Journal of Cell Science a1bnsdrcl oteersde nSP6adta fiin idn eed nteS2adSH3 and SH2 the on depends binding efficient that and SLP76 on residues these to directly binds Vav1 epooeta a1i erie oteI ybnigt L7 n htti neato sciia o h rndcino inl leading signals of transduction flux. the calcium for critical TCR-induced is in interaction function this signalling that and its SLP76 and to Vav1 binding of by IS phosphorylation flux. the the calcium to for recruited to critical is Vav1 are that domains propose two We same the that show (SH3 *rsn drs:FxCaeCne etr hldlha A111 USA 19111, PA Philadelphia, Center, Cancer Chase Fox address: **Present h rgnlwr spoel ie n l ute itiuin ftewr raatto r ujc to subject are adaptation or work terms. the License of distributions Commons further Creative Attribution all same and Commons that cited the provided Creative properly medium is any the work in of original reproduction which the and terms distribution (http://creativecommons.org/licenses/by-nc-sa/3.0), use, the non-commercial License under unrestricted permits distributed Alike IS, article Share Access Non-Commercial Open the an signalling is within This of segregate also Imaging PKC peripheral these example, 1998). pSMAC) that and for al., with, and shown central has et the (cSMAC proteins as (Monks clusters known ring respectively a activation zones by a in supramolecular surrounded with molecules integrins proteins, TCR of of of arrangement IS the ordered accumulation cases an some central In integrin- by 1998). T al., characterised et this stable Dustin is 2010; synapse of a al., immunological et the of (Dustin interface termed (IS) formation been cell-cell has the conjugate The hence stabilised and conjugate. APC cell:APC counter- the their to on molecules activation adhesion receptors to these leading of TCR, binding TCR integrins, the to of from an antigenic pMHC transduction of signal of to surface in binding (TCR) the results Initial on receptor (APC). (pMHC) antigen cell molecules antigen-presenting cell MHC to T binding bound surface the peptide by cell triggered the is cells of T of activation Antigen-specific Introduction words: Key cell T and microclusters in to role recruited critical is a it that plays show factor now CD4 exchange We pathways. nucleotide primary multiple cell- guanine in of the Vav1 at IS activation microclusters The the the to to (IS). proteins to leading signalling synapse signalling, of immunological (TCR) recruitment in receptor the results antigen as cells presenting known antigen interface to cells cell T of binding antigen-specific The Summary §§ `` " USA 94143, CA Francisco, § San California, of ` University address: *Present 5 4 3 2 1 Ksionda Olga TCR in localisation Vav1 signalling of function and Mechanism 5302 o:10.1242/jcs.105148 doi: 5302–5314 125, Science Cell of Journal 2012 August 8 Accepted enfrE Smith-Garvin E. Jennifer rsn drs:Bbaa nttt,Bbaa eerhCmu,CmrdeC2 A,UK 3AT, CB22 Cambridge Campus, Research Babraham Institute, Babraham address: Present rsn drs:Fjfl isnhBoehooisU t,BlssAeu,Bliga S31H UK 1LH, TS23 Billingham Avenue, Belasis Ltd, UK Biotechnologies Diosynth Fujifilm address: Present rsn drs:Cnr ’muooi asil uiy asil,France Marseille, Luminy, Marseille d’Immunologie Centre address: Present TSuhetr eia etr als X730 USA UK 75390, USA 1AA, TX 19104, NW7 Dallas, PA London Center, UK Philadelphia, Research, Medical 1AA, Medicine, Medical Southwestern NW7 of UK for UT London School 1AA, Institute Research, Pennsylvania NW7 National Medical of London MRC for University Research, Biochemistry, Institute Medical Physical National for of MRC Institute Division Structure, National Molecular MRC of Biology, Division Cell Immune of Division rsn drs:Uiest fBitl rso,B81D UK 1TD, BS8 Bristol, Bristol, of University address: Present uhrfrcrepnec ( correspondence for Author 02 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2012. B oan fVv,ado hshtrsns12ad18o h L7 dpo rti.Bohsclmaueet hwthat show measurements Biophysical protein. adaptor SLP76 the of 128 and 112 phosphotyrosines on and Vav1, of domains ) muooia yas,Tcl,Sga rndcin L7,Vav1 SLP76, transduction, Signal cell, T synapse, Immunological 1, ,AeadrSaveliev Alexander *, [email protected] h + n ai on ntecMCand cSMAC the in found talin and n CD8 and 4, * hitp Wu Christoph **, + el.Frhroe eso htti erimn eed nteS2adCtria SH3 C-terminal and SH2 the on depends recruitment this that show we Furthermore, cells. T ) 1, ` oetKo Robert , lfing ¨ 5, `` chl ¨ arnRittinger Katrin , 1 oahnRapley Jonathan , inligeet Bnele l,20;Bnele l,2006). al., et proximal Bunnell TCR initial 2002; the al., study et to move (Bunnell used to events best is unable signalling and system are antibodies, these anti-CD3 the In but immobilised thus by 2001). form bound al., are et microclusters they Bunnell because TCR 2002; Braiman al., the 2005; et studies, to al., Bunnell cases 2006; et some al., (Barda-Saad in et and, glass 1999; TCR on the where immobilised al., to system microclusters CD28, antibodies imaging with et TCR alternative interact 2006). an cells Grakoui in al., T observed et 2005; been Varma also al., 2005; have molecules al., TCR et et which (Campi from Mossman area internalised an within be extinguished are may usually in which is occurs cSMAC, signalling but the active microclusters, that (Yokosuka peripheral shown cSMAC have the the Studies to 2005). reaching centripetally, recruited al., migrate before et also too dissociate are these they 1999; adaptors and though IS, al., SLP76 the and et ZAP70 within LAT the Grakoui microclusters as the 2005; such to and molecules minutes al., Signalling kinase several 2005). et of al., (Campi et course Yokosuka the cSMAC over the synapse to move form TCR the then the of which IS engagement, centre the of ligand the periphery of the in seconds microclusters forms within bilayers lipid that supported shown on has with presented interacting ligands cells integrin T and of pMHC analysis Kinetic respectively. pSMAC 2 o Carter Tom , 1,2,§ utpaFaroudi Mustapha , 3 n itrL .Tybulewicz J. L. Victor and B oan fVv.Fnly we Finally, Vav1. of domains eerhArticle Research 1, " , 1,§§ Journal of Cell Science el ojgtdt Pshssonadvrerneof range diverse a 2009). al., shown et T (Singleton has efficient activation for APCs important cell be may to which in patterning, sensors conjugated spatiotemporal signalling different cells 30 of T imaging 3D cell live Finally, i.1. Fig. e etpg o legend. for page next See oei C inlig rndcn inl htla oan to lead that signals transducing Ca