractwiththreeofthem: EFA6,BRAGs,andcytohesins. ExchangefactorforArf6(EFA6)wasthefirstArf6 specificgefidentified.the generalstructureoftheefa6familyisasec7domain,aphdomainresponsibleforplasma membranelocalization,andacoiled coildomain,whichisinvolvedinactincytoskeletal remodeling(franco,petersetal.1999).theexpressionofefa6isaccompaniedbythe reorganizationofcorticalactin,formingmembranerufflesviatheactivationofrac1.these resultsindicatethatefa6iscoordinatingmembraneandactinremodelingbycatalyzing theexchangeofgdpforgtpthereinactivatingarf6andrac1(franco,petersetal.1999). EFA6alsoplaysaroleincellpolaritydevelopment,however,boththeArf6 specific nucleotideexchangeandcoiled coildomainarenecessaryintightjunctionstabilization (Luton,Kleinetal.2004). Brefeldin resistantarfgefs(brag)arebestcharacterizedbythepresenceofaniqdomain,whichlikethesec7domain,iscatalyticinnatureandisadjacenttothephand coiled coildomain(casanova2007).ofthebragfamily,brag2/arfgep100hasbeen foundtopreferentiallyincreasethenucleotideexchangeofarf6andpartiallycolocalize withthisandendosomalproteineea1attheplasmamembrane.thedepletionofarf GEP100increasesB1integrinlevelsonthecellsurfaceandeffectscelladhesion.These findingsshowthatarf GEP100hasafunctionatthecellperipherybyregulatingArf6 13

25 activation,whichcancontrolendocytosisandintegrininternalization(someya,sataetal. 2001;Dunphy,Moravecetal.2006). TheARNO/cytohesinfamilycontainthecatalyticSec7homologydomain,the pleckstrinhomologydomainthatallowsformembranetargeting,andthen terminal coiled coildomain(casanova2007).cytohesin 2and 3areubiquitouslyexpressedwhile cytohesins 1and 4arefoundprimarilyinspecificcelltypes.TheseArfGEFScanbefound mainlyattheplasmamembraneandcouldberecruitedtothislocationthroughseveral differentmodels.onestudysuggeststhatcytohesinscanberecruitedviaaninteraction withphosphoinositidesandinositolphospholipidsinresponsetopi3 Ksignaling (Klarlund,Guilhermeetal.1997).However,thereisanothermodelthatsuggestsArf6 bindingisresponsibleforarnomembranerecruitment.theinteractionbetweenarno andarf6occursatthephdomainandallowsactivearf6torecruitarnototheplasma membrane(cohen,hondaetal.2007). UnliketheEFAandBRAGfamilies,thecytohesinsareratherpromiscuousintheir substratebinding.althoughitappearsthatarf6andarnocolocalizeattheplasma membranesuggestingarf6isarno sprimarysubstrate,arf1alsohasregulatingrolesat theplasmamembraneandpotentiallyinteractswitharno(santy2001).an overexpressionofarnocanovercomedefectsincellspreadingandthisrescuecanbe reversedbythereductioninarf1orarf6activityimplicatingbothgtpasesinconnectionto ARNO(Goldfinger,Ptaketal.2006).Therefore,ARNOmayactivateArf1inasubsequent GTPasecascadeimplyingthatArf6canbebothaneffectorandasubstrateofthecytohesins (Casanova2007;Cohen,Hondaetal.2007). 14

26 Figure1.3:Arf6GuanineNucleotideExchangeFactors(GEFs).Thecytohesins,EFA6, andbragfamilyareabletoexchangeaguaninenucleotidetoactivatethesmallgtpase Arf6.AllofthesefamiliescontainthecatalyticSec7domainusedduringguanineexchange (adaptedfromcasanova2007). ConnectingR rastoarf6 Thisfamilyofcytohesins,specificallyARNO,hasalsobeenimplicatedinepithelial cellmigration.arnohasbeenshowntohavetheabilitytoaltertheactincytoskeletonand changecellshape.arnooverexpressioninducescellscatteringinmdckcells,which 15

27 mimicsscatteringinducedbyhgf(santy2001).rlip76alsohasaneffectonthearf GEF ARNO.Thetwoproteinsphysicallyassociate,whichleadstoRaclocalizationand subsequentcellspreading.theknockdownofeitherrlip76orarnowassufficient enoughtoblockcellspreadingandlamellipodiaformation.thereforeitappearsthat RLIP76isthekeylinkerofR raswithadhesion inducedarf6andrac1activation (Goldfinger,Ptaketal.2006). Hypothesis WhileitisknownthatRasandARF6arebothrequiredforepithelialmotility,the signalingpathwaylinkingthesecomponentstocellshapechangesremainsunclear. Hepatocytegrowthfactor/Scatterfactorisapotentmotogen,whichcancausecell spreadingandscatteringinmdckcells.thesmallgtpaser rasisabletomimicthis spreadingandscatteringaswellasinducebranchingtubulogenesiswhentheactivated formisexpressedinthissamecelllineimplicatingitasapossibledownstreameffectorof Met.R rasalsohasaroleinadhesion inducedactivationofarf6,agtpasethatisalso downstreamofc Met;however,thetwoGTPaseshaveneverbeenlinkedviaapathway involvinghgf.therefore,ihypothesizethatr RasactivationstimulatedbyHGFwill promotearf6activationdownstreamofc Metandalterthecell sshapeandmorphology. 16

