ARAP, a Novel Adaptor Protein, Is Required for TCR Signaling and Integrin-Mediated Adhesion

12 Pages·2016·1.62 MB·English

ARAP, a Novel Adaptor Protein, Is Required
for TCR Signaling and Integrin-Mediated
Adhesion
Seung Hee Jung, Eun Hye Yoo, Mi Jin Yu, Hyeon Myeong
This information is current as
Song, Hee Yoon Kang, Je-Yoel Cho and Jong Ran Lee
of April 7, 2019.
J Immunol 2016; 197:942-952; Prepublished online 22 June
2016;
doi: 10.4049/jimmunol.1501913
http://www.jimmunol.org/content/197/3/942
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Supplementary http://www.jimmunol.org/content/suppl/2016/06/22/jimmunol.150191 adad
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References This article cites 47 articles, 24 of which you can access for free at: h h
http://www.jimmunol.org/content/197/3/942.full#ref-list-1 ttpttp
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The JournalofImmunology
ARAP, a Novel Adaptor Protein, Is Required for TCR
Signaling and Integrin-Mediated Adhesion
Seung Hee Jung,* Eun Hye Yoo,*,† Mi Jin Yu,*,† Hyeon Myeong Song,* Hee Yoon Kang,*
Je-Yoel Cho,‡ and Jong Ran Lee*,†
AnoveladaptorproteinwasidentifiedbyanalyzingphosphotyrosineproteomesfrommembraneraftsofactivatedTcells.This
proteinshowedsequencesimilaritytoawell-knownTcelladaptorprotein,adhesionanddegranulation-promotingadaptorprotein
(ADAP);therefore,thenovelproteinwasdesignatedactivation-dependent,raft-recruitedADAP-likephosphoprotein(ARAP).Sup-
pression of ARAP impaired the major signaling pathways downstream of the TCR. ARAP associated with the Src homology 2
domain of Src homology 2–containing leukocyte protein of 76 kDavia the phosphorylation of two YDDV motifs in response to
TCRstimulation.ARAPalsomediatedintegrinactivationbutwasnotinvolvedinactinpolymerization.Theresultsofthisstudy
indicatethatanovelTcelladaptorprotein,ARAP,playsauniqueroleinTcellsasapartofboththeproximalactivationsignaling
andinside–outsignalingpathwaysthatresultinintegrinactivationandTcelladhesion. TheJournalofImmunology,2016,197:
D
942–952. o
w
n
A lo
ntigen recognition by T cells occurs at a junction of a (18–21). Upon TCR stimulation, ZAP-70 translocates from the cy- a
d
TcellandanAPCknownastheimmunologicalsynapse toplasm to membrane rafts; there, it phosphorylates signaling mole- ed
(IS)andleadstosignalingeventsinTcells(1–5).After cules that contain several phosphotyrosine sites and protein–protein fro
triggering TCRsignaling, actinpolymerization and inside–out interactiondomains,resultingintherecruitmentofothercytoplasmic m
h
signaling follow at the T cell contact site (6, 7). Adhesion is signalingmoleculestotherafts(18–21). ttp
mediated by the binding of LFA-1 to a family of adhesion mol- Toanalyzethephosphotyrosineproteinsthatareconcentratedin ://w
ecules, including ICAM-1, by an F-actin–dependent mechanism andrecruitedtomembraneraftsataTCR-mediatedactivationstate, w
(8–11), and therefore Tcells become more adhesive. raftswereisolatedbytheultracentrifugationofJurkatTcelllysates w
Previousstudieshavedemonstratedtherolesofmembranemicro- in a sucrose density gradient and the subsequent collection of .jim
domains,alsocalledglycolipid-enrichedmicrodomainsordetergent- floating fractions (18). Phosphotyrosine proteins were then iso- m
u
insoluble rafts, in the initiation of TCR-mediated signaling (12–17). lated by an immunoprecipitation (IP) of the raft fractions using no
ISformationoccursatrafts,andavarietyofsignalingmoleculesare anti-phosphotyrosineAbs.Furtherseparationandidentificationof l.o
rg
concentrated within rafts; these signaling molecules include the Src the proteins were accomplished using liquid chromatography– b/
familykinaseLck,theTCR,theCD4andCD8coreceptors,andthe tandem mass spectrometry (LC-MS/MS)(22,23). y
g
transmembraneadaptorproteinlinkerforactivationofTcells(LAT) Inthisstudy,wereportanoveladaptorproteinthatisrecruitedto u
e
membraneraftsandisinvolvedinearlyTcellactivationafterTCR st o
stimulation. This 83-kDa phosphotyrosine protein shares sequence n
*DepartmentofLifeScience,CollegeofNaturalSciences,EwhaWomansUniver- A
sity,Seoul03760,Korea;†ResearchCenterforCellularHomeostasis,EwhaWomans similarity with the adhesion and degranulation-promoting adaptor p
UlengieveorfsiVtye,teSrienoaurly0M37e6d0ic,iKneo,reSae;oaunldN‡aDtioepnaarltmUnenivteorsfitVy,etSeerionualry08B8i2o6c,hKemoriestary,Col- pderositeginnat(eAdDaActPiv)at(i2o4n,-d2e5p);entdheenret,forraef,t-trheicsruniotevdelAaDdaApPto-lrikperoptheionspwhaos- ril 7, 2
ReceivedforpublicationAugust27,2015.AcceptedforpublicationMay19,2016. protein(ARAP).ARAPcontainsseveralmodulardomains,suchasa 01
9
ThisworkwassupportedbytheNationalResearchFoundationofKoreafundedbythe proline-rich(PR)domain,alysine-richdomain,andanSrchomology
Korean Government (Grants 2009-0074129, 2011-0016543, 2012R1A5A1051354,
(SH)3 domain. It also contains multiple phosphotyrosine sites, in-
and2015R1A2A2A01004209toJ.R.L.).S.H.J.andH.M.S.weresupportedinpart
bytheBrainKorea21ProjectoftheKoreanMinistryofEducation. cludingtyrosinemotifs(YDDV)(seeFig.1A).WeshowthatARAP
ThehumanandmousecDNAclonespresentedinthisarticlehavebeensubmittedto associateswiththeSH2domainofSH2-containingleukocyteprotein
GenBankunderaccessionnumbersNM_001004303andNM_001162980. of 76 kDa (SLP-76) via the phosphorylation of specific tyrosine
AddresscorrespondenceandreprintrequeststoProf.JongRanLee,Departmentof motifsinARAP.WealsodemonstratethatARAPisnotinvolvedin
LifeScience,CollegeofNaturalSciences,EwhaWomansUniversity,ScienceBuild-
actinpolymerizationbutisrequiredforTcellactivationviatheTCR
ingC-407,52Ewhayeodae-gil,Seodaemun-gu,Seoul03760,Korea.E-mailaddress:
[email protected] andforinside–outsignalingduringintegrinactivation.Basedon
Theonlineversionofthisarticlecontainssupplementalmaterial. these results, we propose that ARAP is an important new com-
ponentofTCR-mediatedsignalingandplaysapositiveregulatory
Abbreviations used in this article: ADAP, adhesion and degranulation–promoting
adaptorprotein;ARAP,activation-dependent,raft-recruitedadhesionanddegran- role inTcellactivation.