signalling, intracellular important in an TCR increase plays in which 2005), role (Tybulewicz, GTPases family a1i unn uloieecag atr(E)frRho for (GEF) factor exchange nucleotide guanine a is Vav1 a1lclsto n ucin5303 function and localisation Vav1 2+ climfu) n h ciainof activation the and flux), (calcium Journal of Cell Science ( GPa1wr ojgtdwt H7clsple ihMCppieor peptide MCC with pulsed CD8 cells P14 CH27 (I) with conjugated were EGFPVav1 ( xrsigEFVv eecnuae o i ihOApeptide-pulsed from OVA cells with (B min APCs 7 NP68-pulsed for or conjugated were EGFPVav1 expressing ucino ierltv ofraino ih elculs(igeo tal., et a (Singleton with couples determined cell were * tight differences a of significant as formation Statistically patterns to 2009). couples indicated relative cell the time of with of percentage EGFPVav1 function the of display accumulation graphs displayed The that peptide. gp33 with pulsed indicated 5 are bar: APCs images. Scale against phase asterisks. as antibodies with well with as stained (pY), phosphotyrosine conjugates and cell:APC EGFP T of sections optical rmBALB/c- from 129S8- ahtm on.()ad()Pretg fTclssoigacmlto of accumulation showing ( cells IS. T the for of towards scored Percentage Vav1 cells (F) and of and pY of number (C) min the point. in 5–40 indicates time after either n each or (D), indicated. peptide, and as cognate (A) formation, of in conjugate absence shown the those in as generated such conjugates images from quantified IS, are APCs images. 10 DIC bar: 6 as Scale well asterisks. as with antibodies Vav1, indicated with and stained (pY) conjugates cell:APC phosphotyrosine T against of sections optical single show i.1 oaiaino a1i rmr CD4 primary in Vav1 of Localisation 1. Fig. (SH3 domains SH3 two and domain increased contains C- protein SH2 hence the 2010). Vav1, al., of and one et domains Yu DH-PH-C1 Vav1 the 2000; to al., of terminal et from (Aghazadeh core domains activity acidic catalytic enzymatic acidic and 2010). CH DH-PH-C1 the al., the of within the et dissociation located to Yu leads the residues 2000; domain of tyrosine al., binding of blocking et Phosphorylation sterically to (Aghazadeh binding and substrates by domains Vav1 GTPase PH of and activity DH region to GEF acidic the N-terminal the an regulate and 2008). with domain negatively al., (CH) that et homology associated calponin Rapley catalytic a 2008; a are tightly these al., forming et domains, is (Chrencik C1 core and Dbl which the (PH) in homology domain, resides pleckstrin Vav1 (DH) of activity GEF homology The 2A). (Fig. domains al., et Krawczyk 1998; al., Wu et Holsinger 2002; 1998; and al., al., activation et also et Fischer (Ardouin integrin is 2003; polarisation to cell Vav1 and leading 2005). rearrangements, signals cytoskeletal Tybulewicz, transduce 2002; al., to et al., al., Reynolds required et 1998; et al., (Costello Reynolds et (NFAT) Holsinger 2004; cells 1998; al., T et activated Fischer 1999; of factor factor nuclear nuclear factors Akt and transcription the the (PI3K), and phosphoinositide-3-kinase kinase, kinases, MAP ERK 5304 inligpoen nTcls(yueiz 05.Truhits Through 2005). (Tybulewicz, cells T in an proteins signalling as potentially functions, GEF-independent protein. adaptor has cell demonstrating and cells Vav1 mutation, In GEF activation 2009). this T that by al., ERK affected In not et flux, are (Saveliev functions. polarisation calcium defective of its TCR-induced is is activation integrins of contrast, Vav1 TCR-induced and all of Vav1, Akt activity not Rac1, a GEF-deficient as GEF but a well the some expressing as that adaptor for showed an important we as function Recently may GEF. Vav1 that suggesting A D P E)o YadVv nteTcls esr fplrsto oad the towards polarisation of measure a cells, T the in Vav1 and pY of SEM) , , , tutrlaayi fVv a hw hti otismultiple contains it that shown has Vav1 of analysis Structural a1hsbe eotdt soit ihanme fother of number a with associate to reported been has Vav1 B E .5 ** 0.05, , , C F ciae CD8 Activated ) Vav1 ciae CD4 Activated ) lige l,2000). al., et ¨lfing ora fCl cec 2 (22) 125 Science Cell of Journal 2 + P / 2 el xrsigEF-a1wr ojgtdwt cells B with conjugated were EGFP-Vav1 expressing cells T , Vav1 .1 *** 0.01, ie oddo o ihteN6 etd.(,)Images (A,D) peptide. NP68 the with not or loaded mice) 2 / 2 + + ie oddo o ihteOApeptide. OVA the with not or loaded mice) 5Tclscnuae ihAC Bclsfrom cells (B APCs with conjugated cells T F5 O11 el ojgtdwt Ps( cells (B APCs with conjugated cells T DO11.10 P , m .( m. 0.001. G CD4 ) H , I H CC CD4 5C.C7 (H) ) Vav1 m + .(,)Rcutetidx(mean index Recruitment (B,E) m. O11 rCD8 or DO11.10 2 / 2 ie.Iae hwsingle show Images mice). + n CD8 and + el expressing cells T + + 5Tcells T F5 cells. T k (NF- B A SH3 , t -test; k B B) ), eitiue oteI ncnuae n omicroclusters to SH3 and and SH2 conjugates the that in identify we IS stimulation, TCR the following to redistributes sn rmr CD4 primary using inlig(agadKrtk,19;Krtk ta. 2006; al., SH3 The TCR et 1996). for al., Koretzky et critical Wu 1999; 1996; protein al., Koretzky, et adaptor Tuosto and an (Fang SLP76), and signalling SLP76 Y113 human been to of in corresponding has pY128 SLP76, Y128 Vav1 and mouse of for Wu pY112 given domain 1994; phosphotyrosine (numbering SH2 al., bind the to et Additionally, reported (Katzav 1997). ZAP70 al., by et subsequent Vav1 the for required on of be 315 may phosphorylation (pY) that phosphotyrosine interaction an to kinase, bind ZAP70 can Vav1 domain SH2 C rngncTcls hc r pcfcfrteN6 peptide NP68 the H-2D for specific on are which presented cells, T transgenic TCR hrb eosrtn htbt h E-eedn and correct Results its GEF-dependent require the Vav1 of both localisation. functions flux, that signalling calcium TCR-induced -independent subsequent demonstrating for and and phosphorylation activity, thereby its GEF for of required activation also are Vav1 oivsiaetercuteto a1t h yas nprimary in synapse the to Vav1 CD4 of recruitment the investigate both To in synapse immunological the CD4 to recruited is Vav1 SLP76. of SH3 Y128 and SH2 and the Y112 that on show that we show depends and Furthermore, movement, recruitment this for required the are Vav1 of domains n oteI nTcl:P ojgts(rymne l,2002; but al., et cells, (Groysman resting conjugates stimulation cell:APC in T TCR in cytoplasm following IS membrane the the to plasma in and the located to is recruited Vav1 that ta. 03 hn ta. 98.Hwvrn tde odt have date CD8 to in studies Vav1 no of Zeng However localisation 1998; 1998). 