28 AimsofStudy 1. DetermineR rasinvolvementinthehgf inducedarf6activationsignal transductionpathway. 2. ObservethemorphologicalcellshapechangesstimulatedbyR rasinthe presenceandabsenceofhgf. SpecificAim1 Usingbiochemicalmethods,IwouldliketoshowthatR rashasapartinthearf6activation pathway.inordertoshowthisiwillusedifferentmutantsofr rastoinhibitoramplifythe GTPase sactivityandthenexaminethearf6activationresponsetoeach.thiswill demonstrateifr ras,thedownstreameffectoractivatedbyhgf,canstimulatearf6, establishingtheroleofthetwogtpasesinthecellmotilitysignaltransductionpathway. 17

29 SpecificAim2 IthasbeenfoundthatRas,specificallyR ras,canaltercellshapeandmorphologyin responsetohgf(khawaja,lehmannetal.1998).thisr rasinducedcellmigrationwas reproducedtosupportthefindingsinspecificaim1,showingthatr rashasaregulatory roleinnormalhgf inducedscattering.usingthemutantsofr rasdescribedinspecificaim 1,IwillshowhowtheyeachaffectMDCKcellmorphologyinthepresenceandabsenceof HGF. 18

30 CHAPTER2 MATERIALSANDMETHODS CellcultureandTransfectionMethod TheT23lineofMDCKcellsweremaintainedinDMEMsupplementedwithpenicillin, streptomycin,andfungizone,and10%fetalbovineserum.cellswereculturedat37 Cina 5%CO2incubator.TransienttransfectionofthiscelllinewasdoneusingtheNeon transfectionsystem(invitrogen)accordingtothemanufacturersinstructions.the settingwasusedtoperformtheelectroporation.TheamountofDNAusedwasasfollows: 5µgofT43NandG38VR ras,4µgofha Arf6,and2µgofpcDNA3(controlplasmid).The amountofcellsusedper6cmplatewereasfollows:1x10 6 controlcellsand2x10 6 T43NRrascellsperArf6pulldownexperiment,1X10 6 controlandg38vr rascellsperarf6 pulldownexperiment,1x10 6 foralltransfectionsduringimmunofluorescenceupto4hours and7.5x10 5 control,t43nandg38vr rascellsfor18hourimmunofluorescence.after transfection,cellswereallowedtorecoverfor18hours. Antibodies,PlasmidsandReagents Thefollowingantibodieswereusedforwesternblottingandimmunofluorescence:rabbit anti Arf6(agiftfromJimCasanova,UniversityofVirginia),mouseanti HA(Covance), mouseanti myc9e10(covance),andhorseradishperoxidasegoatanti rabbitandgoat anti mouseassecondaryantibodies(invitrogen).rhodamine conjugatedphalloidinand Alexa488conjugatedanti mouseantibodieswerealsousedduringimmunofluorescence 19

31 andpurchasedfromcovance.recombinanthumanhgfwasobtainedfrompeprotech. SecinH3wasobtainedfromCalbiochem.HA Arf6wasagiftfromJimCasanova.Myc G38V andmyc T43NR RasweregiftsfromLarryGoldfinger,UniversityofCalifornia,SanDiego. T43NR RaswassubclonedintothepCXN2vectorusingEcoR1andXba1sites.Thecontrol vectorusedinallexperimentswaspcdna3. Immunofluorescence MDCKcellsweretransfectedandplatedonaglasscoverslipscoatedwith30µg/ml fibronectin.cellswereallowedtorecoverfor18hoursandwerethenincubatedinthe presenceorabsenceof60ng/mlhgf.cellstransfectedwitht34nr Raswereincubated withhgffor0,4,and18hours.cellstransfectedwithg38vr Raswereincubatedinthe absenceofhgforpresenceof30µmsecinh3forupto32hours.afterincubation,cells werefixedwith4%paraformaldehydeinpbs,blockedwithpbscontaining10%normal goatserumand0.2%saponin,andstainedwithmouseanti mycantibodyfollowedby stainingwithalexa488conjugatedantimouseantibodyandrhodamine conjugated phalloidin.thecellswereobservedwithanolympus1x81microscopeandhamamatsu OrcaCamera.ThesoftwareusedwasSlidebook5.0(IntelligentImagingInnovations, Denver,CO). Arf6PulldownAssay MDCKcellswereincubatedinthepresenceorabsenceof30ng/mlHGFfor5 6hoursand lysedat4 Cin0.65mlof200mMNaCl,50mMTris,pH7.5,10mMMgCl2,1%TritonX 100, 0.5%sodiumdeoxycholate,0.1%SDS,5%glycerol,1mMDTT,andproteaseinhibitors. 20