ulation–promotingadaptorprotein–likephosphoprotein;CMTMR,5-(and-6)-(((4-
chloromethyl)benzoyl)amino)tetramethylrhodamine;CT,C-terminalregion;IB,
immunoblotting;IP,immunoprecipitation;IS,immunologicalsynapse;LAT,linker Materials and Methods
foractivationofTcells;LC,liquidchromatography;LC-MS/MS,liquidchromatog-
Abs andreagents
raphy–tandemmassspectrometry;MS,massspectrometry;NT,N-terminalregion;
ORF,openreadingframe;PLC,phospholipaseC;PR,proline-rich;PV,pervanadate;
Anti-human CD3 mAb (UCHT1; BD Pharmingen, San Diego, CA) was
SEE,staphylococcalenterotoxinE;SH,Srchomology;shRNA,shorthairpinRNA;
SKAP, Src kinase–associated phosphoprotein; SLP-76, Src homology 2 domain– usedforcellstimulation.ForIPandimmunoblotting(IB),weusedanti-LAT
containingleukocyteproteinof76kDa;WT,wild-type. polyclonalAb,anti–phospholipaseC(PLC)g1,andanti-phosphotyrosine
(4G10) mAbs (Upstate Biotechnology, Lake Placid, NY), anti–SLP-76,
Copyright(cid:1)2016byTheAmericanAssociationofImmunologists,Inc.0022-1767/16/$30.00 anti-Akt, anti-ERK2, anti-Rac1, anti-Rap1, anti-GST, anti-Lck, anti-Fyn,
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501913
TheJournal ofImmunology 943
anti–b-actin, and anti–phospho-PLCg1 (pPLCg1, Y783) polyclonal Abs Celllines,cellculture,andTcellactivation
(Santa Cruz Biotechnology, Santa Cruz, CA), anti-HA and anti-FLAG
(M2)mAbs,peroxidase-conjugatedcholeratoxinBsubunitfordetection Human Jurkat T leukemia cells, additional Jurkat derivatives created
of ganglioside GM1, andBSA (Sigma-Aldrich,St Louis, MO),anti–Src usingthepSuperiorvectorsuppressingARAPandpCMS4suppression/
kinase–associated phosphoprotein (SKAP) 1 polyclonal Ab (BD Trans- reexpressionvectorsuppressingARAP/reexpressingvariousmutantforms
ductionLaboratories,FranklinLakes,NJ),andanti–phospho-Akt1(pAkt, ofARAP,andRajiBcellswereculturedinRPMI1640mediumcontaining
S473),anti–phospho-ERK1/2(pERK,T202/Y204),andanti-phosphotyrosine 10%heat-inactivatedFBS,100U/mlpenicillin,100mg/mlstreptomycin,
(p-Tyr100)mAbs(CellSignalingTechnology,Danvers,MA).Wegenerated and20mMglutamine.Celllinesweremaintainedat37˚Cwith5%CO2/
a mouse polyclonal anti-ARAP antisera using purified GST–ARAP–C– 95% O2. For activation, Jurkat T cells and derivatives werewashed and
terminal region (ARAP-CT, aa 546–728) as the immunogen. Secondary resuspendedinRPMI1640mediumat13107cells/mlandincubatedwith
Abs,anti-mouseIgG,anti-rabbitIgGconjugatedwithHRPfromBio-Rad eithercontroloranti-CD3Abforindicatedtimesat37˚C.
(Hercules, CA), and anti-Armenian hamster IgG from Jackson Immuno-
Expression ofplasmidDNA
ResearchLaboratories(WestGrove,PA)wereused.
Ficoll-Paque Plus, ECL reagents, and GammaBind G Sepharose Togeneratestablecelllines,JurkatTcellswereelectroporatedusinga
werepurchasedfromAmershamPharmaciaBiotech(ArlingtonHeights, Gene Pulser (Bio-Rad)at asettingof 250Vand950 mFdincuvettes
IL).CellTrackerOrange5-(and-6)-(((4-chloromethyl)benzoyl)amino) containing23107cellsin0.4mlofcytomixintracellularbuffer(27)and
tetramethylrhodamine (CMTMR) and fura-2 acetoxymethyl ester (fura- 40mgofplasmid.After7dinculture,cellsweresortedforexpressionof
2AM)werepurchasedfromMolecularProbes(Eugene,OR).FITC-ortet- GFPusingaFACSAria(BDBiosciences,MountainView,CA).Afteran
ramethylrhodamine isothiocyanate–phalloidin, polyethylenimine branched, additional2wkinculture,cellsweresortedagaintoobtainstabletrans-
laminin,poly-L-lysine,polyoxyethylene(20)oleylether(Brij98),octyl-b-D- fectants showing GFP fluorescence. Similar levels of surface TCR and
glucopyranoside, glutathione-agarose, and luciferin were obtained from LFA-1expressionwereconfirmed.TransientexpressionofplasmidDNA
Sigma-Aldrich. Superantigen staphylococcal enterotoxin E (SEE) from inHEK293Tcellswaspreviouslydescribed(28).HEK293Tcellswere
ToxinTechnology(Sarasota,FL),humanfibronectinfromBDBiosciences transfectedwithamixtureofeachofthevariousexpressionplasmidswith
(FranklinLakes,NJ),andICAM-1–FcchimerafromR&DSystems(Min- polyethyleniminesolution.
neapolis, MN) were also used. A pan T cell isolation kit II from Miltenyi D
o
Biotec(BergischGladbach,Germany)wasusedfordepletionofnon–Tcells. HumanTcellpurificationandactivation w
n
Plasmidsandcloning Peripheralbloodsampleswerecollectedfromnormalhealthydonorsafter loa
d
written informed consent was obtained from all donors. All studies were e
ToclonetheARAPcDNA,afullARAPopenreadingframe(ORF)clone d
(DKFZp686O04253Q)waspurchasedfromImaGenes(Berlin,Germany). aUpnpirvoevrseidtybyMthedeiIcnasltiCtuetniotenra.lTHhuemiasonlaEttihoincsoRfemvioenwonBuocalerdarofceElwlshaanWdoTmcaenlsl fro
ThehumanARAPcDNA(ARAP–wild-type[WT])wasgeneratedusinga m
PCR with oligonucleotide primers59-CGCGGATCCATGAAGGGGAA- fractionusingFicoll-PaquePlus densitycentrifugation followedby deple- h
GGG-39and59-CCGCTCGAGCTAAGGTGACCAACTTTG-39andligated tionofnon–Tcellswithsuperparamagneticmicrobeadswasdescribedpre- ttp
intopCMV-Tag2B(Stratagene,LaJolla,CA)atBamH1andXhoIsites. vreisotuesdlyat(2397)˚.CI.soFloarteadctTivacteilolns,wceerllesr(e1su3sp1e0n7d/emdl)inwRerPeMinIc1u6b4a0tedmweditihumeitahnedr ://w
PCRproductswereverifiedwithDNAsequencing.TheN-terminalregion w
controlAboranti-CD3Ab(5mg/ml)for5minat37˚C.