2011; al., the al., et examined al., et Xavier Zhang et 2001; 2003; Sylvain al., al., et 2011; et Villalba al., 2000; et al., et Singleton Tamir 2006; al., et Miletic ewe h el n h Ps(i.1–) iial,we Similarly, CD8 1A–C). in (Fig. IS the APCs both to the (IS) Vav1 and of of synapse recruitment cells immunological analysed accumulation T the confocal the at clear by between of Vav1 imaged and and showed mark and points phosphotyrosine Vav1, (a Analysis time against different phosphotyrosine microscopy. and at against signalling) fixed antibodies were active Cells with Vav1 cells. Vav1- stained detect T would used antibody in We anti-Vav1 conjugates. only an that cell:APC peptide so T OVA APCs of deficient with pulsed formation been induce had to antigen- that as (APCs) mice Vav1-deficient cells from presenting cells B primary with mixed elluamccl ieadi os CD4 T mouse Jurkat in the and in line studies also cell imaging may and leukaemic Vav1 Biochemical cell that IS. expectation the the Vav1 to to of led move has association are these, documented of the this some and with IS, for the 2005). at required microclusters al., et domains mechanism Dombroski similar Vav1 2000; al., the a the et with (Bunnell by associates though unknown Vav1 Nck1 addition, kinase, of In Itk 2010). domain al., may SH3 et and through (Barda-Saad an 2001), protein al., adaptor bind et Grb2 (Nishida also the interaction of SH3-SH3 domain atypical SH3 an an to bind can r pcfcfra vlui etd OA2-3)presented (OVA323-339) peptide I-A ovalbumin on an for specific are ciae 5Tclswr ie ihpiayBclsfrom NP68 cells with B pulsed primary been had with that mixed APCs were as cells mice Vav1-deficient T F5 activated h erimn fTRpoia inligmlclsto molecules signalling proximal TCR of recruitment The + + n CD8 and el eue O11 C rngncTcls which cells, T transgenic TCR DO11.10 used we cells T d Mrh ta. 90.AtvtdD1.0Tclswere cells T DO11.10 Activated 1990). al., et (Murphy + rmr cells T primary + b n CD8 and Mmlk ta. 93.Oc again, Once 1993). al., et (Mamalaki + el,w hwta Vav1 that show we cells, T + + el.I hsstudy, this In cells. T el aeshown have cells T A + oano Vav1 of domain el sn F5 using cells T B oan of domains B Journal of Cell Science uha hs hw n() ubro el nlsd() T(5,S2 (103) SH2* (75), WT (n): a analysed with cells determined of Number were (B). in shown those as digitally such was nucleus the from signal L-HBmtnswr ojgtdfr7min 7 for conjugated were mutants NLS-SH3B tie ihatbde gis hshtrsn p)adEF,a ela DI as well as EGFP, and (pY) phosphotyrosine against antibodies with stained inl(L)(47 49 43)aehglgtdwt seik.( asterisks. with highlighted are K493A) R489A (K487N (NLS) signal n a1 swl sDCiae.AC r i are APCs images. DIC as well as Vav1, and sdi hssuy h uain nteS2(66) SH3 (R696A), SH2 the in mutations The study. this in used P8ple Ps( el rm129S8- from cells (B APCs NP68-pulsed uha hs hw n() ubro el nlsd() T(0) H 7) PH* (71); DH* (102); WT (n): analysed cells Vav1. of wild-type Number to (E). different in shown those as such i.2 orc oaiaino uatVv rtisi 5CD8 F5 in proteins Vav1 mutant of localisation Correct 2. Fig. R2G oan shglgtdwt seik.( asterisks. with highlighted as domains (R422G) t ts;* -test; P , .5 ** 0.05, utatda hw.Saebr 5 bar: Scale shown. as subtracted Vav1 P dctdwt seik.Saebr 5 bar: Scale asterisks. with ndicated , 2 / ihN6-usdAC Bclsfo 129S8- from cells (B APCs NP68-pulsed with .1 *** 0.01, 2 E )CD8 ie.Iae hwsnl pia etoso elACconj cell:APC T of sections optical single show Images mice). + P 5Tclsepesn TVv rVv ihmttosi h Ho Hdmiswr ojgtdfr7mnwith min 7 for conjugated were domains PH or DH the in mutations with Vav1 or Vav1 WT expressing cells T F5 , A .0.( 0.001. W3Y,adSH3 and (W637Y), + el eursteS2adSH3 and SH2 the requires cells T D ceai ersnaino a1poen ihmttosi h H(K3A)adPH and (LK334AA) DH the in mutations with proteins Vav1 of representation Schematic ) B m .( m. )CD8 C mgs Psaeidctdwt seik.Frqatfcto fteSH the of quantification For asterisks. with indicated are APCs images. C ,SH3A*(76),SH3B*(63),NLS*(74),NLS-SH3B*(66).Statisticallysignifi rpssoigma ( mean showing Graphs ) + m 5Tclsepesn GPa1wl-ye(T,S2 SH3 SH2, (WT), wild-type EGFPVav1 expressing cells T F5 .( m. B W2K oan fVv n uaindis mutation a and Vav1 of domains (W820K) F rpssoigma ( mean showing Graphs ) 7) h erimn ne nteD n Hmtnswsntsignificantl not was mutants PH and DH the in index recruitment The (70). Vav1 2 / 2 ie.Iae hwsnl pia etoso elACconjugates cell:APC T of sections optical single show Images mice). B 6 domains. E)rcutetidxo GPa1qatttdfo images from quantitated EGFPVav1 of index recruitment SEM) a1lclsto n ucin5305 function and localisation Vav1 gtssandwt nioisaantpopoyoie(pY) phosphotyrosine against antibodies with stained ugates 6 E)rcutetidxo a1qatttdfo images from quantitated Vav1 of index recruitment SEM) ( A ceai representatio Schematic ) bigtepttv ula localisation nuclear putative the abling A fEFVv constructs EGFPVav1 of n ,SH3 3 B B ,NLSand uat h EGFP the mutant, atdifferences cant y(NS) Journal of Cell Science S(i.1,) GPVv a o on ocnrtdat concentrated found CD4 either not in IS was the of EGFP-Vav1 the centre across the 1H,I). distribution (Fig. lamellal the EGFP- or the diffuse IS of define peripheral, most a that had This and to Vav1 first formation time. the conjugate of used in following function occurred min EGFP-Vav1 a 2 was of as recruitment IS peak conjugates that the showed at the EGFP-Vav1 peptides. of of agonist distribution with analysis pulsed APCs Image with conjugated were these etrwsue oifc CC CD4 the 5C.C7 classify infect the retroviral to to expressing at used EGFP-Vav1 and Vav1 was The of time, vector 2009). distribution the al., real the et image of in (Singleton terms IS to IS in protein recruitment the data of fusion to and pattern this 1G, Vav1 used (Fig. of peptide we movement of Initially absence the shown). in not not but APCs, with itiuino a1wsbodysmlrbtenCD4 between similar broadly was CD8 Vav1 of distribution etal nCD8 in centrally ifrne ewe h eltps npriua,tecytotoxic the particular, in types; CD8 of cell responses the between differences htwiepopoyoiewsuulyfudi h peripheral the CD4 note in in also IS found the usually We of regions was 2001). phosphotyrosine al., while et that (Stinchcombe formation conjugate CD8 n 5CD8 F5 and CD4 both in IS the to both recruited is against the CD8 Vav1 at and Thus antibodies accumulated 1D–F). both (Fig. with Image that IS showed conjugates. stained Vav1 cell:APC and conjugates T phosphotyrosine of of formation analysis induce to peptide iia oVv,EF-a1wsas erie oteI of IS the to recruited also was CD4 EGFP-Vav1 primary Vav1, to