32 Lysateswereclearedbycentrifugationat14,000rpmfor2minutesat4 Cinthepresence ofcl 4BSepharosebeads.Arf6 GTPwasisolatedusingglutathioneS transferase(gst) GGA3beads.0.5mloftheclearedlysatewasincubatedwiththe40µgoftheGGA3beads for30minutesat4 C.Theresultsofthisassaywereanalyzedbywesternblotting. QuatificationoftheblotswasdoneusingImageJ.LevelsofactiveArf6werenormalizedto thetotalamountofarf6inthestartinglysate.normalizedlevelsofactivearf6inr ras expressingcellsinthepresenceofhgfwasthendividedbynormalizedlevelsofactive Arf6intheabsenceofHGFincontrolcells.DifferencesinArf6activationwereanalyzedfor significanceusingapairedttest. 21

33 CHAPTER3:TheinvolvementofR RasintheHGF inducedarf6activation signaltransductionpathway. EpithelialcellmigrationistriggeredbygrowthfactorssuchasHGF.OnceHGFis boundtoitscellsurfacereceptorc Met,asignaltransductionpathwayisinitiated.The bindingofhgfandsubsequentc Metactivationcausestheactivationofdownstream effectorssuchasrasandpi3 K.Theseinteractionscantriggeractincytoskeleton rearrangements,lossofcell to cellcontactsandvesiculartransportviatheactivationof smallgtpases,suchasarf6.amemberoftherassuperfamily,r ras,alsohasapartincell migration.whileitis55%identicaltorasandinteractswithmanyofthesame downstreameffectors,r rashassomeuniquefunctionsandeffectorsdifferentfromthe traditionalformofras.r rascanhaveaneffectoncellspreadinginmdckcellsinthat activatedr rascaninducescatteringandtubulogenesissuggestingitisanimportant componentofthenormalcellularresponsedownstreamofc Met.Alloftheseresultspoint tothepossibilityofr rasandarf6actingtogetherinthehgfsignaltransductionpathway. Atpresent,thetwohavenotbeenlinkedviaHGF.Figure3.1showstheproposedpathway. Therefore,theroleofR rasintheactivationofarf6viahgfwillbeexamined.inorderto determiner ras involvement,differentmutantswereused:t43n,thedominantnegative formofr ras,andg38v,theconstitutivelyactiveformofr ras. 22

34 Figure3.1:TheproposedsignalingpathwayleadingfromHGFtoactiveArf6.The pathwayproposedshowsthatc MetcouldactivateRas,which,throughaproteinscaffold andthearf6gefarno,activatesarf6leadingtoepithelialcellmovement. EXPERIMENTONE:USEOFTHET43NR RASMUTANT Inthefirstexperiment,dominantnegativeT43NR raswasusedtodramatically decreaser rasactivityandpotentiallydisrupttheproposedpathway.thedominant negativemutantisonethatisconstantlyintheinactive,gdp boundform.thismeansthat 23

35 theexpressedmutantwillnotbeabletocycletotheactive,gtp boundstate.themutant GTPasehindersendogenousR rasactivationbysequesteringthegefsneededtocontinue cyclingbetweentheactiveandinactivestate.thoughthisdoesdramaticallyreducer ras activity,itisnotacompleteknockdownandsomeresidualendogenousr rasactivation exists.mdckcellsweretransientlytransfectedwitht43nr rasandincubatedinthe presenceorabsenceofhgffor6hours.theearliestsignsofrobustarf6activationin controlcellscanbeseenafter6hoursofincubationinhgf(figure3.2a,b).thecells transfectedwithr rasshowedmarkedlyreducedarf6activationafterincubationwithhgf (Figure3.2A,B).Therefore,theseresultsimplythatR rasplaysaroleinhgf inducedarf6 activation. Figure3.2A:DominantnegativeR Ras(T43N)decreasesArf6activation.MDCKcells weretransfectedwithmyc T43NR RasorpcDNA3(emptyvector).Transfectedcellswere allowedtorecoverfor18hoursandincubatedinthepresenceorabsenceof60ng/mlhgf 24