ofARAP(ARAP-NT,aa1–545)andtheCTofARAP(ARAP-CT,aa546– w
728) were created from ARAP-WT by restriction digests using BamHI/ IPandIB .jim
PstIandPstI/XhoI,respectively,andclonedbetweentherespectivesitesin m
the vector pCMV-Tag2B. The GFP-conjugated plasmids for ARAP-WT, Tcellstimulationwasterminatedbyaddinganequalvolumeofice-cold u
n
-NT, and -CT were generated by restriction digests of pCMV-Tag2B/ medium,andcelllysateswerepreparedin1%NonidetP-40lysisbuffer o
ARAP-WT,-NT,and-CTfollowedbyligationatBglIIandSalIsitesin- containing protease and phosphatase inhibitors, as described previously l.o
troatothrieesm,PamalmoaAlilaton,GCFAP).expression plasmid pEGFP-C1 (Clontech Labo- s(3am0)p.lFeobruIfBfe,r,lybsoaitleesdf,raonmd1su3bje1c0te6dcetolls8,w1e0r,eomri1x4e%dwSiDthS-2P3AGLEa.emFomrlIi’Ps, byrg/
Short hairpin RNA (shRNA) for ARAP (shARAP) was generated by lysatesfrom103106cellsweretumbledwithGammaBindGSepharose g
u
cloning19nucleotidesofhumanARAP(nucleotides979–997,59-CCA- beadsconjugatedwithindividualAbasindicated.Theimmunecomplexes e
s
CATAATTACGAGGCAA-39) into the pSuperior vector (Oligoengine, werethensubjectedtoSDS-PAGE,followedbyIB. t o
Seattle, WA) according to the manufacturer’s instructions. The same nu- n
cleotideswerealsoclonedintothepCMS4-H1-pEGFPvector(agiftfrom Measurementsofintracellularcalcium A
p
Dtlherni.scDipnC.gDM.anSBd4ilrlsaeud-peexpapruer,sesMsisoianoyn/oreo-Cfelxripnersiceis,stsaRinootncchvDeesNctteAorr,.M(A26Nm))uw(tpaaCsntMufsSoer4dm-sfhooArfRsAhARRPNA)A,Parnseid-- lJouarkdaetdTwcitehlls5(2mM31fu0r6a)-w2eAreMwfaosrhe1d,hsuastp3e7n˚dCe.dAinfteRrPtMwIo1w6a4s0hmesewdiiuthm,Caan2d+ ril 7, 2
loadingbuffer(140mMNaCl,2mMKCl,2.5mMCaCl,1mMMgCl, 0
sistanttoshRNA(rARAP)wascreatedusingaQuickChangesite-directed 2 2 1
10mMHEPES,10mMglucose,40mMsucrose,0.05%BSA[pH7.4]),the 9
mutagenesis kit (Stratagene). The mutation did not confer translational cellswereresuspendedinCa2+loadingbuffer(13106cells/ml).Afterin-
changes(59-ccGcaCaaCtaTgaggcaa-39;uppercaselettersindicatemutations
cubatingfor20minatroomtemperature,baselineCa2+levelsweremeasured
introducedtothetargetingsequences).TheFLAG-taggedWT(rARAP),the
for50s.ThecytosolicfreeCa2+concentrationwasmonitoredbytheaddition
proline-richregion (PR,aa1–310), and the SH3 domain (aa591–728)of ofananti-CD3ε mAb(2mg/ml)followedbycrosslinkingwithagoatanti-
ARAPwereclonedintothepCMS4-shARAPplasmidattheMluIandSalI
ArmenianhamsterIgG(10mg/ml)withanRF-5301PCspectrofluorophoto-
restriction sites. Point mutants of single (Y491F, Y521F, Y587F), double
meter(Shimadzu,Kyoto,Japan).Ionomycin(2mM)wasaddedtoinducethe
(Y2F: Y491, 587F), and triple tyrosine residues (Y3F: Y491, 521, 587F)
maximumCa2+increaseasacontrolforthefura-2AMdyeloading.
were generated from pCMS4-shARAP-rARAP using a QuickChange mu-
tagenesiskit.Alloftheconstructswereverifiedbysequencing. Luciferaseassay
PlasmidsofIL-2enhancerregionNF-AT–luciferase,CMV–b-galactosi-
dase, FLAG-ADAP, HA– or FLAG–SLP-76 (WT), and the SH2 domain Thecellsweretransfectedbyelectroporationwithaluciferaseconstructof
mutant FLAG–SLP-76 (RK) were provided by Dr. G.A. Koretzky (Weill NF-ATanda pCMV/b-galactosidase plasmid.After24h,thecellswere
CornellMedicalCollege,NewYork,NY).TheactiveformofLck(Y505F) harvested,platedin24-wellplatesprecoatedwith2mg/mlanti-CD3mAb
andFyn(Y528F)aswellasHA-SKAP1plasmidsweregiftsfromDrs.Y.D. at 5 3 105 cells per well, and incubated for 16 h. The cells were also
Yun(EwhaWomansUniversity),A.Veillette(InstitutedeRecherches incubated with medium alone or with 50 ng/ml PMA and 1 mM ion-
CliniquesdeMontreal,Montreal,Canada),andB.Schraven(Ottovon omycin.Thecellswerelysed,andluciferaseactivitywasmeasuredwithan
GuerickeUniversity,Magdeburg,Germany),respectively.TheGST-RalGDS LB953 luminometer (Berthold, Bad Wildbad, Germany). To control for
andGST-PAK1plasmidswerealsoprovidedbyDrs.D.D.BilladeauandY.S. transfectionefficiency,luciferaseactivitieswerenormalizedtotheactivity
Bae(EwhaWomansUniversity),respectively. ofb-galactosidase.Theexperimentswereperformedtwiceintriplicate.
Polymerasechainreaction Adhesionassay
The ARAP cDNA in a multiple tissue panel and an immune system Flat-bottom96-wellplateswerecoatedwithfibronectin(3mg/ml),laminin
panel(ClontechLaboratories)wasdetectedwithasetofprimers(59- (10mg/ml),ICAM-1–Fc(1mg/ml),or2.5%(w/v)BSAovernightat4˚C
CGCGGATCCATGAAGGGGAAGGG-39plus59-CCGCTCGAGCTAAGGT- andblockedwith2.5%BSAfor2hat37˚C.Tcells(2.53105),either
GACCAACTTTG-39). unstimulatedorstimulatedwithanti-CD3(5mg/ml)andanti-hamsterIgG
944 ANOVELADAPTORPROTEININTCELLACTIVATION
(10 mg/ml) together for 30 min at 37˚C, were added in triplicate to the NonidetP-40,10%[v/v]glycerol,1mMPMSF,10mg/mlleupeptin,5mg/ml
preparedwells.Theplateswereincubatedfor30minat37˚C,andadherent aprotinin,and1mMNaVO)andcentrifugedat18,0003gfor10min.
3 4
cellswereremovedusingEDTA(50mM)andcounted. Clearedcelllysates(500ml)weretransferredtoglutathioneagarosebeads
conjugatedeitherwithPAK1PBD(GST-PBD)fortheRac1activationassay
Rafts isolation orwithRalGDSRBD(GST-RBD)fortheRap1activationassay.Afterin-
cubationwiththelysatesfor10minat4˚C,thebeadsweremixedwith23
TheBrij-insolubleraftsfractionswereseparatedfromthecelllysatesby
Laemmli’sreducingbuffer,boiledfor5minat96˚C,andsubjectedto14%
sucrose density gradient ultracentrifugation as described previously (18,
29).JurkatTcells(13108)werestimulatedwitheithercontrolIgorwith PAGE. After being transferred to a nitrocellulose membrane, IB was per-
formedforRac1orRap1.LoadingcontrolsfortheIBwereshownusingan
anti-CD3Ab(2mg/ml)for5minat37˚C.Celllysateswerepreparedin
anti-GSTAb.
1% Brij-98 lysis buffer containing protease and phosphatase inhibitors.
Brij-insoluble fractions were separated by sucrose density gradient cen- Analyses ofphosphotyrosineproteomes
trifugation.Fractions(1ml)werecollectedsequentiallyfromthetopofthe
gradientandmembraneraftswereanalyzedbydetectionofGM1. The raft fractions were collected and concentrated using n-octyl-b-D-
glucoside.IPwasperformedontheconcentratedraftsusingamixtureof
ISformationandimmunocytochemistry anti-phosphotyrosineAbs(4G10andp-Tyr100),andtheimmunecomplex
was separated using 10% SDS-PAGE. The gel was stained with a Coo-
ConjugationofJurkatTcellsandRajiBcellsasAPCs,followedbyim-
massie brilliant blue solution and excised into 15 pieces. In-gel tryptic
munocytochemistry, was described previously (29). Jurkat T cells were
digestionandLC-MS/MSanalysisusinga ThermoFinniganProteomeX
transfectedwithexpressionplasmidsencodingGFP-ARAPconstructs,and
workstation LTQ linear ion trap MS (Thermo Electron, San Jose, CA)
RajiBcellswerestainedwithCellTrackerOrangeCMTMR.
equipped with electrospray ionization sources (San Jose, CA) were per-
Actin polymerization formedoneachoftheexcisedproteinbands,aspreviouslyreported(22).
