Cha�ter

�General introduction

Chapter 1

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IntroductionThe �a�� ��u�er�a�il�� con��i��t�� o� a large grou� o� �ono�eric G �rotein�� �hich c��cle bet�een GDPboundinactiveandGTP-boundactivestate.ThisswitchfunctionofRas-likeproteinsi�� regulated b�� guanine nucleotide exchange �actor�� (G����) and GTPa��e�acti�ating �rotein�� (GAPs).GEFsinducethereleaseofG-proteinboundnucleotide,allowingrebindingofthecellularmoreabundantGTP.GTP-bindingchangestheconformationofthesmallG-proteinin the ���itch � and ���itch 2 region�� (���), �hich are in�ol�ed in nucleotide�binding and allows theassociationwith itseffectors.GTPaseactivatingproteins (GAPs)stimulate theintrinsicGTPaseactivityoftheG-protein,whichresultsinthehydrolysisofGTPtoGDPandtherebyterminatessignallingoftheG-protein.

Rap family Rap family proteins were first identified in a screen for Ras homologous genes (4) andcompriseofRap1A,Rap1B,Rap2A,Rap2BandRap2C.Independently,Rap1wasidentifiedasasuppressoroftheK-Rastransformationphenotype(5),andthisfindinginvokedattentionstoRap proteins and the signalling pathways theymight be involved in.Due to the high��i�ilarit�� bet�een the e��ector region�� o� �a� and �a��, �a� �a�� h���othe��i�ed to antagoni�e RassignallingbytrappingRaseffectorproteinsinaninactivecomplex.Indeed,RapbindstoRaseffectorswithsimilaraffinities(6-8).Moreover,overexpressionofactiveRapinterfereswithERKactivationinfibroblasts(9).However,itwasshownthatRapmightalsobeabletoactivateRaseffectorproteinsandinduceoncogenictransformationofSwiss3T3fibroblasts(10,11). Although overexpression of Rap may interfere in Ras signalling, endogenousRap1isunlikelyabletoefficientlytitrateRaseffectormolecules(12,13).ThisindicatesasignallingfunctionofRap,whichisindependentofRas.Indeed,RapwasfoundtoregulateintegrinmediatedcelladhesioninlymphocyteTcells(14).Subsequentstudiesdemonstratea crucial role �or �a�� in the regulation o� in��ide�out ��ignalling to integrin��, �hich �ill be discussedinmoredetaillater.MorerecentlyRap1hasbeenimplicatedinE-cadherin(15,16)

Fig. 1 Regulation of the Rap1 GTPase. �a� acti�it�� i�� u�regulated b�� a nu�ber o� �a�G���� and do�nregulated b�� RapGAPs.Rap1GEFsarecontrolledby��econd �e����enger�� or �rotein t��ro��ine kinases.PTK:proteintyrosinekinase.

Rap1

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GEF GAP

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cAMP EpacPTK C3G ? PDZ-GEFcalcium/DAG CalDAG-GEF

Rap1

General introduction

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andVE-cadherin-mediatedjunctionformation(17-20).InthiscontextRapwasfirstshowntopositivelyregulatetheadhesionofcellstoE-cadherin(16).Asubsequentreportshowedthat C�G, a guanine nucleotide exchange �actor (G��) �or �a��, regulate�� the ��ignalling mediatedbyE-cadherinandnectininepithelialcells(15,21).Inhu�an u�bilical �a��cular endothelial cell�� (HUV�C), the ��ac��a�� �ath�a�� regulate�� V��cadherin��ediated cell�cellcontacts(17-20).

Activation of Rap by guanine nucleotide exchange factorsAvarietyofGEF-proteinsareable tocatalysenucleotideexchangeofRap (Fig.1).Likeother GEF proteins for G-proteins of the Ras family, they contain a CDC25-homologydo�ain and a �a�� exchanger �otif (REM)domain.TheCDC25-homologydomain is thecatalyticdomain.Studiesrevealedthattheisolateddomainissufficienttoinducenucleotideexchange (22,23).REMdomainsalwaysoccur togetherwithCDC25-holomolgydomains(24) and ha�e a �ainl�� ��tructural �unction b�� ��hielding the h��dro�hobic ��ur�ace area o� theCDC25-homologydomain(25).Exceptforthesecommonelements,RapGEFsvaryindomaincompositionaswillbedescribedinthefollowingsections.EpacThe ob��er�ation that c�MP induce�� �a� acti�ation inde�endent o� �rotein kina��e � (PK�) encourageddeRooij andco-workers to search the sequencedatabase forgenesencoding�rotein�� that contain both a c��clic nucleotide binding(CNB)domainandaCDC25homologydomain.Thisresultedintheidentificationofthe�xchange �rotein directl�� acti�ated b�� c�MP (��ac) and indeed ��ac �a�� ��ho�n to be directl�� acti�ated b�� c�MP in vivo and in vitro(26).In an independent screen,Epacwas identifiedas aproteindifferentially expressed in thebrainandcontainingaputativesecondmessengerbindingdomain(27).TheEpacproteinfamilyconsistsof3members:Epac1,Epac2andRepac(RelatedtoEpac)(Fig.2).Epac1is

Fig. 2 Domain structure of Epac and PDZ-GEF. DomainorganizationofEpac1,Epac2,Repac,PDZ-GEF1andPDZ-GEF2areshown.DEP,Disheveled,Egl-10andPleckstrindomain;CNB,cyclicnucleotidebindingdomain;REM,Rasexchangermotif;RA,Rasassociationdomain;CDC25-HD,CDC25-homologydomain;aCNB,atypicalcyclicnucleotidebindingdomain;PDZ,PSD-95,Dlg,ZO-1domain.