36 for6hours.arf6activitywasassessedusingthegst GGA3pulldownassay,whichisolates onlyactivearf6.theresultsofthisassaywereanalyzedviawesternblotdetecting endogenousarf6andmyc.3.2ashowsarepresentativegelfromapulldownexperiment quantifiedin2b. Figure3.2B:DominantnegativeR Ras(T43N)decreasesArf6activation.Activationof Arf6inMDCKcellsexpressingT43NR RasorPCDNA3(emptyvector)inthepresence(+) orabsence( )ofhgf.datashownismean±standarderrorof5independentexperiments. 25

37 EXPERIMENTTWO:USEOFTHEG38VR RASMUTANT ThesecondexperimentusedaconstitutivelyactiveformofR ras,g38v,inorderto furtherexaminetheroleofr rasintheproposedsignaltransductionpathway.thisformof R rasisconstantlyinthegtp boundstate.mdckcellswereco transfectedwithg38vand HA Arf6inordertoobservestrongArf6activation.Thesecellswereincubatedinthe presenceorabsenceofhgffor5hours.controlcellsshowedstrongactivationofha Arf6 after5hoursofhgfincubation.theg38vexpressingcellsinthepresenceofhgfshowed adecreaseinha Arf6activationwhencomparedtotheHGF+control.ThefactthatArf6 activationisnotincreasedsimilarlytothecontrolcellsinthepresenceofhgfcouldbedue tothedownregulationofpathwaycomponentsasaresultofr rashyperactivation. AdditionaltrialsinthepresenceandabsenceofHGFinG38VR rasexpressingcellsare necessarytofullyverifyifthereisasignificantincreaseinarf6activation.theseresults alongwiththeg38vr rasmorphologicalstudypresentedinchapter4alludetothe conclusionthatr rashasaroleinthehgf inducedarf6activationsignaltransduction pathway. 26

38 Figure3.3A:ConstitutivelyactiveR ras(g38v)decreasesarf6activationinthe presenceofhgf.mdckcellsweretransfectedwitheithermyc G38VorpcDNA3(empty vector).transfectedwereallowedtorecoverfor18hoursandincubatedinthepresence (+)orabsence( )of30ng/mlhgffor5hours.arf6activitywasassessedusingthegst GGA3pulldownassay,whichisolatesonlyactiveArf6.Theresultsofthisassaywere analyzedviawesternblottargetingha Arf6andMyc.3.3Ashowsarepresentativegelfrom apulldownexperimentquantifiedin3.3b. 27

39 Figure3.3B:ConstitutivelyActiveR ras(g38v)decreasesarf6activationinthe presenceofhgf.quantificationofwesternblotsshowin3.3a.shownistheactivationof Arf6inMDCKcellsexpressingG38VR rasorpcdna3(emptyvector)inthepresence(+) andabsence( )ofhgf.datashownismean±standarderrorof5independent experiments. 28

40 CHAPTER4:ThemorphologicaleffectsofR Rasoncellularmotility. Epithelialcellmotilityischaracterizedbyanepithelial to mesenchymaltransition. Thisallowsthecelltomovefromarigid,fixedstructuretoamotile,moreinvasivecell phenotype.duringthisprocess,thecellwilldissolveitsnetworkofjunctions,lose polarization,andtakeonaspindlelikeshape.onceemtiscompleted,thecellcanform leadingedgeprotrusions,adheretosurroundingecmthatwillactastractionforthe movingcell,translocatethecellbodyinthedirectionofcellmovement,andretractthetail endtoaccomplishcoordinatedmotility.thesmallras relatedgtpase,r ras,hasbeen implicatedinthisprocessofcellspreadingandmigration.r rascanpromotecelladhesion byregulatingintegrinfunctionandfocaladhesionformation(kwong,wozniaketal.2003). Leadingedgemembraneprotrusionsformedbyactincytoskeletalrearrangementsarealso regulatedbyr rasanditscontroloverrac1andrhogtpases(wozniak,kwongetal. 2005;Ada Nguema,Xeniasetal.2006).ActivatedR rascanalsoinducescatteringand tubulogenesisinmdckcellsinthesamemannerashgf inducedscatteringinthesamecell line(khawaja,lehmannetal.1998).therefore,iwantedtoobservethemorphological changesinducedbyr rasinmdckcellsinthepresenceorabsenceofhgf. 29