Tandemmassspectrawereextracted,andthechargestatewasdeconvo-
JurkatTcellsconjugatedwithCMTMR-stainedRajiBcellswereprepared luted and deisotoped by Sorcerer 3.4 beta2 (Sorcerer software 3.10.4,
on poly-L-lysine–coated slides. Slides were fixed in 3.7% paraformalde- SorcererWebinterface2.2.0r334andTrans-ProteomicPipeline2.9.5)as D
hydefor20minatroomtemperature,permeabilizedin0.1%TritonX-100, previouslyreported(23).AllMS/MSsampleswereanalyzedusingSequest o
blocked,andstainedwithFITC-ortetramethylrhodamineisothiocyanate– (ThermoFinnigan,SanJose,CA;version27,revision11).Scaffold(ver- wn
conjugated phalloidin. F-actin was imaged using a Zeiss Axiovert 200 sion Scaffold-01_07_00, Proteome Software, Portland, OR) was used to lo
fluorescencemicroscopeequippedwitha340objectivelens(CarlZeiss, validateMS/MS-basedpeptideandproteinidentifications. ad
e
Oberkochen, Germany). Background fluorescence levels in T cells that d
expressedvector-associatedGFPwereminimal.Correctionofbackground Results fro
fluorescencewasusedforTcellfluorescencemeasurements.Allimages m
wereacquiredandanalyzedwithZeisssoftware. Identificationandcloningofanoveladaptorproteininthe h
TCRsignalingpathway ttp
GTPaseactivationassay Weanalyzedthephosphotyrosineproteomesthatareconcentrated ://w
Jurkat T cells (5 3 106) were either unstimulated or stimulatedwith an w
in and recruited to membrane rafts following TCR cross-linking. w
apnotiin-tCaDf3terAsbti(m2umlagti/omnl,)caetl3ls7˚wCerfeorlythseedinidnic6a0t0edmdluoraftiGonTsP.aAseteaacctihvattiimone Raft fractions were isolated from either resting orTCR-activated .jim
buffer (50 mM Tris, pH 7.5, 500 mM NaCl, 5 mM MgCl, 0.5% [v/v] Jurkat T cells using centrifugation of sucrose density gradients m
2 u
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FIGURE1. Aminoacidsequence,domainstructure,andexpressionofhumanARAP.(A)Thepredictedaminoacidsequencesofhuman(Hs)andmouse
(Mm)ARAP.Theputativemodulardomainsareindicatedbythick,thin,anddottedlinesabovetherespectivesequences.Theconservedtyrosinemotifs
YD(E)DVareindicatedbysquareboxes,andtyrosineresiduesareindicatedbyasterisks.(B)SchematicpresentationoftheputativedomainsofARAPand
ADAP.ThePR[thicklinein(A)],lysine-rich[thinlinein(A)],andSH3[dottedlinein(A)]domainsareshownwiththeircorrespondingnumbersinthe
aminoacidsequenceofARAP.Thesequencesforthetwoputativenuclearlocalizationsites,aninternalSH3domain,andabindingsiteforenabled/
vasodilator-stimulatedphosphoproteinhomology1areexclusivetoADAP.Theputativetyrosine(Y)-basedmotifsandsequencehomologies(—,∼50%;…,
∼28%)arealsoindicated.(CandD)ThepresenceofARAPmRNAinvarioustissuesasdetectedbyPCRofthemultipletissuecDNApanels(Clontech
Laboratories)isshown.(E)Westernblotsofvarioushumancelllines(ERK2isaloadingcontrol).(F)WesternblotsoflysatesfromJurkatTcells(50and
100mg),purifiedprimaryhumanTcells(80mg),andHEK293TcellstransfectedwithARAPcDNA(5mg).
TheJournal ofImmunology 945
asdescribedinMaterialsandMethods.Phosphotyrosineproteomes polyclonal anti-ARAP antiserum was generated as described in
were separated from the rafts using IPs with anti-phosphotyrosine MaterialsandMethods.ARAPwaswidelyexpressedinthevarious
Abs.Theimmunecomplexeswereseparatedonagel,andin-gel humantissuesand celllinestested(Fig.1C–E).ARAPtranscripts
tryptic digestion and LC-MS/MS analyses were carried out as weredetectedbyPCRincDNApreparedfromheart,liver,kidney,
described inMaterials andMethods. pancreas, and lymphoid tissues, including thymus, lymph node,
In this study, we identify one protein that is recruited to and tonsil (Fig. 1C, 1D). ARAP expression levels varied in dif-
membrane rafts and undergoes tyrosine phosphorylation follow- ferent batches of cDNA panels. ARAP expression was not de-
ingTCRactivation.ThecorrespondinghumancDNA,containing tected in bone marrow in any batch of cDNA panels. ARAP
chromosome1ORF168(NC_000001.11,http://www.ncbi.nlm.nih. expression in spleen, thymus, lymph node, and peripheral blood
gov/gene/199920),encodedanovelprotein.ThehumancDNA leukocyte was observed, although the expression level varied in
clone (GenBank accession no. NM_001004303) contained an different batches ofcDNApanels. The expression of ARAP pro-
ORF encoding a protein of 728 aa with a predicted molecular tein was verified in purified primary human T cells as shown in
massof83kDa.ThehomologousmousecDNAclone(GenBank Fig.1F.Althoughweonlyshowedthebandthatmatcheswiththe
accession no. NM_001162980) showed a 70% overall identity expressionoffullgenesequenceinHEK293Tcells,fourshorter
withthehumancDNAcloneandcontainedachromosome4ORF variantformsknowninthedatabasecouldbedetectedbyamouse
encodinga90-kDaproteinof793aa(Fig.1A;alignmentfromthe polyclonal anti-human ARAP antiserum that was generated by
genedatabaseisshown).Thisnewlyidentifiedproteinalsoshares usingtheARAP C-terminal immunogenic peptides.
sequence homology with a known T cell adaptor protein, ADAP
ARAPistyrosinephosphorylatedandisrecruitedtomembrane
(24, 25), and we therefore named the novel protein ARAP. As a
raftsandtheISafterTCRstimulation
novel adaptor protein, ARAP contains modular domains, such as
D
N-terminalPRsequences(228–286),alysine-richsequence(527– WeconfirmedthetyrosinephosphorylationofARAPwithanIP o
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662),andanSH3domain(667–728)neartheCterminus(Fig.1A, of Jurkat T cell lysates after stimulation of the TCR for 5 min n
lo
1B).SeveralputativetyrosinemotifsarealsofoundinARAP,and followedbyIBforphosphotyrosineproteins(Fig.2A).Toclearly a
d
some of these motifs are homologous to those (EVYDDV) in demonstrateARAPphosphorylation,primaryhumanTcellswere ed
ADAPthatmediateitsinteractionwiththeSH2domainofSLP-76 stimulated withanti-CD3 Abfor 5 min, and proteins wereimmu- fro
(31–34) (Fig.1A,1B). noprecipitated using anti-phosphotyrosine Absfollowed byWest- m
h
ThehumanARAPcDNA(ARAP-WT)wasclonedbyPCRusinga ernblottingwithanti-ARAPantiserum,anti-ADAP,andanti-LAT ttp
clonecontainingthefullORFofARAPasatemplate,andamouse Abs(Fig. 2B). A low levelof tyrosine phosphorylationof ARAP ://w
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FIGURE2. InductionofARAPtyrosinephosphorylationandrecruitmenttomembranerafts.(A)ARAPtyrosinephosphorylationafterTCRstimulation
inJurkatTcells.Cellswereunstimulated(2)orstimulatedfor5minviaCD3(+).ARAPwasimmunoprecipitatedfromlysatesandanalyzedbyIBto
visualizetyrosine-phosphorylatedARAP(top)andtotalARAP(bottom).(B)ARAPtyrosinephosphorylationafterTCRstimulationinpurifiedprimary
humanTcells.Cellswereunstimulated(control)orstimulatedfor5minwithanti-CD3Ab.IPwasperformedusingamixtureofanti-phosphotyrosine(pY)
Abs(4G10andp-Tyr100)andanalyzedbyIBtovisualizetyrosine-phosphorylatedARAP(top),ADAP(middle),andLAT(bottom).(C)ARAPrecruitment
tomembraneraftsafterTCRstimulation.MembraneraftswereisolatedfromJurkatTcelllysatesafterstimulationwithcontroloranti-CD3Absfor5min
andresolvedbySDS-PAGE.IBforARAP,SLP-76,LAT,andGM1followed.(D–F)LocalizationofARAPtotheIS.ARAP-WT,NT-,and-CTwere
conjugatedwithGFP(D).JurkatTcellsexpressingGFP-ARAP-WT,-NT,or-CTwereincubatedwithCMTMR-labeledRajiBcellsintheabsence(2)or
presence(+)ofthesuperantigenSEEfor15min,andlocalizationofGFPtotheISwasvisualized(originalmagnification3400).Fluorescenceandphase-
contrast micrographs are shown for each GFP-ARAP construct expressed (E). Maximum fluorescence intensity at the IS in the presence of SEE was
quantified,andtheintensityvalueswerecomparedbetweenTcellsexpressingGFP-ARAP-WT,-NT,or–CT.*p,0.05,unpairedttest(F).Alldatashown
arerepresentativeoftwo(primaryTcells)orthree(JurkatTcells)separateexperiments.