RAEpac2 REMDEP

RAREMDEP CDC25-HDEpac1 CNB

CNB-BCNB-A

RAPDZ-GEF2 REM CDC25-HDaCNB aCNB PDZ

CDC25-HD

RAPDZ-GEF1 REM CDC25-HDaCNB PDZ

regulatory region catalytic region

RAREM CDC25-HDRepac

Chapter 1

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widelyexpressedwithhighexpressionlevelsinkidney,ovary,brainandskeletalmuscles,�herea�� ��ac2 ex�re����ion i�� �ainl�� re��tricted to the brain, li�er, adrenal gland, �ituitar�� and β-cells of the pancreas (27-30). Epac consists of a regulatory and a catalytic region(Fig.2).ThemajordifferencebetweenEpac1andEpac2isanadditionalN-terminalCNBdomaininEpac2.ThisCNBdomainhasaloweraffinityforcAMPthanthesecondCNBdomainandisnotrequiredtokeepEpac2intheauto-inhibitedstate(28,31).BothEpac1and��ac2 contain a Di��he�elled, �gl��0 and Pleckstrin (DEP)domain,which isnot required�or the regulation o� ��ac b�� c�MP but �ediate�� it�� locali��ation at the �la���a �e�brane (28,32).Anubiquitin-likefolded�a�� a����ociation (��) do�ain i�� in��erted bet�een the ��M domainandtheCDC25-homologydomain,whichforEpac2wasshowntobindH-Ras((33),addendum1ofthisthesis).The anal����i�� o� ��ac��ediated e��ect�� �a�� �acilitated b�� the de�elo��ent o� the ��ac ��electi�e c�MP analogue 8��CPT�2’�O�Me�c�MP (007), �hich i�� not able to bind to or activatePKA.With theuseof007,Epac1was linked to integrin-mediatedandcadherin-mediatedadhesion(16,34).Epac2wasshowntofulfilapredominateroleinthepotentiationof insulin secretion and the regulation of neurotransmitter release (35,36).The effects ofEpaconinsulinsecretionweresuggestedtobepartiallyRapindependent.UnlikeEpac1 andEpac2,Repac lacks a regulatory region and is therefore constitutivelyactive(28).ItiscurrentlyunclearwhetherRepacisregulatedbytheinteractionwithotherproteins,whichcouldtakeoverthefunctionoftheregulatoryregion.C3GC3GisubiquitouslyexpressedandwasoriginallyidentifiedasaproteinboundtotheSH3domainofc-Crk(37).C3GcanactonRap-1,Rap-2,andR-RasviaitsC-terminalcatalyticregion(37-40).TheCrk-C3Gcomplexisrecruitedtotheplasmamembraneuponactivationof receptor tyrosine kinases by several growth factors and cytokines. In epithelial cellsC�G interact�� �ith the c��to�la���ic do�ain o� ��Cadherin, �hich i�� re���on��ible �or �a�� activationduringcell-cellcontactformation(15,41).PDZ-GEFTwoisoformsofPDZ-GEF,PDZ-GEF1and2,existinmammalians(Fig.2).Bothproteinscontain a PSD-95, Dlg, ZO-1 (PDZ) domain, a putative RA domain, a REM-domain, aCDC25-homologydomainandanatypicalCNB(aCNB)domain,whichisnotabletobindcAMP(42,43).PDZ-GEFshavebeenreportedtobeRapspecificexchangefactors(42,43),however,thisspecificitywaschallengedbythesuggestionthatPDZ-GEF1canalsoactivateRas (44). It is currently unclear, howPDZ-GEFs are regulated.Recently the function ofPDZ-GEFwasanalysedgenetically inC.elegans and Drosophila. In C.elegans, thePDZ-G�� (pxf-1)/Rappathwayisrequiredforthemaintenanceofepithelialintegrity(45)andinDrosophilaPDZ-GEF(Dizzy) i�� in�ol�ed in �a���ediated integrin�de�endent cell adhe��ion duringcellmigrationinembryonicdevelopment(46).

General introduction

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CalDAG-GEFTheCalDAG-GEFs(orRasguaninenucleotidereleasingproteins,RasGRPs)areregulatedby the second messengers diacylglycerol and calcium. They differ in their specificitiestowardsG-proteins.CalDAG-GEF1actsonRapproteins(47,48),CalDAG-GEF2onH-RasandK-RasbutnotonRap(48-50)andCalDAG-GEF3onbothRapandRasproteins(48,51).CalDAG-GEF4hasnotbeenstudiedextensively,butitshowsspecificityforRas(52).