similar 5306 EF)adVv EF-a1.Iaigo O11 CD4 protein DO11.10 of fluorescent a Imaging expressing green (EGFP-Vav1). vector Vav1 enhanced retroviral and a between (EGFP) generated protein we IS, fusion the to Vav1 essigfrlne tteI nCD4 in IS the at longer for persisting oalwfrhraayi ftemcaimo erimn of recruitment of mechanism the of analysis further allow To + + el,tekntc eesmwa ifrn,wt Vav1 with different, somewhat were kinetics the cells, T el Fg BCEFHI.Ti a elc functional reflect may This 1B,C,E,F,H,I). (Fig. cells T + ora fCl cec 2 (22) 125 Science Cell of Journal rmr cells. T primary + + el netdwt hsvrlvco hwdthat showed vector viral this with infected cells T n CD8 and + + el,i gemn ihpeiu eot of reports previous with agreement in cells, T el cu eyrpdy ihn45mnof min 4–5 within rapidly, very occur cells T + + el,i a eeal otabundant most generally was it cells, T el nppiespecific-conjugates peptide in cells T + rCD8 or + rP4CD8 P14 or + el oprdto compared cells T + el.Wiethe While cells. T + el and cells T + and + + fetwsol ata.I otatteSH3 the contrast In partial. only was effect uain(HA)aoihsbnigo h SH3 the of binding abolishes W637Y (SH3A*) the peptides; domain mutation containing SH2 phosphotyrosine the bind of to ability Vav1 of the abolishes (SH2*) mutation R696A oan fVv eerqie o erimn oteI.We which point IS. SH3 SH2, with the determine the to EGFP-Vav1 in to recruitment expressing mutations for vectors protein required retroviral EGFP-Vav1 were generated Vav1 the of used domains we Next SH3 and SH2 The uaint nciaeteNS rvoswr a eee eight deleted novel had a work designed Previous we NLS. this, the inactivate address to To mutation recruitment. IS in domain rtis xrsino hs uatEF-a1poen in proteins EGFP-Vav1 mutant these CD8 of Expression proteins. rti otences ahrta ietrl o h SH3 the for the role of direct some of a localisation than to rather due nucleus, be the might to EGFP-Vav1- had IS of protein recruitment the authors of reduced domain to the the PH SH3B* the work, if in wondered this (NLS) We signal In Vav1. localisation 2002). nuclear a al., defined et (Houlard nucleus a1t h -emnlS3dmi fGb Nsiae al., et (Nishida the of Grb2 ability the of abolishes domain SH3 (SH3B*) mutation SH3 W820K the C-terminal 2001); the to Vav1 erimn oteimnlgclsynapse immunological the to recruitment Ba ta. 09 ekn ta. 09 ’ef ta. 2004; al., et O’Keefe 2009; al., et 2001). al., Jenkins et 2009; Stinchcombe al., et (Beal a1(i.2) ale tde a hw htdlto fthe of deletion that wild-type shown to had contrast studies in nucleus, SH3 Earlier the 2B). in (Fig. was Vav1 protein the of 3A–D). or much (Fig. DO11.10 APCs between peptide-pulsed agonist conjugates and of cells imaging T by 5C.C7 shown as cells togyrdcdrcuteto h rti oteI Fg 2B,C). (Fig. IS the SH3 the to of protein Mutation the of recruitment reduced strongly a lorqie o erimn oteI fCD4 of IS the EGFP-Vav1 to of recruitment domain for SH2 required The IS. also the was to recruitment on effect ciesgaln ntecMCo CD8 of cSMAC the in signalling active niaigteS3*mtn GPVv,w oie that noticed we EGFP-Vav1, mutant SH3B* the imaging In B B + oano a1t idpl-rln oisi target in motifs poly-proline bind to Vav1 of domain oano a1cue h rti omv nothe into move to protein the caused Vav1 of domain 5Tclssoe htmtto fteS2domain SH2 the of mutation that showed cells T F5 ojgtdwt H7Bcl ypoaAC usdwt 10 with pulsed APCs lymphoma cell B CH27 with conjugated oanwr ojgtdfr7mnwt V peptide-pulsed BALB/c- SH2 OVA from the with cells in min mutation (B 7 APCs a for with conjugated Vav1 were or domain Vav1 WT expressing cells T H uaini ihihe iha seik ( asterisk. an The with study. highlighted this is in mutation used constructs SH2 mutant SH2 and WT EGFPVav1 uniae rmiae uha hs hw n() ubrof ( Number (57). (B). SH2* ( in (50), shown WT those analysed: as cells such images from quantitated 5 bar, ( images. Scale mean DIC asterisks. showing as with well indicated as are antibodies EGFP, APCs with and stained (pY) conjugates phosphotyrosine cell:APC against T of sections optical t5mn t7mn.Saitclysgiiatdfeecswere differences significant Statistically min). a 7 with at determined of (11 7 initiation SH2* min, (14), after 5 WT min at analysed: 7 cell of and Number 5 formation. imaged conjugate and peptide, agonist MCC i.3 h H oano a1i eurdfrcorrect for required is Vav1 CD4 of in domain localisation SH2 The 3. Fig. 6 E)rcutetidxo GPa1i CC CD4 5C.C7 in EGFPVav1 of index recruitment SEM) B B oan fVv r eurdfor required are Vav1 of domains oanas erae erimn,btthe but recruitment, decreased also domain 6 E)rcutetidxo EGFPVav1 of index recruitment SEM) t ts;* -test; + A cells. T n SH3 and P , Vav1 .5 ** 0.05, ( A ceai ersnainof representation Schematic ) 2 B / 2 oan Fg A.The 2A). (Fig. domains D P ie.Iae hwsingle show Images mice). + , rp hwn mean showing Graph ) 0.01. el Fg 1A,D) (Fig. cells T A uainhdno had mutation B m CD4 ) .( m. A + oanof domain C rmr T primary + Graph ) DO11.10 + cells T m M B Journal of Cell Science oovossrcua oe hncmie ihteSH3B* the with combined play that When NLS role. the R489A, In in structural (K487N, residues 2008). obvious acids basic al., only amino no unpredictable the et three are with which Rapley just K493A), domain, mutated 2002; the we al., contrast, mutation of et the unfolding (Houlard thus and consequences in Vav1, of result domain a PH play could residues the these in of role several however, structural NLS; the in acids amino i.4. Fig. confounding of absence the in mutation SH3B* allowing the thus 2A,B), of (Fig. nucleus study the localisation to abolished EGFP-Vav1-SH3B* (NLS*) of mutation NLS novel this mutation, e etpg o legend. for page next See eue erimn Fg BC.Tu ecnld htthe that EGFP-Vav1 conclude on showed we still effect Thus mutant no 2B,C). double SH3 (Fig. the had the recruitment whereas own that IS, reduced mutant showed the its to conjugates of on recruitment cell analysis T mutation Image F5 NLS* in localisation. proteins nuclear EGFP-Vav1 from effects uain nteedmis(i.2) h 34 K335A L334A Vav1 The of activity 2D). GEF (Fig. the abolishes domains point mutation bearing DH*) mice these (LK334AA, of use in made we IS, mutations the to recruitment in Vav1 oaayeaptnilrl o h HadP oan of domains PH and DH the for role potential a analyse To B oano a1i eurdfrnra Srecruitment. IS normal for required is Vav1 of domain a1lclsto n ucin5307 function and localisation Vav1 Journal of Cell Science ooaiaino GPa1wt L7 n ievra ( vice-versa. and ( SLP76 mean with EGFPVav1 of colocalization *** tie o F.Saebr 5 CD4 bar: Scale GFP. for stained GPa1(re)adSP6(e)wt