946 ANOVELADAPTORPROTEININTCELLACTIVATION
and ADAP was observed in resting T cells, which was enhanced Several major proteins that are known to be tyrosine phosphory-
after TCR stimulation. In this experiment, tyrosine phosphoryla- lated and involved in TCR-mediated signaling pathways were
tion of LATwas examined to determine whether isolated human examined after the stimulation of TCRs in ARAP-suppressed
TcellsrespondedproperlytoTCRstimulation.Wealsoconfirmed Jurkat T cells using IB with a protein-specific anti-phosphotyrosine
that ARAP translocated to the fractions of membrane rafts that Ab. TCR-mediated tyrosine phosphorylation of the enzymes
include proteins such as GM1 and LAT(Fig. 2C). Similarly to PLCg1, ERK2, and Akt was dramatically reduced in ARAP-
SLP-76,thetranslocationofARAPwasTCRstimulation-dependent suppressed Jurkat T cells (Fig. 3C). Other cellular functions
(Fig. 2C). To examine the structural requirements of ARAP in that require the tyrosine phosphorylation and activation of
TCR-mediated T cell activation, the following GFP-tagged con- theseenzymeswerealsoexamined.Adramaticreductioninthe
structswereprepared:ARAP-WT,theNTofARAP(ARAP-NT, cytosolic Ca2+ level following TCR stimulation in ARAP-
aa1–545),andtheCTofARAP(ARAP-CT,aa546–728)(Fig.2D). suppressed Jurkat T cells was observed (Fig. 3D); conse-
The recruitment of ARAP to the IS of T cell–APC (Raji B cells) quently, the NF-AT luciferase activity, which is the readout of
conjugateswasvisualizedusingfluorescencemicroscopywithGFP- TCRsignaling(35),wassignificantlyreducedinJurkatTcells
taggedconstructsexpressedinJurkatTcellspriortoconjugation when the ARAP gene was suppressed (Fig. 3E). In contrast,
with APCs (Fig. 2E). In the presence of the superantigen (SEE), ARAP-suppressedcellshadsimilarresponsestotreatmentwith
green fluorescence from T cells expressing GFP-ARAP-WTand ionomycin or a combination of PMA and ionomycin because
GFP-ARAP-NT, but not GFP-ARAP-CT, localized to the IS; these treatments induce signaling mechanisms that bypass the
moreover, yellow fluorescence overlapped with red fluorescence TCR (Fig. 3D, 3E).
in APCs only when GFP-ARAP and GFP-ARAP-NT constructs
ARAPisrequiredfortheactivationofintegrin,butnotforthe
wereexpressed(Fig.2E).Maximumfluorescenceintensitytothe
polymerization ofactin D
IS in each T cell–APC conjugate in the presence of SEE was o
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quantifiedandtheintensityvalueswerecomparedbetweenTcells TheinitiationofTCRsignalingattheISbycross-linkingoftheAg n
expressingGFP-ARAP-WT,GFP-ARAP-NT,andGFP-ARAP-CT affects cytoskeletal properties, such as actin polymerization and loa
d
(Fig. 2F). integrinsignaling,whichfurtherstrengthenTCRactivation(6–11). ed
ARAPisrequiredforTCR-mediatedactivationofsignaling Therefore, we also examined cytoskeletal rearrangements after fro
TCRstimulationinARAP-suppressedJurkatTcells.F-actinwas m
pTTahctehewlflsuanybscytisounpaplressigsinnigficAanRcAePotfhrAouRgAhPexwparessseixoanmoifneadshiRnNJAurkfoart lcbaoebfmeolpreeadreaudnsdinbgeatfwFteIeTreCnc-occnoojnnujtguraogtlaitoaennddwpAhitaRhllAotPihd-eisnuA,pagpnrde(SsflsEeuEdo)r–TesAccPeenCllcse(bRwoaatjhsi http://w
w
ARAP (Fig. 3A). Suppression of ARAP did not seem to affect B cells) complex. The results demonstrated that actin polymeri- w
expressionofADAPandSKAP55(Fig.3A).InARAP-suppressed zationwasnotinfluencedbythesuppressionofARAPinTcells .jim
Jurkat T cells, tyrosine phosphorylation of proteins was remark- (Fig.4A).Theseresultswerequantitativelycomparedbycounting m
u
ably reduced following TCR stimulation compared with the re- the number of conjugates that showed F-actin staining in the IS n
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sponseincellstreatedwithascrambledcontrolshRNA(Fig.3B). (Fig.4B).Inagreementwiththeseresults,theactivationkineticsof l.o
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FIGURE3. SuppressionoftheARAPgeneandinhibitionofTCRsignaltransduction.(A)ThesuppressionoftheARAPgenebytheexpressionofan
shRNAforARAPwasvisualizedbyWesternblotting.TheexpressionofADAPandSKAP1wasdeterminedinARAP-suppressedJurkatTcells.Loading
controlisshownbyIBwithanti–b-actin.(BandC)InhibitionofproteintyrosinephosphorylationinARAP-suppressedJurkatTcells.ControlorARAP-
suppressed Jurkat T cells were stimulated with an anti-CD3 Ab for the indicated durations. The lysates were resolved by SDS-PAGE, and tyrosine-
phosphorylated proteinswere detectedby IB (B). Thesame blotwas used to analyzethe tyrosinephosphorylation ofPLCg1, ERK2,andAkt. IB for
tyrosine-phosphorylatedprotein(top)andtotalprotein(bottom)(C)isshown.Datashownarerepresentativeofthreeseparateexperiments.(D)Reduced
calcium response in ARAP-suppressed Jurkat T cells. Control or ARAP-suppressed Jurkat T cells were loaded with fura-2, and intracellular calcium
elevationafterTCRstimulation(anti-CD3)orionomycintreatmentwasdetectedbyspectrofluorometry.Calciummeasurementswererepeatedwithsimilar
responses.(E)InhibitionofNF-ATluciferaseactivitybyARAPsuppression.ControlorARAP-suppressedJurkatTcellsweretransfectedwithanNF-AT–
luciferasereporterplasmid.Cellswereeitherunstimulated(control)orstimulatedasindicatedandsubsequentlyassayedforluciferaseactivity.Theresults
areshownasfoldstimulationcomparedwithactivityinunstimulatedcellsandarenormalizedbyb-galactosidaseactivityfromthecotransfectedpCMV–b-gal
plasmid.Thevaluesareexpressedasthemean6SDofthreeindependentexperiments.***p,0.001versuscontrolIg,###p,0.001.