Inactivation of Rap by GTPase activating proteinsThe intrin��ic GTPa��e acti�it�� o� �a� �rotein�� i�� �er�� lo� and there�ore ra�id inacti�ation ofRaponlyoccurswith theaidof specificGAPproteins (Fig.1).Membersof theSPA-� (��ignal�induced �roli�eration a����ociated gene��) �a�il�� contain a catal��tic do�ain called G�P�related domain (GRD), which is responsible for stimulating the intrinsic GTPaseactivityofRapproteinsbyseveralordersofmagnitude.Originall�� SP��� �a�� �ound a�� a �itogen�inducible nuclear �rotein (4�) and �a�� later �ound tobeaprincipalRap1-specificGAPinthelymphohematopoietictissues(53).Thestructurallyrelated �rotein�� o� SP���, �6TP� (�6�targeted �rotein �), SP�� (���ine�a����ociated �a�G�P),

Fig. 3 Domain structure of AF6, afadin, Canoe and Ce-AF6. DomainstructureofthethreehumanAF6isoforms(AF6i1,AF6i1 andAF6i3), the two its rat homologue afadin (long l-afadin and short s-afadin), itsDrosophilahomologueCanoeanditsC.eleganshomologueCe-AF6areshown.Therelativepositionsofindividualdomainsareshown.RA1,2,Ras-associatingdomain1and2;FHA,forkhead-associateddomain;DIL,adilutedomain;PDZ,PSD-95,Dlg,ZO-1domain;pro,proline-richregion,ABS,actin-bindingsite.

RA1,2 FHA DIL PDZ Pro Pro16

11

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Chapter 1

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andseveralSPA-1-likeproteins(SPA-Ls)makeuptheSPAfamily,butallshowauniquecellulardistribution invarious tissues (53-56).Spa-1was reported tobe regulatedby therecruitmentofAF6andsubsequentdownregulateRapmediatedadhesion(57).Rap1GAP,aproteinwithamolecularweightof73KDa,wasthefirstidentifiedGAPforRap(58).ItexistsasthetwosplicevariantsRap1GAPIandRap1GAPII,withthelattercontainsanadditionalN-terminalregion.Theα-subunitoftheGi-familyproteinsbindsspecificallyto �a��G�PII and recruit�� it to the �e�brane, �hich re��ult�� in the attenuation o� �a�� activation(59).Morerecently,Rap1GAP2,whichexhibits50%similaritywithRap1GAPIwasidentifiedinhumanplatelet(60).

The Rap binding protein AF6��� a re��ult o� chro�o��o�e tran��location, the �LL�� (�cute L����hobla��tic Leukemia)geneisfusedtoothergenesonpartnerchromosomes,whichresultsinchimericproteins.Originallyidentifiedasan�LL�� �u��ion �artner on chro�o��o�e 6 (6�), ��6 i�� a �rotein con��er�ed �ro� humantonematodeandfly,althoughitisknownundervariousnamesindifferentspecies(Fig.3).AF6containstwoRAdomains,a�orkhead�a����ociated (�H�) do�ain, a dilute do�ain (DIL),aPDZdomain,andaproline-richregion.ThetwoRAdomainsmediatetheinteractionofAF6withtheGTP-boundformsofRapandRas(62,63).ThePDZdomainwasshowntobeinvolvedintheassociationwithnectinandSpa-1(57,64,65).RecentlyanewhumanAF6isoformwascloned(66),namelyAF6isoform3(AF6i3).AF6i3differsfromAF6isoform� (��6i�), �hich �a�� the onl�� hu�an i��o�or� being ��tudied, b�� an additional C�ter�inal F-actin-bindingregion.Thisactin-bindingregionofAF6i3shareshighsimilaritywiththeC-terminusfromoneofitsRathomologs,l-afadin.Theactin-bindingwassuggestedtofacilitateAF6i3topositivelyregulateadhesion(66).EventhoughAF6caninteractwithbothRapandRas,mostofitsbiologicaleffectswereassignedtoRapsignalling.Canoe, the Drosophila ortholog o� ��6, �unction�� a�� an e��ector o� �a�� during e�br��onic de�elo��ent and it i�� requiredfortheproperdorsalclosureprocess(67).Inaddition,Canoe act�� do�n��trea� o� the ��ider�al Gro�th �actor (�G�) rece�tor and �a�� b�� controlling o��atidal rotation during �lanar cell �olarit�� (PCP) e��tabli��h�ent in the Drosophilaeye(68).At subcellular levelAF6 is localized at epithelia adherens junction. It binds to severaljunction �rotein��, including Zona Occluden�� �(ZO-1)(69),Junctional �dhe��ion Molecule (JAM)(70),profilin(62)andNectin(71).SeveralstudiessuggestthatAF6isimportantfortheformationand/orstabilityofcellularadherensjunctions.InthecontextofE-cadherin-mediatedadhesion,thePDZdomainofAF6hasbeenshowntointeractwithC-terminusofnectinandthisinteractionfacilitatesE-cadherin-dependentcell-celladhesion(64,65).Takaiandco-workershaveshownthattheRap/AF6complexpromotestheinteractionbetweenE-cadherin and ��20�catenin in a �a���de�endent �anner and ��tabili��e�� ��cadherin��ediated adhesion (71). By contrast, recent study shows thatAF6i3 is associated with the actin

General introduction

��

cytoskeletonandisrequiredforthestabilizationofE-cadherin-dependentadhesioninaRas/Rap independentmanner (66). Interestingly, in thecontextof integrin-mediatedadhesion,AF6 plays a role as a negative regulator ((57) and chapter 2). In humanfibroblastsAF6enhancestheinhibitionofSpa-1onRap1inducedcelladhesion(57).Theoppositeeffectso� ��6 on ��cadherin� and integrin��ediated adhe��ion indicate a �otential role �or ��6 a�� abalancekeeperbetweenthecell-celladhesionandcell-matrixadhesion.ItseemspossiblethatAF6playsasignificantroleincadherin-mediatedcell-celladhesionbykeepingRap1awayfromintegrinmediatedcell-matrixadhesion.