ooaiainvsaie ya by 5 of visualised bar, overlay colocalization Scale an with colour. show (red) yellow images SLP76 merged and The (green) SLP76. EGFPVav1 or GFP for stained nlsd() TTcls–EFVv 8) Y(9;SLP76 (39); pY (80), cells EGFPVav1 of – Number cells microclusters. T any WT formed (n): that analysed cells in only calculated n niC2 nioisadsandfrGP cl a:5 bar: Scale anti-CD3 GFP. with for coated stained ( coverslips and on antibodies min anti-CD28 3 and for activated were Cells mutants. ircutr.Nme fclsaaye n:EFVv-T(46), any EGFPVav1-WT EGFPVav1-SH3 formed (n): (13), that analysed EGFPVav1-SH2* cells cells in of only Number calculated microclusters. microclusters, by occupied fraction r hw fclswtot( without ( cells of shown are GPa1(5,p 8) ( (84). pY (15), EGFPVav1 ttsial infcn ifrne eedtrie iha with determined were differences significant Statistically i ncvrlp otdwt anti-CD3 with coated coverslips on min 3 CD4 in EGFP of i ncvrlp otdwt anti-CD3 with coated coverslips on min 3 ttsial infcn ifrne eedtrie ihFse’ xc test; exact Fisher’s with determined *** were differences significant Statistically p)idpneteprmns ubro el nlsd() TTcls- cells T WT SLP76 (n): (67); analysed pY two cells (163), and of EGFPVav1 (EGFPVav1) Number five experiments. from independent are (pY) Data microclusters. phosphotyrosine or hshtrsn p) cl a:5 anti- bar: Scale on (pY). min phosphotyrosine 3 for activated were CD3 Cells EGFPVav1. wild-type expressing no ( caused parameters. CD4 these anti-CD28 in of of either inclusion in microclusters; change any significant only formed calculated that area), cells clusters/cell of in (area microclusters by occupied fraction a1famnsadppie rmSP6(Y1,p18ad2pY) and calorimetry. pY128 titration (pY112, isothermal SLP76 by from determined peptides and fragments Vav1 eemndwt ihrseatts;*** test; exact Fisher’s were with differences determined significant Statistically (n): (73), (100). analysed EGFPVav1-SH3A* EGFPVav1-SH3B* cells (43), of EGFPVav1-SH2* Number (72), experiments. EGFPVav1-WT independent two from are microclusters to recruitment i.4 erimn fVv nomcolsesrqie h H and SH2 the requires microclusters into Vav1 SH3 of Recruitment 4. Fig. CD4 in EGFP-Vav1 microscopy image fluorescence reflection to internal at total microclusters (TIRFM) using to recruited by is IS Vav1 the whether examined we Next SH3 and SH2 The of Prisco binding CD8 1998; F5 al., abolishes of et analysis (Han Image (PH*) 2005). domain al., al., mutation PH et et the (Saveliev R422G to domain phosphoinositides DH the the of 2009); folding perturbing without Si rmr el,weesteD,P n SH3 and PH DH, the whereas cells, T primary in IS 5308 hwn rtclrl nti process. this in role critical no show Fg EF.Tkntgte ecnld htteS2and IS SH2 the the that to conclude Vav1 we of together SH3 recruitment Taken that showed affected 2E,F). APCs (Fig. with mutation conjugates in Vav1 neither mutant PH* or DH* nJra el ltdot anti-CD3 onto anti-CD3 plated onto microclusters cells TCR plated antibodies. T of stimulatory induction Jurkat the with in shown coated had studies coverslips Previous onto minutes 3 6 F enpretg fCD4 of percentage Mean ) E)pretg fCD4 of percentage SEM) P P B e + B , , oan fVv n 12adY2 fSLP76. of Y128 and Y112 and Vav1 of domains el omn GPa1mcolsesadma ( mean and microclusters EGFPVav1 forming cells T n niC2 otdcvrlp n tie o F or GFP for stained and coverslips coated anti-CD28 and 6 .01 ( 0.0001. .0.( 0.001. oan fVv r eurdfrnra erimn othe to recruitment normal for required are Vav1 of domains + E)dsoito osat ( constants dissociation SEM) el xrsigEFVv-T H,SH3 SH2, EGFPVav1-WT, expressing cells T ora fCl cec 2 (22) 125 Science Cell of Journal G E en( Mean ) + IF mgso To SLP76 or WT of images TIRFM ) el xrsigEFVv.Clswr ciae for activated were Cells EGFPVav1. expressing cells T B oan fVv r eurdfor required are Vav1 of domains 6 e H E)ae rcinocpe ymicroclusters, by occupied fraction area SEM) m + 2 n niC2 niois(Barda-Saad antibodies anti-CD28 and + .Gah hwtema ( mean the show Graphs m. IF mgso CD4 of images TIRFM ) m el omn GPa1mcolses Data microclusters. EGFPVav1 forming cells T e n ih(v)EFVv microclusters. EGFPVav1 (+ve) with and ve) el W rSLP76 or (WT cells T .Gah hwma ( mean show Graphs m. 2F m .FrSLP76 For m. el GPa1(6) Y(123). pY (169), EGFPVav1 - cells T K P A d fbnigbtenteindicated the between binding of ) 3) EGFPVav1-SH3 (33), * , e e .0.( 0.001. 6 6 niC2 nioisand antibodies anti-CD28 niC2 nioisand antibodies anti-CD28 e + B ri rmr cells T primary in or 2F el xrsigthe expressing cells T IF mgso EGFP of images TIRFM ) + D 2F el,eapeimages example cells, T 2F en( Mean ) el ltdfor plated cells T + 6 omn EGFPVav1 forming ) CD4 A el ciae for activated cells T E)pretg of percentage SEM) 6 ( rSH3 or A I D percentage SD) t rp shows Graph ) + IF images TIRFM ) ts;** -test; 6 m 2F cells T 6 E)area SEM) .( m. A E)area SEM) el – cells T B B C domains (66). * domain P Mean ) , 0.01, e on htteS2dmi on oppie otiigeither containing We peptides to (2pY). bound from residues domain peptides both SH2 to the or bind that pY128 to found pY112, ability containing its assess SLP76 to (ITC) calorimetry a oioe yTRM nlsssoe htwiethe while that showed Analysis TIRFM. protein by SLP76 fusion the monitored of recruitment was the and EGFP-Vav1 expressing hs w eiusw aeueo el rmSLP76 to from cells Vav1 T of of phosphorylation of use the made recruitment on we adapter dependent residues the is SLP76 two IS these whether the the of at examine bind microclusters pY128 to To reported and previously protein. 112 been has (pY) Vav1 phosphotyrosine of domain SH2 The SLP76 Y112 of on depends Y128 microclusters and to Vav1 of recruitment The arigmttosi L7 hr ohtrsn eiushad al., residues SLP76 et (Jordan tyrosine 2008). Y128F) both (Y112F, phenylalanines where with replaced SLP76 been in mutations carrying xlr hsbnigfrhrw xrse h H domain SH2 To the SLP76. expressed on we pY in one further Vav1 to binding of binding this domain each explore either molecules SH2 between of binding Vav1 co-operative Vav1 mutation is One two there why association. that the Vav1-SLP76 clear is of explanation not loss for possible to is lead would site it alone 1994), residue binding al., both et and consensus similar (Songyang very the are sequences SLP76 the of fit that protein Y128 Given and adaptor 1999). Y112 the Koretzky, surrounding of and interaction (Fang Vav1 the with abolished SLP76 coefficient, of Y128 correlation Pearson’s 4H, (Fig. r anti-CD3 anti-CD28 either analysis and onto EGFP-Vav1 with TIRFM plated of Indeed cells co-localisation in