TheJournal ofImmunology 947
Rac1GTPaseswascomparableinbothcontrolandARAP-suppressed NF-ATactivitywasanalyzedinARAP-suppressedJurkatTcells
TcellsfollowingstimulationoftheTCR(Fig.4C).Incontrast, re-expressing ARAP-WT, ARAP-PR, or ARAP-SH3. The results
celladhesiontomatrixproteinssuchasfibronectinandlaminin demonstratedthatneitherthePRnortheSH3domainofARAPis
(Fig.4D)andintegrin-mediatedadhesiontoICAM-1(Fig.4E) sufficientforthefullrecoveryofNF-ATactivityinTcells(Fig.
weresignificantlyreducedinARAP-suppressedJurkatTcells.Cell 5C).Theseresultsalsosuggestedthatotherregionsinadditionto
adhesion occurring via TCR-bypassed signaling using PMAwas the PR and SH3 domains are requiredfor ARAPto transduce ac-
normalinARAP-suppressedcells.ThesedefectsinTCR-mediated tivationsignalsafterTCRstimulation.TheregionbetweenthePR
celladhesioncorrespondwiththeinhibitionofTCR-inducedRap1 and SH3 domains includes many tyrosine residues, and the con-
GTPaseactivityinARAP-suppressedTcells(Fig.4F). served tyrosine motif YD(E)DV was found at Y491, Y521, and
Y587.ThesespecifictyrosinemotifswerealsoconservedinADAP
ARAPfunctioninTcellsignalingismediated bythe
andwerepreviouslyshowntomediateADAPassociationwithSLP-
phosphorylationofspecifictyrosinemotifsthatassociatewith
76 (33, 34). To examine the functional significance of these three
theSH2domainofSLP-76
tyrosine motifs in ARAP, single tyrosines or combinations of ty-
TofurtherexaminethemechanismsbywhichARAPfunctionsin rosines were mutated, and these mutants were re-expressed in
Tcellactivation,therecoveryofARAP-suppressedTcellfunction ARAP-suppressedJurkatTcells.Singletyrosinemutantsateither
in Jurkat T cells was monitored following reconstitution with Y491 or Y587 did not fully restore the TCR-mediated NF-AT
severalARAPmutants.Asdescribed,avectorsystemwasusedto activity, but the single tyrosine mutant at Y521 fully recovered
suppressARAPwhilealsore-expressingthemutantARAP.The activityincontrolTcells(Fig.5D).Furthermore,ARAP-suppressed
re-expressedARAPconstructscontainedbothaFLAGepitope Jurkat T cells that re-expressed double tyrosine ARAP mutants at
andmutationsinthetargetingsequencesforshARAP(Fig.5A). Y491andY587(Y2F)didnotrespondtoTCRstimulationanddid
D
Re-expression constructs of ARAP included WT, truncation mu- notinduceNF-ATactivation(Fig.5D). o
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tants,ARAP-PRandARAP-SH3,andtyrosinemutantsofARAP The TCR-activated cytosolic Ca2+ response was restored in n
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atY491,Y521,andY587.Allofthetyrosinemutantswerelocated ARAP-suppressed Jurkat T cells that re-expressed WTARAP or a
d
within YD(E)DV motifs (Fig. 5A). The expression of these con- theARAPY521Fsingletyrosinemutant(Fig.6A).Incontrast,re- ed
structsinARAP-suppressedJurkatTcellsisshownbyIBwithan expressionwithneithertheY491FnortheY587Fmutantrestored fro
anti-FLAGAb (Fig. 5B). the TCR-mediated Ca2+ response to the same extent as the re- m
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FIGURE4. SuppressionoftheARAPgeneandimpairedintegrinactivationresultinimpairedcelladhesion.(AandB)Actinpolymerizationisnot
affectedbyARAPsuppression.ControlorARAP-suppressedJurkatTcellswereincubatedwithCMTMR-labeledRajiBcellsintheabsence(2)and
presence(+)ofthesuperantigenSEE.Thecellsweretransferredtopoly-L-lysine–coatedslides,fixed,permeabilized,andstainedwithFITC-conjugated
phalloidin.Imagesobtainedusingfluorescencemicroscopyareshown(originalmagnification3400),andthelocalizationofF-actintotheISwasvisu-
alizedusingfluorescencemicroscopy(A).TheF-actinlocalizedintheISfromconjugates(.60)wasquantifiedinthreeindependentexperiments.The
valuesareexpressedasthemean6SD(B).(C)Rac1GTPaseactivationwasassayedusingtheGST-PBDfusionprotein.ControlorARAP-suppressed
JurkatTcellswerestimulatedwiththeanti-CD3Abfortheindicatedtimes,andthelysateswereincubatedwithGST-PBDbeadsfor10min.Theproteins
fromthebeadswereresolvedbySDS-PAGEandweresubjecttoIBforRac1andGSTforequalamountsofeachfusionprotein.ERK2IBisshownfortotal
protein. Data shown are representative of three separate experiments. (D and E) Cell adhesion is inhibited by ARAP suppression. Control or ARAP-
suppressedJurkatTcellswerestimulatedfor30minasindicatedandaddedtothewellsthatwerecoatedwithBSA,fibronectin,andlaminin.After30min
incubation,adherentcellswereremovedandcounted.Thevaluesareexpressedasthemean6SDofthreeindependentexperiments.*p,0.05,**p,
0.01,***p,0.001versuscontrolIg;##p,0.01,###p,0.001(D).ControlorARAP-suppressedJurkatTcellsthatwerestimulatedas in(D)were
incubatedinICAM-1–coatedwellsfor30min,andadherentcellswereremovedandcounted.Thevaluesareexpressedasthemean6SDofthreein-
dependentexperiments.***p,0.001versuscontrolIg,###p,0.001(E).(F)Rap1GTPaseactivationwasassayedusingtheGST-RBDfusionproteinasin
(C).IBwasperformedforRap1andGSTforequalamountsofeachfusionprotein.Rap1IBisshownfortotalprotein.Datashownarerepresentativeof
threeseparateexperiments.
948 ANOVELADAPTORPROTEININTCELLACTIVATION
sponseinARAPWTre-expressedcells(Fig.6A).Asexpected,a (Fig. 7A). When we induced tyrosine phosphorylation in the
Ca2+responsewasnotobservedafterTCRstimulationinARAP- transfectedcellsbypervanadate(PV)stimulation,SLP-76protein
suppressed Jurkat T cells; moreover, it was not observedin cells was immunoprecipitated with both ARAP and ADAP (Fig. 7A).
withthedoubletyrosinemutantsofre-expressedARAP(Fig.6A). We also tested for the association of the endogenous proteins by
SimilarresultswerealsoobservedforICAM-1bindingafterTCR performingIPinJurkatTcells.AsshowninFig.8Aand8B,TCR
stimulation (Fig. 6B). stimulation with anti-CD3 Ab induced protein tyrosine phos-
Theseexperimentsdemonstratedthattyrosinephosphorylationof phorylation, and IP with anti–SLP-76 Ab immunoprecipitated
ARAP,especiallyatY491andY587,isrequiredforARAPfunction bothADAPandARAP.Abasallevelofinteractionwasobserved,
in T cell activation after TCR stimulation. Therefore, the level of and the level of interaction increased after TCR stimulation
tyrosinephosphorylationwasexaminedinARAP-suppressedJurkat (Fig. 8B).
TcellswhereoneofWTARAPoratyrosinemutantofARAPwas SKAP1 plays a central role in ADAP-mediated signaling to
re-expressed.Tyrosinephosphorylationofre-expressedWTARAP integrins,andtheidenticalmotifsoftheARAPPRregionoccurin
or mutant ARAPswas detected by IP with an anti-FLAG Ab fol- theprimarysiteofmouseandhumanADAPthatbindstoSKAP1
lowedbyIBwiththeanti-phosphotyrosineAb4G10.Reducedty- (36, 37). Therefore, we examined whether ARAP also binds to
rosine phosphorylation in the Y491F and Y587F mutants and an SKAP1 through the ARAP PR region. FLAG-tagged ADAP or
evenfurtherreductioninthedoubletyrosinemutantswereobserved ARAP was transiently overexpressed in HEK 293T cells in
(Fig. 6C). The patterns of tyrosine phosphorylation in ARAP cor- combinationwithHA-taggedSKAP1,andtyrosinephosphorylation
roborated the patterns of ARAP association with SLP-76. SLP-76 inthetransfectedcellswasinducedbyPVstimulation(Fig.7B).