Rap in T cellsLymphocytes are blood cells that are responsible for the adaptive immune response. In�a��al�� t�o cla����e�� o� l����hoc��te��, the B cell�� and T cell�� account �or the antibod�� response and cell mediated immune responses, respectively. T cells derive their namesbecausetheydevelopinthethymus.Incell-mediatedimmuneresponses,activatedTcellsrecogni��e �oreign antigen�� that are �re��ented b�� the Major Hi��toco��atibilit�� Co��lex (MHC)proteinsonthecellsurfaces.Tcellsaredividedintotwomainclasses:thecytotoxicTcellsandhelperTcells.Onceactivatedbybinding to theMHCcomplexofanantigenpresentingtargetcell,cytotoxicTcellsinduceapoptosisofthetargetcell.Insteadofkillingthetargetcellsdirectly,helperTcellsactivatemacrophages,BcellsandcytotoxicTcells.Inactivelymphocytescirculatecontinuouslybetweenthelymphandblood.Inresponseto��ti�uli, l����hoc��te�� ���itch ra�idl�� �ro� a non�adherent ��tate to an adherent ��tate and thi�� processrequiresinside-outsignallingmediatedbyintegrins.Rap in T-cell adhesionStimulationofcelladhesionisoneofthebest-studiedeffectorpathwaysofRap1(14)(Fig.4).In l����hoc��te��, ��ti�ulation o� the T cell rece�tor (TC�) i�� �ollo�ed b�� ra�id �a� acti�ation (72).WorkfromdifferentgroupssuggestedthatRapiscrucialforthecontrolof integrin-mediatedcelladhesion(14,73,74).“Inside-out”signallingistheregulatedintegrin-mediatedadhesion by intracellular signals. It has been proposed thatRap1 couples diverse stimulisuchasTCR(75),phorbolester (76),CD31(14),CD98 ligation (77)andSDF-1 (78,79)tointegrinactivationthrough“inside-out”signalling.OntheotherhandRapisatargetof��ignal�� generated in re���on��e to the binding o� integrin�� to their extracellular ligand��, ter�ed “outside-in”signalling(80).Thissuggestsapotentialpositivefeedbackloopthatcanfurtherenhanceintegrinfunction.Inagreementwiththestudiesintissueculturecells,lymphocytesfromRap-/-miceshowimpairedcelladhesion(81).Rap in T-cell migrationThe l����hoc��te tran���igration �ro� �e����el�� into ��urrounding ti����ue�� i�� controlled b�� adhesive interactionwith thevascularendothelium.Chemokinesandadhesionmolecules,suchasintegrins,mediatetheadhesiveinteractionsandsubsequentlyinfluencetherolling,

Chapter 1

�4

firmadhesionandtransmigrationprocesses.DuringlymphocyteemigrationRapisactivatedupon chemokines stimulation within seconds and triggers rapid integrin activation (78).�n acti�e �a� �utant can tran���or� l����hoc��te�� into �olari�ed cell�� and ��ti�ulate cell migrationovertheadhesionmoleculesICAM-1andVCAM-1.Rapactivationwasshowntobecriticalforfirmattachmentoflymphocytesandthesubsequenttransmigrationthroughthevascularendothelium.ApivotalroleofRap1wassuggestedinregulationofchemokine-inducedlymphocyteextravasation(82).

ROS suppression�eacti�e Ox��gen S�ecie�� (�OS) re�re��ent a heterogeneou�� grou� that include�� ox��gen anion�� and radicals or hydrogenperoxide (83).Diverse stimuli increase the intracellular oxygenradical�� and �ro�ote cellular e�ent��, ��uch a�� �roli�eration, gene acti�ation, cell c��cle arre��t andapoptosis(84).InTlymphocytes,transientproductionofROSmodulatestranscriptionaland proliferative responses to TCR signalling (85). In contrast, chronic oxidative stressresults ina reducedproliferative responseandanenhanced transcriptionof inflammatorygeneproducts.Ras/Ralsignallingpathwayhasbeenshowntoberequiredandsufficienttomediatephorbolester-inducedROSproduction(85).Rap1isalsoactivatedinTlymphocytesuponTCRstimulation.Inaphosphatidylinositol3’-kinase(PI3K)-dependentmanner,Rap1suppressesagonist-andRas/Ral-inducedROSproduction,whereasithasnoeffectonbasalROSproduction(85).AlthoughthePI3K-dependentROSsuppressionwasdemonstratedbyu��ing a PI�K inhibitor (LY2�4002), no direct a����ociation bet�een �a�� and PI�K in T cell�� hasbeenobserved.ThissuggestsaninvolvementofaPI3K-dependentunknownRapeffectorforROSsuppressioninTlymphocytes.Rap1 effectors in T-cellsRAPL�egulator o� �dhe��ion and cell Polari�ation enriched in L����hoid ti����ue�� (��PL) �a�� i��olated inayeasttwo-hybridscreenfromahumanleukocytecDNAlibraryusingRap1A-G12V,aconstitutivelyactiveformofRap,asabait(86).RAPLencodesa256aminoacidsproteinthat con��i��t�� o� a �a�� binding domain(RBD)andaC-terminalcoiled-coildomain.RAPLwasfirstidentifiedasanalternativespliceproductoftheRassF5genethatispredominantly

Fig. 4 Rap1 downstream signaling effects in T cells. �I�M and ��PL ha�e been re�orted to inter�actwithRapandfunctionaseffectorsonadhesion.Requirement for Rap1 activity in several cellular�roce����e�� ha�� been re�orted ��uch a�� �eacti�e Ox���gen S�ecie�� (�OS) ��u��re����ion, cell adhe��ion and cellmigration.