of similar. degree SLP76 is large be high Vav1 very should a a If proteins showed distributions two the 4E–G). caused SLP76, (Fig. the to it Vav1 of binding via of microclusters phosphotyrosine, to recruitment recruited for the in staining reduction by judged swt erimn oteI ngnrl h pcfcrecruitment specific the general, in IS microclusters Thus, on the 4B–D). effect to showing (Fig. recruitment no microclusters with cells had decreased as to mutation of EGFP-Vav1 SH3A* in of the percentage recruitment resulted contrast, the In had it 4B–D). but on (Fig. as microclusters, effect EGFP-Vav1 partial by no occupied was microclusters, area into effect fractional The recruitment the 4B–D). affected decreased (Fig. however also microclusters strongly mutation EGFP-Vav1 fractional SH3B* percentage the by mutant and the occupied EGFP-Vav1 by of area SH2* microclusters measured showing as cells the of microclusters that into recruitment showed Vav1 Movies material 2). by supplementary changed 1, 4A; not (Fig. was anti-CD28 clusters of the of inclusion size the microclusters or anti-CD3 microclusters, to onto Vav1 recruited plated rapidly cells is al., in EGFP-Vav1 et that with Raab found interacting 1996; We cells al., T et compared signals Nunes anti-CD3 we transduce 2000; 2005), CD28 al., Tybulewicz, and et 2001; TCR Since (Michel both 2002). both Vav1 and through al., via CD28 stimulation et and to Bunnell TCR response 2006; the in al., phosphorylated et is Braiman Vav1 2005; al., et fVv omcolsesas eursteS2ad oalesser a to and, SH2 the requires SH3 the also extent, microclusters to Vav1 of 5 rvossuishdsonta uaino ihrY1 or Y112 either of mutation that shown had studies Previous xmnto fTclsepesn uatfrso EGFP- of forms mutant expressing cells T of Examination 0.959 2F 6 e uainddntafc h omto fmcolsesas microclusters of formation the affect not did mutation .0 r0.960 or 0.002 ntepeec rasneo niC2 antibodies. anti-CD28 of absence or presence the in E.coli 2F B CD4 domains. uiidi n sdiohra titration isothermal used and it purified , + el eeifce iharetrovirus a with infected were cells T 6 .0,respectively). 0.003, e n h rqec fclswith cells of frequency the and , e ln ranti-CD3 or alone 2F mice e Journal of Cell Science a omaual idn ihteupopoyae peptide. SH3 unphosphorylated the the whether we with identify as unlikely binding To is outside measurable latter peptide, no the the although saw and pocket, Vav1 binding SH2-pY between the the or between fragments, interactions of Vav1 additional co-operativity This of two of is result there a fragment shown). as that perhaps 3-domain suggests binding, peptide detectable (not the 2pY no the using peptide and was when Vav1 unphosphorylated affinity there in Again an enhancement shown). to not data binding and 4I, (Fig. n SH2-SH3 and SH3 SH3 two of 14 Mad15 Mfrtep12adp18peptides pY128 was peptide and 2pY pY112 a the with to the stronger binding significantly for However 4I). nM Fig. 1353 respectively; and nM (1846 n 9 M epciey(i.4) h idn fteSH2 the of a showed binding pY128 and The pY112 both K 4I). containing peptide (Fig. a to respectively domain nM, 990 and Y1 rp18wt iscaincntn ( constant dissociation a with pY128 or pY112 rgeto a1t idt hs etds gi idn othe to with binding occurred Again peptides. peptides these pY to single bind to Vav1 of fragment 03n o Y1 n Y2,rsetvl)adslightly and respectively) pY128, and pY112 for nM 1043 hsehne idn,w esrdteafnte fSH3 of affinities the measured we binding, enhanced this ekrafnt o h igep etds( peptides pY single the for affinity weaker oan ihasalehneeto fiiyi h w pY two not data the and 4I, in (Fig. affinity peptides pY of single SH2 the enhancement to Vav1 small compared the peptide bind a independently with could domain, pY each that implying hw) hr a odtcal idn oappiei which shown). in (not peptide unphosphorylated a were to Y128 binding and Y112 detectable both no was There shown). d etw xmndteaiiyo -oanSH3 3-domain a of ability the examined we Next f50n n tihoer f21(H domain:peptide), (SH2 2:1 of stoichiometry a and nM 520 of A S2famn eae ieteioae H oanwith domain SH2 isolated the like behaved fragment -SH2 A B -SH2-SH3 -oanfamnsfrtep etds The peptides. pY the for fragments 2-domain B oeue idn ooepeptide one to binding molecules K A d rSH3 or f27n,adastoichiometry a and nM, 227 of K d ntemcooa range micromolar the in s B oan otiue to contributed domains K d K 02n and nM 1062 : d f14 nM 1640 of ) A -SH2-SH3 A -SH2 B oanfamn ihwae idn otesnl Ypeptides pY single the to binding ( weaker with fragment domain htteS2 uaincue opeels ftyrosine of loss complete a caused found mutation We SH2* protein. the fusion the that of phosphorylation TCR-induced Pic ta. 05 aeive l,20) hstesame the Thus 2009). process al., this et for critical Saveliev not 2005; is al., domains et these that In suggesting (Prisco of Vav1, 5B). mutations of function (Fig. DH* phosphorylation and the tyrosine phosphorylation PH* affect the not that in shown did had reduction we mutation studies partial SH3B* previous the whereas a effect, no mutation caused had contrast, domain In SH3A the 5A). of (Fig. EGFP-Vav1 of phosphorylation phosphorylation nacdbnigt h p etd ( peptide 2pY the to binding enhanced a1 eue ervrlifcint xrs idtp and wild-type express to CD4 infection primary in of To retroviral EGFP-Vav1 phosphorylation 2005). mutant used on (Tybulewicz, mutations we activity domain on Vav1, of GEF phosphorylated effects its the is of required evaluate activation Vav1 process a full IS. stimulation, for TCR the following at residues tyrosine microclusters of recruitment to of consequences Vav1 functional the examined we Next SH3 and SH2 The domain. u togyehne idn o2Y( 2pY to binding enhanced strongly but otat h SH2-SH3 the contrast, etd fSP6i nacdb h rsneo h SH3 the of presence the by 2pY the enhanced to molecules is Vav1 SLP76 of two pY128 of of and binding pY112 peptide Vav1 show both the to results that 2 binds These and Vav1 was SLP76, shown). of domain interaction (not SH2 peptide the the that 2pY of one stoichiometry to fragments the again Once K d 35n n 02n o Y1 n Y2,respectively), pY128, and pY112 for nM 1062 and nM 1325 : a1lclsto n ucin5309 function and localisation Vav1 B oan fVv r eurdfrits for required are Vav1 of domains B rgetbhvdsmlryt h 3- the to similarly behaved fragment To H uatEFVv,or EGFPVav1, ( mutant SH2 or WT ( with needn experiments. two independent least at from EGFPVav1, of phosphorylation percentage ( mean indicated. show as Graphs pErk2 and Vav1, Erk2 to pY, antibodies with probed immunoblots were the and times, the