associationwas alsoreduced inthe Y491Fand Y587Fmutants of SKAP1 protein was immunoprecipitated with both ARAP and
ARAP,anditwasalmostblockedinthedoubletyrosinemutantsof ADAPregardlessofPVstimulation(Fig.7B).IPwithanti-SKAP1
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ARAP(Fig.6C).Thus,theassociationofARAPwithSLP-76may AbinJurkatTcellsdetectedbothADAPandARAP(Fig.8A,8C). o
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have been mediated by the ARAP phosphotyrosines at Y491 and The association was notchanged after TCRstimulation withanti- n
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Y587 and by the SH2 domain of SLP-76. This association was CD3 Ab (Fig. 8C). To confirm that SKAP1 binding to ARAP is a
d
furtherconfirmedbyanexperimentwhereSLP-76–deficientJurkat mediated bytheARAPPR regionbutnot bytheARAP tyrosine ed
TcellswerereconstitutedwithWTSLP-76orwithaSLP-76SH2 motifs, we overexpressed FLAG-tagged ARAP mutants, ARAP- fro
mutant(RK)(Fig.6D).ARAPassociationwithWTSLP-76was PR, or ARAP-Y3F in HEK 293T cells together with HA-tagged m
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strongly induced after TCR stimulation, but the association of SKAP1 (Fig. 7C). Irrespective of the tyrosine phosphorylation ttp
ARAPwithSLP-76RKwasnotchangedbyTCRstimulation,al- inducedbythePVstimulationofthetransfectedcells,SKAP1 ://w
thoughtyrosinephosphorylationofWTandSH2mutant(RK)SLP- proteinwasimmunoprecipitatedwithbothARAP-PRandARAP- w
76wasinducedbyTCRstimulation(Fig.6D). Y3F (Fig. 7C). w
To clearly demonstrate ARAP interaction with SLP-76 and .jim
ARAPdirectlyassociateswithSLP-76,SKAP1,Lck,andFyn SKAP1,primaryhumanTcellswerestimulatedwithanti-CD3Ab m
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TodeterminewhethertheinteractionofARAPwithSLP-76occurs for 5 min, and cell lysates were immunoprecipitated using anti- no
directly, we transiently overexpressed FLAG-tagged ADAP or ARAP antiserum followed by Western blotting with anti-ARAP l.o
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ARAPincombinationwithHA-taggedSLP-76inHEK293Tcells antiserum,anti–SLP-76,andanti-SKAP1Abs(Fig.8D,8E).The b/
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FIGURE5. FunctionalanalysisofARAPmutantsinTcellsignaling.(A)SchematicpresentationofFLAGepitope-taggedARAPmutants.Themu-
tationsinthetargetingsequencesofshARAPareindicated( ).(B)IBofre-expressionconstructsinARAP-suppressedJurkatTcellsusingananti-FLAG
^^^^
mAb(SLP-76isaloadingcontrol).(C)NF-ATluciferaseactivitywasassayedincontrolcells(EV),ARAP-suppressedJurkatTcells(shARAP),orARAP-
suppressedcellsthatre-expressedARAP-WT,ARAP-PR,orARAP-SH3.ThesecellstransfectedwithanNF-AT–luciferasereporterplasmidwereplacedto
thewellscoatedwithcontrolIg(unstimulated)oranti-CD3Ab.Luciferaseactivitywassubsequentlyassayedandtheresultsareshownasfoldstimulation
comparedwithactivityinunstimulatedcellsandarenormalizedbyb-galactosidaseactivityfromthecotransfectedpCMV–b-galplasmid.Thedataarethe
means6SDofthreeindependentexperimentsperformedintriplicate.Statisticalsignificancewasanalyzedbytwo-wayANOVAwithBonferronimultiple
comparisontests.*p,0.05,***p,0.001versuseachcontrolIg;###p,0.001versusshARAP(anti-CD3).(D)NF-ATluciferaseactivityassayincontrol
cells(EV),ARAP-suppressedJurkatTcells(shARAP),orARAP-suppressedcellsthatre-expressedvarioustyrosinemutantsofARAP.Thedataarethe
means6SDofthreeindependentexperimentsperformedintriplicate.Statisticalsignificancewasanalyzedbytwo-wayANOVAwithBonferronimultiple
comparisontests.***p,0.001versuseachcontrolIg,###p,0.001versusEV(anti-CD3).
TheJournal ofImmunology 949
FIGURE6. FunctionalanalysisofARAPtyrosinemutantsinTCRsignaltransduction.(A)IntracellularcalciumelevationafterTCRstimulation(anti-
D
CD3) in fura-2–loaded control cells (EV), ARAP-suppressed Jurkat T cells (shARAP), or ARAP-suppressed cells that re-expressed various tyrosine o
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mutantsofARAP.Thedataarerepresentativeofthreeseparateexperiments.(B)AdhesionassayinICAM-1–coatedwells.AfterTCRstimulation(anti- n
CD3),controlcells(EV),ARAP-suppressedJurkatTcells(shARAP),andARAP-suppressedcellsthatre-expressedvarioustyrosinemutantsofARAP loa
wereincubatedinICAM-1–coatedwellsfor30min,andadherentcellswereremovedandcounted.Thedataarethemeans6SDofthreeindependent de
e0x.0p0e1rimveernsutsspeearcfhorcmoendtrionltIrgi;pl#ipca,te.0S.0ta1t,is#t#ic#pal,sig0n.0ifi0c1anvceerswusasEaVna(laynztei-dCbDy3t)w.(oC-)wAayRAANPOasVsAocwiaittehsBwointhfeSrrLoPn-i7m6ublytiptlheecAomRApaPripsohnostpehstost.y*r*ospin,es0a.0t1Y,4*9*1*pan,d d from
Y587.ControlorARAP-suppressedJurkatTcellsthatre-expressedvarioustyrosinemutantsofARAPwereunstimulatedorstimulatedviaCD3for5min.Re- h
expressedARAPWTandtyrosinemutantswereimmunoprecipitatedfromlysateswithananti-FLAGmAbandanalyzedbyIBtovisualizeSLP-76(top), ttp
tyrosine-phosphorylatedARAP(middle),andtotalARAP(bottom).Theintensityofthebandswasquantified,andthenormalizedvalueswerecalculatedbased ://w
ontheintensityofthetotalARAPbands.TheSLP-76interactionandtyrosinephosphorylationofARAPtyrosinemutantsafterCD3stimulationwerecompared w
withthoseoftheARAPWT.Thedataarerepresentativeofthreeseparateexperiments.(D)AssociationofARAPwithSLP-76ismediatedbytheSH2domain w
ofSLP-76.SLP-76–deficientJ14cellsthatwerereconstitutedwiththeFLAG-taggedWTorSH2mutant(RK)ofSLP-76wereunstimulatedorstimulatedvia .jim
CD3for5min.ThereconstitutedSLP-76WTandRKmutantwereimmunoprecipitatedfromlysateswithananti-FLAGmAbandanalyzedbyIBtovisualize m
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ARAP(top),tyrosine-phosphorylatedSLP-76(middle),andtotalSLP-76(bottom).Asin(C),theARAPinteractionwasnormalizedandcomparedwithvalues n
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ofunstimulatedSLP-76WT.Thedataarerepresentativeoftwoseparateexperiments. l.o
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b/
IPwithanti-ARAPAbrevealedconstitutivebindingofARAPand tyrosine-phosphorylated after TCR stimulation, such as the TCR y
g
SKAP1aswellastyrosinephosphorylation-dependentbindingof z-chain,ZAP-70,PLCg1,LAT,andSLP-76(18–21)wereamong u
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ARAPandSLP-76 (Fig.8D,8E). theproteins identifiedfrom thephosphotyrosine proteome analy- st o
We next examined whether ARAP also binds to Lck or Fyn. sis in TCR-stimulated Jurkat cells. These results confirmed that n
A
FLAG-taggedADAPorARAPwasoverexpressedincombination the methods used for the analysis of TCR signaling complexes p
withconstitutivelyactiveformofLck(Y505F)orFyn(Y528F)in were effective and that novel candidate proteins can be found to ril 7
HEK293Tcells(Fig.7D).BothARAPandADAPproteinswere better understand themechanisms ofTcellactivation. , 2
0
immunoprecipitated with a constitutively active from of Lck ARAP, the novel protein that we identified by analysis of the 1
9
(Y505F) or Fyn (Y528F) (Fig. 7D). These interactions were phosphotyrosine proteome in activated Jurkat T cells, shares se-
irrespective of the tyrosine phosphorylation induced by a consti- quence homology (Supplemental Fig. 1) with the T cell adaptor
tutively active form of kinases, because both ARAP and ADAP protein, ADAP (∼50%), and also with promyelocytic leukemia–
alsoimmunoprecipitatedwithaninactiveformofLckorFyn(data retinoic acid receptor a–regulated adaptor molecule-1 (∼40%)
not shown). thatismainlyexpressedinmyeloidcells(38,39).FollowingTCR
stimulation, ADAP is well known to mediate specific signaling
Discussion pathwaysforinside–outactivationoftheintegrinmoleculeLFA-1
To better understand the initial stages of TCR signaling and the (24, 25).Similar toADAP, ARAP contains a PR region atthe N
subsequent mechanisms of T cell activation, we analyzed the terminus,anSH3domain attheCterminus,andseveraltyrosine
proteinsinvolvedintheTCRsignalingcomplexformedattheIS motifs between these domains (Fig. 1A, 1B). However, the se-
afterTcellactivation.Toisolatesignalingcomplexeslocalizedto quencesforthetwoputativenuclearlocalizationsites,aninternal
theIS,membraneraftswerepurifiedfromactivatedJurkatTcells SH3 domain, and a binding site for an enabled/vasodilator-
using sucrose density gradient centrifugation. The tyrosine- stimulated phosphoprotein homology 1 domain are exclusive to
phosphorylated proteins from the isolated membrane rafts were ADAP (39, 40). The absence of these domain sequences may
collectedbyIPusinganti-phosphotyrosineAbsandwereseparated explainthedifferentialregulationofF-actinorganizationbetween
with SDS-PAGE. In-gel tryptic digestion was applied to each ADAP and ARAP. ADAP regulates TCR-mediated F-actin orga-
excisedproteinband,andpeptideswereidentifiedbyLC-MS/MS nizationbymediatinginteractionsbetweenSLP-76andWiskott–
as described in Materials and Methods. To exclude endogenous AldrichsyndromeproteintogetherwithNckadaptorprotein(41).