RAPLRIAM

Adhesion

Rap1-GTP

?

Migration ROS production

Ras-GTP

RAL

General introduction

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expressedinlymphoidtissues(87).InTcells,uponRap1activationRAPLformsacomplex�ith L���� and re�locali��e�� together �ith it�� e��ector M��t�� to the leading edge o� �olari�ed T cells(88).PlasmamembranelocalisedMst-1increasestheadhesivenessoftheTcellintegrinLFA-1toICAM-1.ARAPLmutantthatisdeficientinRap1-bindingblocksRap1-,TCR-,and chemokine-dependent adhesion. In agreementwith the results obtained from studiesbasedonoverexpressionofRAPL,lymphocytesderivedfromRAPLknock-outmiceshowreduced adhe��ion, de�on��trating the e����ential role �or ��PL in integrin��ediated adhe��ion (89).Inadditiontointegrinactivation,RAPLwasalsoreportedtobecapableofinducingcell�olari�ation o� l����hoc��te�� and enhancing �a��de�endent �igration o� �a��cular endothelial cells (90). Interestingly, the restricted expression of RAPL indicates that it is unlikelyresponsibleforRap1inducedadhesioninnon-haematopoieticcells.RIAM�a���Interacting �da�tor Molecule (RIAM) was identified in a yeast two-hybrid screenusinganotherconstitutivelyactivateformofRap,Rap1A-Q63E,asabaittoscreenaJurkatcDNAlibrary(91).RIAMcontainsanRAdomain,apleckstrinhomology(PH)domainandseveralproline-richsequences.InJurkatcells,over-expressedRIAMinducesRap-inducedintegrin-mediatedadhesion.ThisadhesionwasabolishedbytheknockdownofRIAMwithsiRNA.InterestinglyRIAMalsointeractswithprofilinandEna/VASPproteins,whicharemodulatorsofactindynamics.InRIAMknockdowncellsthecontentofpolymerizedactinisreducedandRap1isnotrecruitedtotheactincytoskeletonatthesitesofadhesionanymore,indicatingaroleforRIAMinactindynamics.Insights from mouse modelsTo study the Rap effects on lymphocytes in living organisms, Rap1 knockout mice ortransgenicmiceoverexpressingconstitutivelyactiveRap1mutantsweregenerated.StudiesusingRap1A-G12VtransgenicorRap1AknockoutmicedemonstratedthatRapaugmentsTcellactivationviatheenhancementofintegrin-dependentadhesion(74,81).TlymphocytesfrommicethatconstitutivelyexpressRap1A-G12VshowanenhancedTCRresponse(74).InRap1Adeficientmicenosignificantdefectsinlymphoidcellsdifferentiationormaturationwere observed (81). Intriguingly, studies on tissue culture T cells from either SPA-1-deficientorRap1A-Q63E transgeneticmicemodels suggested thatRap1 is involved inTcell�� unre���on��i�ene���� and anerg��, a ��tate in �hich T cell�� beco�e re�ractor�� to ��ti�ulation (92,93).SPA-1deficientmicedevelopedanage-dependentTcelldysfunctionprecedingthemyeloiddisorders(92).HelperTcellsfrommicethatexpressRap1A-Q63EshowanincreaseofCD4+CD103+regulatoryTcellsfraction.These T cells exhibit a potent inhibition of T cellsThe��e T cell�� exhibit a �otent inhibition o� T cell�� proliferation and IL-2production (93-95).Together, these evidences suggested that Rap1Together, the��e e�idence�� ��ugge��ted that �a�� playsaroleasanegativeregulatorofTcellsfunction.Thediscrepancyfromdifferentmice�odel�� could be due to di��erent T cell�� u��ed (�6), e��ect�� �ro� di��erent �a� �utant�� (��) or differenttransgeneticsystemsusedineachstudy(96).