indicated for antibodies anti-CD28 anti-CD3 and with stimulated were cells yae fCD4 of lysates cell total and immunoprecipitates anti-Vav1 of Immunoblots n SH3 and SH3 and SH2 intact requires Vav1 of phosphorylation TCR-induced 5. Fig. B ihvrssepesn T SH3 WT, expressing viruses with ) + A el,adte examined then and cells, T K ovrs rvrsexpressing virus or virus, no ) B d 7 M Fg I.By 4I). (Fig. nM) 579 : uat fEFVv.The EGFPVav1. of mutants K d 1 M Fg 4I). (Fig. nM) 111 : + el transduced cells T 6 B SEM) domains. e B A Journal of Cell Science xrclua acu,tu iiigclimfu orlaefo nrclua trs h iea hc el eesiuae ihcosikn a crosslinking with stimulated were cells which at time The stores. intracellular from ( arrow. release vertical to a flux by calcium indicated limiting thus calcium, extracellular loecne esr fitaellrCa intracellular of measure a fluorescence, lefursec,amaueo nrclua Ca intracellular of measure a fluorescence, blue uat,o ih()rtoiue xrsigEF-T rNS,S3*adNSS3*mtns ne itgasso GPepeso nTclstransdu cells GFP T distinguish in to expression used EGFP gates show show histograms Inset markers mutants. and NLS-SH3B* protein, and WT SH3B* EGFPVav1 NLS*, expressing or retroviruses EGFP-WT, with expressing retroviruses (C) with or mutants, odmntaeteclimfu nVv-eiin el,GFP cells, T Vav1-deficient in flux calcium the demonstrate To ae,ol GFP only cases, rgesrlaeo Ca of release triggers xeietoto he sshown. is three of out experiment xrs idtp rmtn GPVv rtisi Vav1- in proteins to infection EGFP-Vav1 retroviral mutant used or we Vav1, wild-type of express function this of perturb 6A). (Fig. presence stores the intracellular calcium from the in limit release thus to stimulated and flux calcium were extracellular chelate cells to EGTA when flux, calcium ta. 02.A xetdfo hs ale bevtos Vav1- observations, earlier these from CD4 expected deficient As 2002). al., et i.6 h H n SH3 and SH2 The 6. Fig. TCR- for (IP required PLC is inositol-3,4,5-trisphosphate of Vav1 activation that flux. indicated induced calcium had to GEF- studies signals has Previous TCR also transducing Vav1 functions, functions GEF-dependent independent its to addition In SH3 and SH2 The SH3 and SH2 the in mutations ircutr,as as opeeo ata osof loss partial activity. GEF IS hence or of and to the phosphorylation, activation for to for and required Vav1 complete is IS domains of these recruitment through the cause that to suggesting phosphorylation, recruitment also reduce microclusters, partially or completely nue acu flux calcium induced 5310 oeaut hte uain nteS2o H domains SH3 or SH2 the in mutations whether evaluate To ora fCl cec 2 (22) 125 Science Cell of Journal + + el r hw.Tetm twihclswr tmltdwt rslnigatbd sidctdb etclarw n representative One arrow. vertical a by indicated is antibody crosslinking with stimulated were cells which at time The shown. are cells n CD8 and B oan fVv r eurdfrTCR- for required are Vav1 of domains B 2+ + oan fVv r eurdfrnra C-nue acu flux. calcium TCR-induced normal for required are Vav1 of domains B el hwadfcieTCR-induced defective a show cells T , rmitaellrsoe (Reynolds stores intracellular from C c n ec otepouto of production the to hence and 1 a1dfcetCD4 Vav1-deficient ) 3 ,ascn esne that messenger second a ), B 2+ oan ht respectively, that, domains safnto ftm in time of function a as 2+ safnto ftm nVv-eiin CD4 Vav1-deficient in time of function a as + el eetasue ih()rtoiue xrsigEFVv To H* HA n SH3B* and SH3A* SH2*, or WT EGFPVav1 expressing retroviruses (B) with transduced were cells T 2 el r hw rmclue netdwt iu xrsigEF a1W.I l other all In WT. Vav1 EGFP expressing virus a with infected cultures from shown are cells Vav1 +/+ gen or (green) eiin CD4 deficient sciia o h blt fVv otasueTRsgast the to signals TCR SH3 the transduce while to calcium, Vav1 partially intracellular domain of in to SH2 ability rise again recruitment the the for phosphorylation, with critical once TCR-induced is as with flux, and is conclusion, IS calcium In the mutant TCR-induced 6C). (Fig. NLS*-SH3B* affect compromised the not the does whereas that show mutation and studies NLS* TCR-induced NLS* These the the expressing EGFP-Vav1. cytoplasmic cells compared mutant a T NLS*-SH3B* also to Vav1-deficient we in or mutant flux domain, localisation, calcium SH3B* nuclear this the partial for expressing its function cells to T due the in was whether seen flux understand that To calcium 6B,C). above reduced (Fig. flux cells calcium T in Vav1-deficient SH2* increase in the no in whereas resulted flux, mutation calcium TCR-induced reduced partially oh5.7CD4 5C.C7 both ehv hw ht ncmo ihmn te signalling other CD4 many primary recruited of with is IS Vav1 the common signals, to TCR in proximal transduce that, that molecules shown have We Discussion role. detectable, yet minor, SH3 Vav1 A 2 oanhdn fet uaino h SH3 the of mutation effect, no had domain + / 2 el xrsigidctdcntut n ae so histograms. on as gated and constructs indicated expressing cells T bak CD4 (black) + + el.Oc gi efudta uaino the of mutation that found we again Once cells. T n GFP and + ( A n 1 CD8 P14 and rpsso enrtoo no1violet/blue Indo-1 of ratio mean show Graphs ) + rCD8 or 2 + el.Gah hwma ai fId- violet/ Indo-1 of ratio mean show Graphs cells. n CD8 and + el ntepeec fET ochelate to EGTA of presence the in cells T + el hwdta a1was Vav1 that showed cells T + el.Dnmciaigof imaging Dynamic cells. T B oanpasamore a plays domain tbd is ntibody B domain ced Journal of Cell Science H oano a1i eurdfrisrcutett SLP76- SH3 to the recruitment of its mutation for that required the and that is microclusters showed which Vav1 containing results cells, of Our T IS. Jurkat domain the in SH2 study to recent recruitment a for with that required agree show not data is our binding however this binds 1998), also al., Vav1 et of (Han membrane domain phosphoinositides PH for The required 2008). is (Lemmon, recruitment interaction this and phosphoinositides Koretzky, and (Fang phosphotyrosine SH2 either Vav1 to 1999). the bind of ability loss to causes the domain Y128 despite explain or association, also Y112 Vav1-SLP76 either may of of and mutation microclusters, that to observation and the IS the to recruitment hti rmr os el h a1Nkitrcinmybe Vav1. may of interaction movement Vav1-Nck the the for cells critical T less mouse primary in that ta. 01.Frhroe nCD4 (Singleton in Cdc42 Furthermore, active 2011). and F-actin al., of et that to correlated closely tblsdb utpedmi neatos oee h akof SH3 lack the the cells, of however T certainly effect interactions, in is any domain formed It