41 MorphologicalStudyPart1 EffectofT43NR ras Initially,MDCKcellsweretransfectedwithT43NdominantnegativeR rasto determinewhetheradefectincellmigrationwouldoccur.thetransfectedcellswerethen incubatedinthepresenceorabsenceofhgffor0,4,or18hours.thisallowedforthe MDCKmorphologychangestobeobservedovertimeascomparedtothecontrolcells. After0hours,boththecontrolandT43Ntransfectedcellsshowednomigrationattempt andremainedstationaryinsmall,confinedislands(figure4.1a). After4hoursinthepresenceofHGF,controlcellsshowmoderatecellspreadingand thebeginningsofcellscattering.thecellsappearflattenedandhaveformedleadingedge projectionsawayfromthegroupingofcellsindicativeofcellspreading.somecellshave evenbegunscatteringinwhichtheyhavecompletelyseparatedandmigratedawayfrom othercells.however,t43nr rastransfectedcellsshowverylittlecellspreadingandno cellscatteringafter4hoursofincubationwithhgf.themajorityofthecellsremainpart ofthecellularislandwhilefewhaveformedflattened,front endprojections.notonlyare theseforwardprojectionsscarcebuttheyalsoappearabnormalwhencomparedtothe control(figure4.1b). After18hoursinthepresenceofHGF,controlcellsshowedalmostcompletecell scattering.ofthesecells,65%werecompletelyscattered(figure4.3).cellswerenolonger confinedtoislandsandweremigratingawayfromoneanother.thet43ntransfected cells,however,showedonlyalimitedamountofcellspreading,thoughmoreabundantthat after4hoursofincubationwithhgf(figure4.1c).thespreadingobservedinthisgroup didappearmorepronouncedinthatthefront endprojectionshadmigratedfurtheraway 30

42 fromcellgroupings(figure4.2).only5%ofthet43ncellsalsoshowedscattering,even after18hoursofincubationwithhgf(figure4.3).therefore,adecreaseinr rasactivity impairsthecellsnormalmigratoryfunctionandaltersthecellsmorphologyandshape duringthisprocess. Figure4.1A:DominantnegativeR Ras(T43N)inhibitscellspreadingandscatteringin MDCKcells(0hours).MDCKcellsweretransfectedwithT43NR rasorpcdna3and platedonfibronectin coatedcoverslips.cellswerethenincubatedintheabsenceofhgf andpreparedforimmunofluorescencethroughfixingandstainingwithmouseanti myc antibodyfollowedbyalexa488conjugatedanti mouseantibodyandrhodamine phalloidin. 31

43 Figure4.1B:DominantnegativeR Ras(T43N)inhibitscellspreadingandscatteringin MDCKcells(4Hours).MDCKcellsweretransfectedwithT43NR rasorpcdna3and platedonfibronectin coatedcoverslips.cellswereallowedtorecoverfor18hoursand thenincubatedinthepresenceofhgffor4hours.immunofluorescencewasdonethrough fixingandstainingwithmouseanti mycfollowedbyalexa488conjugatedanti mouse antibodyandrhodaminephalloidin. 32

44 Figure4.1C:DominantnegativeR Ras(T43N)inhibitscellspreadingandscatteringin MDCKcells(18hours).MDCKcellsweretransfectedwithT43NR rasorpcdna3and platedonfibronectin coatedcoverslips.cellswereallowedtorecoverfor18hoursand thenincubatedinthepresenceofhgffor18hours.immunofluorescencewasdone throughfixingandstainingwithmouseanti mycfollowedbyalexa488conjugatedantimouseantibodyandrhodaminephalloidin. 33

45 Figure4.2:DominantnegativeR ras(t43n)inhibitstypicalcellmigrationinthe presenceofhgf.displayedarefourrepresentativepicturesofcellsexpressingt43nrras.theirabilitytomigrate,eveninthepresenceofhgffor18hours,isimpaired comparedtothecontrolcells.theyexhibitdelayedcellspreadingandinhibitedcell scattering. 34

46 Figure4.3:DominantnegativeR ras(t43n)inhibitscellscatteringafter18hoursof HGFincubation.MDCKcellsweretransfectedwithT43NR rasorpcdna3(control)and wereincubatedinthepresenceofhgffor18hours.thepercentscatteredcellswas quantifiedasthenumberofscatteredcellsdividedbythetotalamountofcells.ascattered cellisdefinedasacellthathasformedatailendprocesspointingawayfromthedirection ofmovement,whichisahallmarkofascatteredcell.datashownismean±standarderror of3independentexperiments. 35

47 MorphologicalStudyPart2 EffectofG38VR ras Next,MDCKcellsweretransfectedwiththeconstitutivelyactiveR rastodetermine ifcellspreadingandscatteringcouldbeinducedintheabsenceofhgf.cellswere transfectedwithg38vr rasandincubatedintheabsenceofhgffor32hours.after32 hours,transfectedcellshadbeguntoscatterwhencomparedtothecontrol(figure4.4). ThisresultimpliesthatR rasisapartofthenormalfunctioningcellmigrationpathwayand onceactivatedcaninducecellspreadingandscattering. TheG38VR rascellswerealsoincubatedinthepresenceofsecinh3,acytohesin inhibitor,totestcytohesininvolvementincellmovementdownstreamofr rasactivation. IntheabsenceofsecinH3,cellsareabletospreadandscattereasilyafter32hours.Inthe presenceofsecinh3,itappearsthatcellsareabletospreadbutexperienceinhibitedcell scattering.figure4.5showsthesecinh3treatedcellsformingleadingedgelamellipodia butarestillmaintainingcellislandcontacts,evenafter32hours.thedisruptioninnormal cellmigrationshowsthatcytohesinshaveafunctiondownstreamofr rasactivation. 36