Chapter 1

�6

ERM proteins The ERM family consists of three closely related proteins, Ezrin, Radixin andMoesin.Radixinwasoriginallyisolatedasaconstituentofadherencejunctionsinratliver(97);Ezrinwasoriginally identifiedasacomponentof intestinalmicrovilli (98,99);andMoesinwasidentifiedas aheparin-bindingprotein inbovineuterus smoothmuscle cells (100). ��M proteins belong to the erythrocyte protein 4.1 super-family,which are characterized by acon��er�ed N�ter�inal �our��oint one, ��rin, �adixin, Moesin(FERM)domain.Theglobular���M do�ain, �hich ha�� been re�orted to bind �rotein�� and li�id�� (�0�), contain�� three ��ub�domains(F1,F2andF3)(Fig.5).F1adoptsanubiquitin-likefold,F2isstructurallysimilartotheacyl-CoAbindingproteinandF3showsstructuralhomologytoPHdomains.InERM�rotein��, the ���M do�ain i�� �ollo�ed b�� an al�ha�helical and a charged C�ter�inal region withafilamentousactin(F-actin)bindingsite.Bio�h����ical and ��tructural anal����i�� ha�e ��ho�n that the C�ter�inal region o� ��M �rotein�� caninteractwiththeN-terminalFERMdomain(102-105).Thisinternalinteractionresultsinaclosedconformationreferredtoas“inactive”.DisruptionofthisinteractionswitchstheERMproteinstoanopenor“active”state.TheopenconformationallowsERMproteinstointeractwithotherproteinsandtofunctionasscaffolds(Fig.6).Theequilibriumbetweentheopenandtheclosedconformationisinfluencedbyseveralregulatorysignals.Regulation of ERM proteinsPhosphorylationofthreonine558ofMoesinwasfirstnotedduringthrombinactivationofplatelets (106).Thephosphorylationof thecorrespondingresidues,T564forRadixinandT�67 �or ��rin, �ere �ound to reduce the interaction bet�een the ���M�do�ain and the C-terminalregion.IndeedT558islocalisedwithintheinteractionsurfacebetweenthetwohalves of the protein.Overexpression of phosphorylation-mimickingmutants induces theformationofcellsurfacestructures(107).Rhokinase(ROCK)(108,109),thetwoisoformsofproteinkinaseC(PKC),PKCa (��0) and PKCq (111),andProteinkinaseB(PKB)(112),ha�e been ��ho�n to �ho���hor��late thi�� con��er�ed threonine and ��tabili�e ��M in the o�en form.The ���M do�ain bind�� �ho���hatid��lino��itol 4, ��bi���ho���hoate (PIP2), �hich i�� in�ol�ed intheactivationofERMproteins(113,114).Itiscurrentlyunclearwhetherthis“activation”

Fig. 5 Domain structure of ERM proteins. ��M �rotein�� contain a ���M do�ain on the N-terminus followed by an α-helix anda C�ter�inu�� tail do�ain (CTT), �hich can associate with the FERM domain. The last~�0 re��idue�� in the CTT are in�ol�ed in actin�binding. FERM, Four-point one, Ezrin, Ra�dixin,Moesin.F1,F2andF3aresub-domainso� the ���M do�ain and the�� are ��tructurall�� similar to an ubiquitin-like fold, acyl-CoAbindingproteinandPHdomain,respectively.

CTTFERMABRF1 F2 F3

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General introduction

�7

i�� due to the induction o� the o�en con�or�ation or due to a PIP2 induced tran��location o� ��M �rotein�� to PIP2enrichedmembranecompartments.Forexample,evidenceexiststhatthe ���M do�ain can bind PIP2 e�en though it i�� bound to the C�ter�inal actin binding domain.(113).ComparedtotheactivationofERMproteins,littleisknownabouttheirinactivation.Myosinlight chain �ho���hata��e ha�� been ��ho�n to bind to Moe��in and de�ho���hor��late the critical carboxy-terminalthreonine(115).Inaddition,apoptosisinducesdephosphorylationofEzrin (116). Besides dephosphorylation it has been shown that Ezrin is sensitive to calpain, aproteaseregulatedbyintracellularcalcium(117).MorerecentstudieshaveshownthatinTcellsstimulationbychemokinessuchasstromalderivedfactor1alpha(SDFa�) or ��econdar�� lymphoidtissuecytokineresultinthedephosphorylationofERMandalossofmicrovilliinaRac1dependentmanner(118,119).Biological function of ERM proteinsThelocalizationofERMproteinshasbeenanalyzedinvarioustissuesandculturedcelllines.In �o��t cell�� all three �a�il�� �e�ber�� o� ��M �rotein�� are co�locali��ed at actin�rich ��ur�ace structures suchasmicrovilli, lopodia andmembrane ruffles (120,121). It is interesting tonotice that �adixin i�� al��o locali��ed to adherent junction and �ocal contact ��ite��, �here ��rin andMoesinareabsent(122).TheexpressionpatternofERMproteinsvariesamongtissues.��rin i�� �ound �ri�aril�� in e�ithelial and �e��othelial cell�� in inte��tine, ��to�ach, lung and kidney.Moesinisenrichedinendothelialandhomatopoieticcellsandfoundonhighlevelsinlungandspleen.Radixinishighlyexpressedinliverandintestine(122).

Fig. 6 A working model for the activation and function of ERM proteins. �ollo�ing activation to unmask binding sites, FERMdo�ain and the C�ter�inu�� region are a�ail�able to bind to �e�brane �rotein�� or actin, respectively.A,Throughscaffoldmolecules��uch a�� �BP�0 and ��K��P, ��M �rotein�� linkmembranetoactincytoskeleton.B,Ac�ti�ated ��M �rotein�� can al��o direct �ro�ide alinkagebetweenactinandtransmembraneproteins. NHE, Na+/H+ exchanger; PIP2,Phosphatidylinositol 4, 5-bisphosphoate;CFTR, cystic fibrosis transmembrane con�ductanceregulator;C,carboxy-terminaldo�main.Modifiedfrom(138).