phosphotyrosine proteins, the same procedures were also applied Actin polymerization and Rac1 activation (the small GTPase
toananalysisofthephosphotyrosineproteomesfromJurkatcells thatregulatesactinpolymerization)wereunaffectedinARAP-
subjected to mock stimulation. Numerous proteins known to be suppressedTcellsafterTCRstimulation(Fig.4A–C).Itremains
950 ANOVELADAPTORPROTEININTCELLACTIVATION
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FIGURE7. DirectinteractionofARAPwithSLP-76,SKAP1,Lck,andFyn.Flag-taggedADAPorARAPincombinationwithHA-taggedSLP-76(A) ://w
orSKAP1(B)wereoverexpressedinHEK293Tcells,andtyrosinephosphorylationwasinducedbyPVstimulationfor5min.Proteinexpressionwas w
w
dTehteecintetderbacytiIoBnwbeitthwaenetni-SHLAP-o7r6a,nStKi-FALPA1G,AARbA.PT,haendamAoDuAntPswofaIsPdfertoemcteudnbtryeaItBedus(icnogntarnotli)-FoLrAPVG-tAreba.te(Cd)lyHsEatKes2w93eTrecdeeltlesrwmeinreedtrabnysIfBectwedithwaitnhtip-HlaAsmAidbs. .jim
m
carryingARAPmutants,eitherARAP-Y3F(FLAG)orARAP-PR(FLAG),alongwithSKAP1(HA).ProteinexpressionwasdetectedbyIBwithanti-HA u
n
oranti-FLAGAb.TheamountsofIPfromuntreated(control)orPV-treatedlysatesweredeterminedbyIBwithanti-HAAb.TheinteractionbetweenSLP- o
76andARAPmutantswasdetectedbyIBusinganti-FLAGAb.(D)ARAP(FLAG)orADAP(FLAG)wasexpressedinHEK293Tcellsinthepresenceor l.o
adbesteernmceinoefdLbcykI(BYw50it5hFa)notir–Fby-nac(tYin5.2T8yFr)o.sPinreotpehinosepxhporerysslaiotinonwiansdduecteedctbeydLbyckIBoruFsyinngeaxnptrie-Lssciokn,awntais-Fdyente,romriannetdi-bFyLAIBGwAitbh.4EGqu1a0l.aTmheouanmtsouonftlsyosaftIePswweerree byrg/
determinedbyIBwithanti-FLAGAb.TheinteractionofARAPorADAPwithLckorFynwasdetectedbyIBusinganti-Lckoranti-FynAbs. gu
e
s
t o
to be determined whether TCR-driven assembly of the Bcl-10– TCRsignaling.Anotablesimilaritybetweenthetwoadaptorsisthe n
A
CARMA1complexupstreamofNF-kBactivationandconsequent phosphotyrosine motifs that are conserved in both ARAP and p
IkB degradation regulated by ADAP (42) is due to these unique ADAP. ARAP contains specific tyrosine residues, which are lo- ril 7
ADAPdomains,andwhetherARAPmaynotparticipateinthese catedwithinYDDVmotifsatY491andY587(Fig.1A,1B).TCR- , 2
0
TCR-mediatedphysiologicalfunctionsduetotheabsenceofthese mediated phosphorylation of these motifs is required for the 1
9
unique domains. functionofARAP(Figs.5,6).SimilartoADAP,ARAPassociates
TheARAPtissuedistributionismuchbroaderthanthatofADAP with the SH2 domain of SLP-76 following TCR stimulation via
(Fig. 1C–E) whereas ADAP expression is restricted to T and the phosphorylation of tyrosines Y491 and Y587 (Fig. 6C).
myeloid cells (42). Further studies are needed to determine the Considering the results shown in Figs. 2D–F and 6A, Y491 ap-
biologicalsignificanceofARAPforintegrinactivationindistinct pears to be strongly required for the TCR-mediated proximal
cell types and to clarify the difference in upstream and down- signaling event as compared with Y587. These results may sug-
stream signaling pathways for ARAP and ADAP in T cells. A gest that ARAP participates with Y491 phosphorylation in the
comparison of ARAP expression levels in the immune system proximalsignalingeventbyothermechanismsinadditiontoSLP-
shows almost no ARAP in the bone marrow, but a strong ex- 76binding. The intervening phosphorylationsite Y521in ARAP
pression in the thymus, lymph node, and tonsil. It remains to be may have a different tyrosine consensus sequence, perhaps more
determined whether ARAP expression is developmentally regu- similar to the YDGI in ADAP that was previously identified to
lated or enhanced by TCR signaling. Four shorter variant forms mediate the association of ADAP with the Src kinase Fyn (43).
areoftenidentifiedinthedatabase,andtheexpressionpatternsof ThismightexplainwhyY521wasessentiallyirrelevantforSLP-
alternativelysplicedisoformsindifferenttissuesmaycontributeto 76 binding in the present studies. Using the HEK 293T cell ex-
cell-specificfunctionalregulation.Otherimportantquestionsare: pression system, we confirmed that ARAP directly binds Fyn
why do two homologous adaptor proteins (ADAP and ARAP) and Lck, which subsequently phosphorylate tyrosine residues in
existinTcells,ifthetwoproteinscross-regulateeachother,and ARAP(Fig.7D).AnothersimilarityisthattheN-terminalARAP
whether ADAP expression increases in response to ARAP sup- PR region appears to show almost complete identity with the
pression, orviceversa. primary ADAP site responsible for SKAP1 binding (36). As
ThesimilaritiesanddifferencesbetweenARAPandADAPmay shown in Fig. 7B and 7G, both ARAP and ADAP constitutively
indicate that they have similar yet critically distinct functions in interactwiththeadaptorproteinSKAP1.AlthoughSKAP1playsa