48 Figure4.4:ConstitutivelyactiveR ras(g38v)caninducecellspreadingandscattering intheabsenceofhgf.mdckcellsweretransfectedwithg38vr rasorpcdna3and platedonfibronectin coatedcoverslips.transfectedcellswereallowedtorecoverfor32 hoursandwerepreparedforimmunofluroescence.cellswerestainedwithmouseantimycfollowedbyalexa488conjugatedanti mouseantibodyandrhoadminephalloidin. 37

49 Figure4.5:Thecytohesininhibitor,SecinH3,reducescellmigrationinducedby constitutivelyactiver ras.mdckcellsweretransfectedwithg38vr rasandplatedon fibronection coatedcoverslips.transfectedcellswereallowedtorecoverfor12hoursand werethenincubatedwith30µmsecinh3for24hours.preparationfor immunofluorescencewasasfollows:fixingandstainingwithmouseanti mycfollowedby Alexa488conjugatedanti mouseantibodyandrhodaminepalloidin. 38

50 Chapter5:Discussion In2007,cancerwasthe2 nd leadingcauseofdeathintheunitedstatescausing 23.2%ofalldeaths(Jemal,Siegeletal.2010).Themostcommontypesofcancerareofthe lung,stomach,breast,colon/rectum,anduterinecervix,whichareallcancersofthe epitheliaorcarcinomasthataccountfor90%ofhumancancers(alberts,lewisetal.2002). Ifthesecancersprogresstoastageofmetastasis,chanceforsurvivalsignificantly decreasesandisresponsiblefor90%ofcancerdeaths(christofori2006).metastasisisa processinwhichtheprimarytumorwillleaveitsinitialsite,invadesurroundingtissue, enterthebloodstream,andtraveltoasecondarysite.whilecellmigrationiscriticalfor cancercellmetastasis,itisalsonecessaryforembryonicdevelopmentandwoundhealing. Therefore,someofthebasicunderlyingmolecularmechanismsinvolvedarecommonto bothnormalandcancerouscellmovement(yamazaki,kurisuetal.2005).germlineand somaticmutationsandgeneamplificationsincellmigrationcomponentscanleadtocell transformation,whichcausesanormallymigratingcelltobecomeinvasiveandmetastatic. Forexample,anincreaseinc Metactivityarefrequentlyfoundinvariouscancertypesand areconsideredkeyinitiatorsofinvasivegrowthandtumorprogression(christofori2006). TheHGF c Metreceptor signalingpathwayisalsoimportantfortypicalepithelialcell movementandchangeincellmorphologyinanormallyfunctioningcell.therefore,an understandingofcellmigrationinnon neoplasticcellswillleadtohelpinunderstanding invasionandmetastasisincancerouscellsandcoulduncoverpotentialtherapeutictargets. AnumberofpotentialtargetsaremembersoftheRassuperfamilyofGTPases, whicharecommonlymutatedinmalignancies.severalproteinsofthisfamilythatare 39

51 frequentlymutatedinhumancancersandleukemiasaretheisoformsn ras,k rasandhras.besidesthese3rasproteins,anotherrassubfamilymember,r ras,hasalsobeen implicatedintransformation,invasion,andmetastasisofseveraldifferentcancertypes (Ehrhardt,Ehrhardtetal.2002).R rashasbeenimplicatedinmalignanttransformationof fibroblasts,initiatingmigrationandinvasioninbreastcancercells,andpromoting metastasisincervicalcancerepithelialcells(cox,brtvaetal.1994;keely,rusynetal. 1999;Mora,Rosalesetal.2007).Untilrecently,littlewasknownaboutR ras downstream effectorsanddistinctcellularfunctions.ithasbeenuncoveredthatr rashasaroleincell migrationandmorphologythroughintegrinregulationandendocytosis.theexact mechanismofhowr rasispromotingtransformationandsubsequentmetastasisis relativelyunknown(gawecka,griffithsetal.2010).thoughsomeprogresshasbeenmade inthisfield,morestudyofr rasanditsfunctioninnormalcellmigrationisnecessaryto understandhowthisprocesshasbecomeaberrantincancerouscells. Aswellascontrollingintegrinendocytosisandactivation,R rashasafunctionin promotingcelladhesionandspreadingduringmigrationbycouplingwithpi3 Kandits ownuniqueeffectorrlip76.thisallowsfordownstreamracactivationthroughthe activationofarf6gtpaseanditsgefarno.subsequentactincytoskeletal rearrangementsresultfromthisactivationandproduceleadingedgeprotrusionsand progressivecellmovement.thoughweknowthatallofthesecomponentsareinvolvedin cellmotility,thesignalingpathwaylinkingthemtogetherisunknown. 40