Multiple pass membrane proteini.e. NHE3, CFTR

Single pass membrane proteini.e. NHE1, CD44,ICAM2

FERM

C

EB

P50 Plasma membrane

Actin filaments

C

FERM

FERM

C

FERM

C

Activation signalsi.e. PIP2 binding and phosphorylation

A B

Chapter 1

�8

Insight from genomic and geneticsKnockdownoftheindividualERMproteinsinculturedcellsinducesnosignificantphenotypicchangesonmicrovilli,cell-cellcontactsorcell-substratecontacts.Furthermore,micethataredeficientinoneoftheERMproteinsdonothaveanydiscernablephenotype(123).Theseresults indicate similar or redundant functions between the ERM familymembers. ERMproteins are conservedduringevolution.HumanEzrin,Radixin andMoesin share ahighdegree o� a�ino acid identit�� to the ��ingle �or� o� ��M �rotein�� in Drosophila(Dmoesin)and C.elegans (ERM-1) (124,125). Without the influence from functional redundancy,C.elegans and Drosophila ha�e been u��ed a�� alternati�e ������te��� �or the genetic ��tud�� o� ��M proteins.InC.elegans,ERM-1isrequiredforapicaljunctionremodellingandtubulogenesisintheintestine(125).InDrosophila,Dmoesinisinvolvedindevelopmentalprocessessuchaspolaritycontrol,cytoskeletalorganizationandadhesion(124).Cytoskeleton-membrane linkingERMproteinswereoriginallyproposedtobecytoskeleton-membranelinkingproteinsbecauseoftheirenrichmentincell-surfacestructuresandactin-bindingability.TheinteractionbetweenERMproteinsandtheactincytoskeletonhasbeenoriginallystudiedwithEzrin(117,126,127).Subsequently,theF-actinbindingsitewithinthecarboxy-terminal30residueswasidentifiedtobeacommonfeatureofERMproteins(108,128-130).Ontheamino-terminusofERMproteins,FERMdomainisresponsibleforthemembranebinding.Bothdirectassociationof���M do�ain �ith the c��to�la���ic tail�� o� tran���e�brane �rotein�� and indirect a����ociation with transmembraneproteins throughadaptorproteinshavebeenreported.ManyproteinsthatinteractwithERMproteinsareadhesionreceptors.Forexample,EzrininteractswiththeIntercellular �dhe��ion Molecule�2 (IC�M�2) and thi�� interaction �acilitate�� the recruit�ent ofICAM-2intouropod,thecellrearofmigratingleukocytes(131,132).Inadditiontothedirect a����ociation �ith the c��to�la���ic tail�� o� �e�brane �rotein��, ��M �rotein�� can al��o interactwithmembraneproteinsindirectlyviawithEBP50/NHEandE3KARP,whichwillbediscussedlater.ERM proteins participate in Rho signallingERM proteins play a crucial role in the cellular cytoskeletal response to Rho-pathwayactivation. It was first observed that in response to Rho activation, the soluble pool ofc��to�la���ic ��M �rotein�� redi��tribute to the �la���a �e�brane and induce �icro�illu�� formation (133-135).The activation of ERM proteins can be achieved by a rise of PIP2

concentration.PIP2��roducing �ho���hatid��lino��itol 4��ho���hate � kina��e (PI4P�K) i�� a direct Rhoeffector(136).IthasbeensuggestedthatRhoactivationactivatesPI4P5Kandincreasesthe PIP2concentration.Moreover,theinteractionbetween�ho GDPDi����ociation Inhibitor (RhoGDI),apotentsequesteringfactorofRho,andERMproteinswasshown(105).ThisinteractionsuppressestheGDIactivityandreleasesinactiveRhofromRhoGDI,allowingRhotobecomeactivatedbyitsexchangefactors.Inaddition,ERMproteinspromoteRho