52 BiochemicalStudyofR rasanditseffectonarf6activation ThisstudyhasconnectedR rastoarf6activationviathehgf c Metsignal transductionpathwayinepithelialcellmigration.whentheactivityofr raswasreduced usingadominantnegativemutant,theactivationofendogenousarf6wasreducedinthe presenceofhgfwhilearf6activationwaspromotedinthecontrolmdckcellsinthe presenceofhgf.theseresultsleadustobelievethatr rasisdownstreamofthec Met receptortyrosinekinase. Tofurthersubstantiatethisresult,theinvolvementofcytohesinsinthispathway mustbeexplored.thecytohesinfamily,morespecificallycytohesin2/arno,hasbeen foundtobenecessaryforβ1integrinrecyclingthereinpossiblyconnectingr ras,however, thetwohaveneverbeendirectlylinkedincellmigration(ohandsanty2010).totestthis biochemically,arnoactivitywouldneedtobedecreasedoreliminatedandarf6activation inthepresenceofhgfwouldbeexaminedviaapulldownassayandwesternblot.we hypothesizethat,afterthereductionofarnoactivity,arf6activationwouldbedecreased inthepresenceofhgf.thisresultwouldsupportarno sinvolvementinthehgf c Met signalingpathway. Also,previousdatashowsthatcytohesinsaredownstreamofR rasthroughthe interactionbetweenarnoandther raseffectorrlip76(goldfinger,ptaketal.2006). However,thisinteractionisweakanddoesnotappeartobedirect.Weproposethatthis bindingintermediateiscnk3,ascaffoldingproteinthathastheabilitytointeractwiththe coil coileddomainofarnotoproducearf6activationandsubsequentracactivation. ThoughtheinteractionbetweenARNOandRLIP76wasdiscoveredinanadhesion induced 41

53 migrationassayandcouldbetheresultofincreasedintegrinsignaling,thisremainstobe confirmed.therefore,aninvestigationoftheinteractionbetweenrlip76andcnk3is necessarytofullyestablishtheconnectionbetweenr raswitharnodownstreamofc Met. AlsoexaminedinthisstudywastheeffectoftheconstitutivelyactiveformofR ras onarf6activation.surprisingly,adecreaseinarf6activationcouldbeseeninthepresence ofhgf.asaresultofthehyperactivationofr ras,pathwaycomponentssuchasessential GEFsorrecruitmentproteinscouldhavebeendownregulatedoverthecourseofthe32 hourexperimenttimeline,reducingarf6activationinthepresenceofhgftolessthanthat ofthecontrol.furtherexperimentsexaminingexpressionandlocalizationofr rasand othereffectorsinvolvedinthepathwayareneededinordertoconfirmthatr rasisa componentofthehgf c Metsignalingpathway.Figure5.1showsthenewproposed pathwaycontainingr rasandotherpotentialcomponentsdownstreamofc Met. 42

54 Figure5.1:NewlyproposedsignalingpathwayleadingfromHGFtoArf6activation. ThenewproposedpathwayshowsHGFactivatingR rasandsubsequentlyactivatingarf6 throughaseriesofinteractionsthatinvolvether rasspecificeffectorrlip76,the scaffoldingproteincnk3andthearf6gefarno. 43

55 MorphologicalStudyofR ras InvolvementinCellSpreadingandScattering ThemorphologicalstudywasdonetoobservetheeffectsR rascanhaveonthecell s abilitytospreadandscatterwhentreatedwiththeepithelialmotogenhgf.notonlydidrrashaveaneffectonarf6activationbutitalsoaffectedtheirmotility.first,themdckcells weretransfectedwithdominantnegativer rasandincubatedinthepresenceorabsence ofhgffor0,4,or18hours.thecontrolcellsgroupedinanislandat0hoursspreadaway fromeachotherstartingatthegroupedgesandfinishedbyscatteringandcompletely separatingby18hours.thecellsexpressingdominantnegativet43nr ras,however,had begunverylittlescatteringandexperiencedlimitedspreadingevenafter18hoursofhgf incubation.only5%ofthet43nexpressingcellshadbegunscatteringafter18hoursof HGFexposurecomparedtothe65%cellscatteringseeninthecontrolcells.TheT43Ncells thatweresuccessfulinspreadingwerelocatedalongtheoutermostedgeofanisland, whichwassimilartoinitialcontrolcellmovement. ThedefectsinspreadingandscatteringofthecellsexpressingdominantnegativeRrascouldnotonlybearesultofblockingtheHGF c Metsignalingpathwaybutalsofroma lackofsufficientintegrinsignaling.integrinsarecellsurfacereceptorsthatbindboththe cell scytoskeletonandcomponentsoftheextracellularmatrixsuchasfibronectin,collagen, andlaminin.thisessentiallinkallowsintegrinstorelaysignalsfromtheseecm attachmentstopromoteactincytoskeletalrearrangementsandactivateintracellular signalingcalledoutside insignaling.conversely,integrinscanalsobeactivatedby intracellulartriggersinaprocesscalledinside outsignalingwheretheintegrinwill experienceanallostericrearrangementofitsextracellulardomainsandincreasesligand 44

56 bindingaffinity(kinbara,goldfingeretal.2003).therefore,theactivationofintegrinsand inductionofsignalingisbi directionalandhighlyregulated. Cell ECMadhesionisakeyeventinepithelialmigration.Onceacelladherestothe extracellularmatrix,integrinsignalingcanresultintheinductionofn cadherinexpression andsupportsthestimulationofemt(yilmazandchristofori2009).thispermitsthecell totakeonmesenchymal likepropertiesandbegintheformationofleadingedge projectionssuchaslamellipodia,whichareanchoredtotheecmviaintegrincontacts. Scatteringandincreasedmotilitycanbepromotedbyenhancedintegrinadhesion.Thisis, however,nottheonlytypeofadhesionnecessarytoinducescatteringsupportingthe notionthatepithelialmigrationisacomplexprocesswithmanyimportantregulators(de Rooij,Kerstensetal.2005). Oneregulatorofthebi directionalintegrinactivationandsignalingandconsequent migrationisthegtpaser ras.thisgproteinwasfirstshowntoregulateintegrinfunction byincreasingintegrinaffinity(zhang,vuorietal.1996).itwaslaterfoundthatexpression ofactivatedr rascouldstimulateintegrin dependentmigrationinbreastcancercellsby signalingthroughtheintegrinalphacytoplasmicdomain(keely,rusynetal.1999).ithas alsobeensuggestedthataprimarymechanismfordrivingtumormetastasisisenhanced endocytotictraffickingofintegrinsthereinlinkingr rastotheseprocesses(gawecka, Griffithsetal.2010). Asaresultofthesestudies,itwasdiscoveredthatR rascanactasanactivatorof integrinsandalsobeactivateddownstreamofintegrin mediatedadhesion.theseroles werediscoveredintheabsenceofhgforothermigrationstimulants.however,ithasbeen foundthathgfcanupregulateintegrin mediatedadhesionsuggestingthatintegrin 45

57 adhesionandthehgf c Metsignalingpathwaycouldbeworkinginconcerttopromote motility.thiscouldexplainthehindered,butsomewhatfunctionalcellspreadingobserved inthet43nr rasmorphologicalstudy.thet43nexpressingcellsformlamellipodiafrom theedgesofislandswhereintegrincontactsandsignalingwouldbethemostabundant. ThisintegrinsignalingcombinedwithresidualendogenousR rasactivationviahgfcould besufficienttopromotesomecellspreadingbutnotcellscattering.inordertoinduce scattering,thiscombinationofsignalswouldneedtogenerateahigherdegreeofr ras activityinorderforthecelltocontinuetomoveforward.aninvestigationofintegrin activityandlocalizationafterthedepletionofr raswouldneedtobeconductedinorderto testthishypothesis. AmorphologicalstudyofconstitutivelyactiveR ras effectoncellmigrationwas alsoconducted.mdckcellsweretransfectedwithg38vr rasandwereallowedtorecover foratleast32hours.evenintheabsenceofhgf,cellspreadingandscatteringwas induced.thoughhgfwasabsentfromtheg38vr rassamples,themovementinducedby constantr rasactivationmimickedthecellscatteringstimulatedbyhgfimplyingthatrrasactsasakeyregulatorofcellmigrationdownstreamofc Met. CellstransfectedwithG38VR raswerealsoincubatedinthepresenceofsecinh3,a cytohesininhibitor.itwasfoundthatcellmigrationinducedbytheconstantactivationof R raswasdisruptedinthepresenceofsecinh3.thecellswereabletospreadnormallybut wereunabletocontinuemigratingandscatter.thesedefectsincellmigrationweresimilar tothoseobservedinthet43nr rasexpressingcellsinthepresenceofhgfwhere lamellipodiaareformedfromtheedgesofcellgroupingsbutthecellsareunabletomigrate anyfurther.thissomewhatactivemigrationandlamellipodiaformationalsosupportthe 46