General introduction

��

activation through interactionwith the tumour suppressorhamartin (137).Together, thesefindings suggested that ERM proteins participate in the activation of Rho, which againactivatesERMproteinsasapositive feedbacksystem.Therefore,ERMproteinsarebothdownstreamandupstreamofRho.Involvement of ERM in Na+/H+ exchanger function and membrane traffickingActivatedEzrin,RadixinandMoesinhavebeenshowntoconnectactinfilamentstomembranechannelsandreceptors(138,139).Na+/H+ exchanger�� (NH���) are Na+ channel�� that �la�� a crucial role in Na+absorption,acid-basehomeostasisandcellvolumeregulation(140).NHEtype3kinaseAregulatoryprotein(E3KARP),��rin binding �ho���ho�rotein (�BP�0) and it�� rabbit ho�olog Na+/H+exchangerregulatoryfactor(NHE-RF)arePDZdomaincontainingproteins,whichbindthroughthePDZdomaindirectlytotheFERMdomainofERMproteins(104,138).TheyfunctiontherebyasscaffoldproteinsandprovidealinkbetweenNHE3andEzrin.EzrincanalsointeractwithPKA(141).ItwassuggestedthatEzrinrecruitsPKAinthe �icinit�� o� the c��to�la���ic do�ain o� NH�� �here PK� �ho���hor��late�� and inhibit�� NHE3actively(142).Recently,anothermechanismwassuggested, inwhichP38 triggersthe �ho���hor��lation o� PKB through M�PK��cti�ated Protein Kinase-2 (MAPKAPK-2).Thi�� re��ult�� in a PKB�de�endent �ho���hor��lation o� ��rin on T�67 and the acti�ated ��rin facilitatesitsmembranetranslocationandtheactivationofNHE3(112,143).Interestingly,inde�endent o� it�� �unction a�� an ion exchanger, NH�� ha�� been re�orted to directl�� interact with ERM proteins and regulate cortical cytoskeleton and PKB-dependent cell survival(144,145).Be��ide�� the in�ol�e�ent in the regulation o� the Na+/H+ exchanger, ��rin �a�� recentl�� connectedtoadrenergicreceptorrecycling(146).ThedirectinteractionbetweenEzrinandadrenergicreceptorcontributestoreceptorrecyclingtotheplasmamembrane.Adhesion The functionofERMproteinsonadhesionwasfirstnotedby theadhesiondeficiencyofepithelialcellsthatweredepletedofERMproteins(147).Moreover,overexpressionofERMproteins enhances cell adhesion (148). ERM proteins can regulate cell adhesion throughdifferentmechanisms.Asmentionedbefore,ERMproteinsareinvolvedintheregulationofRhosignalling,whichcontrolstheactincytoskeletonremodelling.ThephosphorylationofEzrinbyROCKisrequiredforRho-dependentfocaladhesionassembly(109).Furthermore,the binding bet�een the TSC� tu�our ��u��re����or ha�artin and acti�ated ��M �rotein�� i�� requiredforfocaladhesionformationuponRhoactivationsignal(137).ERMproteinscanalsointeractwiththecytoplasmictailsofadhesionmoleculessuchasCD44,ICAM-1andICAM-2(131,149)andrecruitthemtothecellmembrane.ERM proteins and human diseaseRadixinknockoutmicedisplayabreakdownofhepatocyteapicalmicrovilli,whichleadstoamildliverinjury(150).HamartinhasbeenlinkedtoERMproteinsincelladhesionregulation

Chapter 1

20

andhamartomadevelopment(137).RecentlyEzrinwasidentifiedasacrucialmoleculeinthedisseminationoftwopediatriccancermetastasis(151,152).Ezrinexpressionlevelishigherin metastatic cells compared to non-metastatic control cells (153,154). OverexpressionofEzrin in low-metastatic cells significantly enhanced themetastatic capacity.Moreover,knockdownofEzrinbyRNAidemonstratesthatahighlevelofEzrinexpressionissufficientformetastaticprogression(151,152).AlthoughtheimportanceforEzrinintumourmetastasisisunambiguouslydemonstrated,theunderlyingmechanismsareunknown.Furthermore,thisfeatureisnotsharedbytheothertwoERMfamilymembers.RadixinandMoesinwerefoundto be do�n�regulated in lung adenocarcino�a��, ��ugge��ting a tu�our ��u��re����or �unction o� thesetwomolecules(155).

Scope of this thesisThe ���all G��rotein �a� centrall�� regulate�� integrin �ediated cell adhe��ion and the �or�ation ofE-cadherinbasedcell-celljunctions.Tothisend,Rapintegratestheinputofavarietyofsignals,which activateRap viaRap specific guanine nucleotide exchange factors.TheseGEFsareinpartregulatedbysecondmessengersandarepartofaprotein-proteinnetwork,whichregulatestheirtemporalandspatialactivities.SeveraldownstreameffectorsofRapmediatethecellularfunctionofthissmallG-protein.TheworkdescribedinthisthesisdealswiththeanalysisoftheRapsignallingnetwork.Chapter2andchapter3focusonthedownstreameffectsofRap.Inchapter2therelationbetweenAF6andRapisanalysed.AF6wasreportedtobeaneffectorofRapduetoitsabilitytobindspecificallyto theactiveGTP-boundformofRap.Herewedemonstrate thatAF6�unction�� to bu��er GTP��a� in re��ting cell��, �aintaining it in a non��roducti�e co��lex and therebynegativelyregulatesRapfunctioninT-cells.Inchapter3theanalysisofthreeRap-likepseudogenes(mRap1A-retro1,mRap1A-retro2andhRap1B-retro)inmouseandhumangenomeisdescribed.Allthreeretrogenesareexpressedandencodefunctionalproteins.Theseproteinshavean increasedGTP/GDPbindingratiocomparedtowildtypeRap1.Moreinterestinglytheyexhibitcleardifferencesintheirabilitytoinducecelladhesionandspreading.To gain more insight in the protein interaction network, which controls the spatial andtemporalorganisationofRapspecificGEFsweperformedyeasttwo-hybridscreensusingEpacandPDZ-GEFasbaits.Addendum1givesageneraloverviewoftheresultsobtainedfromthisapproach.RadixinandEzrinbelongtotheproteinsthatwereidentifiedtointeractwithEpac1inthetwo-hybrid.This interaction is characterised in detail in chapter 4,which focuses on theinteractionbetweenEpacandEzrin.AshortRadixin/Ezrinbindingregionwasidentifiedinthe N�ter�inu�� o� ��ac� and it �a�� ��ho�n that ��ac can onl�� interact �ith the o�en, acti�e conformationofEzrin.Furthermore,we show thatEzrin couples the activationof the β-

General introduction

2�

adrenergicreceptortoRap1signallingviatherecruitmentofEpac1.Chapter5focusesontheinteractionbetweenEpacandRadixin.AnovelRadixinmutantischaracterized.ThisRadixinmutant fulfils the classicalbiophysical criteriaofbeing in anactivestate.

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