multiple is results. by complex our stabilised Vav1-Nck-SLP76 to SH2 a contrast the for that in role possible process, no this provides in but recruitment, domain its for important is oorrsls h aesuyas on ata oefrthe for role partial a found also study same the contrast SH3 In results, 2011). our al., et to (Sylvain movement this inhibits partially n a1 uhamdlpeit htteSH3 the that predicts model a Such Vav1. and opeeydpneto h H oano a1adpartially and was Vav1 SH3 of functional microclusters a domain to on SH2 dependent and the IS on the dependent completely to recruitment This antibodies. with anti-CD3 stimulation following immobilised membrane plasma the at microclusters eurmn o h H Ho SH3 or PH DH, the for requirement SA itiuin iia itiuinwsas enin seen also was distribution the similar to CD4 diffusely DO11.10 closely A or most IS distribution. corresponding the pSMAC interface, of cell-cell periphery the the across to predominantly localised L7,adtruhisCtria H oanbnst the to binds domain SH3 on C-terminal pY128 its and pY112 SH3 through to binds and Nck proposed domain, SLP76, authors SH2 The same in its Nck. through SLP76 the protein data with that adaptor However Vav1 our the of on 2010). with association dependent cells al., was agreement Jurkat in et in that showed (Barda-Saad SLP76, study cells of mutations T by Y128 abrogated primary and was Jurkat SLP76 in Y112 to study Vav1 another in SH3 of In the binding 2001). to the al., et cells, binding we (Nishida abolish one Grb2 to the mutation of predicted and different domain are mutation a this (W637Y) of both used use that have the cells note to Jurkat we or between though cells, differences (P657A), T to mouse due primary be and may but unknown, is ohteS2adSH3 and SH2 why explains the potentially molecule both SLP76 one to molecules Vav1 oan,adta hsbnigwssrnl nacdb the by enhanced strongly was SH3 binding the SH2 pY128, of Vav1 this and two presence that pY112 recruit and both can and to peptide Vav1 domains, phosphorylated binds doubly of Vav1 was a of co-localisation showed that domain studies interpretation SH2 of biophysical the addition, degree that an In microclusters. high once in Such residues a SLP76 to these due by phosphorylated. to be domain supported may become SH2 this Vav1 that the they suggesting of SLP76, binding of direct on Y128 dependent was and microclusters Y112 to recruitment Vav1 that showed nsm Hdmi-otiigpoen h Hdmi binds domain PH the proteins domain-containing PH some In A A oani hsrcutet h esnfrti discrepancy this for reason The recruitment. this in domain oano a1 hrb rdigtebnigo SLP76 of binding the bridging thereby Vav1, of domain + el hr h itiuino a1wsmost was Vav1 of distribution the where cells T B e A oan hscoeaiebnigo two of binding cooperative This domain. ntepeec rasneo anti-CD28 of absence or presence the in uaino a1rcutetsuggests recruitment Vav1 on mutation B oan fVv r eurdfor required are Vav1 of domains B oan ncnrs efudno found we contrast In domain. + A el a1wsfudin found was Vav1 cells T oan.Frhroe we Furthermore, domains. A oano Vav1 of domain B domain oani eurdfrhg fiiybnigt SLP76. to binding affinity high for required is domain hs C-nue a1popoyaini eue nSLP76 kinase. in reduced is bring the with phosphorylation to agreement In required Vav1 it. be by TCR-induced phosphorylates may that this, turn kinase phosphorylated the in by to which close caused SLP76, Vav1 be be to bind could on to to phosphorylation pY315 failure to reduced bind hence the to SH2* Alternatively, Vav1 the and of failure of cells by ZAP70 Jurkat phosphorylation caused in Defective be might microclusters 2011). mutant to al., Vav1 et did of Y174) (Sylvain recruitment Y160, (Y142, affect of sites not mutation is phosphorylation because tyrosine phosphorylation unlikely three is all that localisation, correct possibility, for required reverse GEF-activating this suggesting The for critical is phosphorylation, modification. Vav1 of TCR-induced localisation correct its that inhibit partially otencesi h HB on uatsget htthis that in suggests Vav1 retains mutant which point protein, SH3B* cytoplasmic a the localisation binding Vav1 in is in domain nucleus change dramatic in the 2005; 1996; The al., to al., reported et 1998). Romero et al., 1995; been et al., Blanchet et Romero has 1998; Clevenger 2010; al., Vav1 al., et et Brugnoli of (Bertagnolo localisation studies known, currently several nuclear is physiological little a which the about nucleus, defining though the in into in Vav1 useful Vav1 of more of be role This entry NLS. may the block mutation defining to restricted precisely able more was thereby nucleus, just domain the the PH on than located the mutation K493A) that of other R489A, (K487N, show surface acids functions we amino study three perturb the just present of may the for of In deletion acids, important localisation. a mutation, nuclear amino probably their that thus eight are and nuclear showed domain, all residues PH for Vav1 the eight of required of stability these structure was NLS of the that acid some amino of domain eight Inspection PH an localisation. defined the had 2002). within authors al., et located the (Houlard study line cell this mast In a cells, RBL-2H3 of nucleus neato.Atraiey h SH3 SH3-polyproline canonical the a Alternatively, through perhaps interaction. cytoplasm, the u eut niaeta hog t H oan a1bnsto binds Vav1 PLC domain, phosphorylates SH2 that its kinase through that Itk indicate the results binds protein, Our adaptor turn PLC an in GEF-independent as of which This be binding to 2011). the proposed al., been stabilising et has Vav1 (Sylvain of cells corroborated function recently T finding (Miletic a Jurkat activity 2009), GEF al., in cells al., its et T Saveliev of 2009; mouse et independent al., primary is et in (Reynolds Vav1 that of shown 6A) function have (Fig. this of others release and stores triggering We intracellular 2002). by flux from calcium calcium to leads which messenger eurdfrtepopoyaino PLC of phosphorylation is the Vav1 because for likely most required Vav1 This on flux. depends calcium pathway TCR-induced signalling inhibit partially or fully domains also SH3 would the possibility for a requirement Such partial 2008). the explain al., et (Jordan thymocytes tutr ftewoeo a1wudhl drs this address help would 3D Vav1 A of 2002). al., whole et the the masks possibility. (Houlard that of suggested conformation structure previously a in as resulting NLS, Vav1 in sequences SH3 oaiet h ulu.Ti ssmlrt eut naprevious a in results to SH3 similar of is deletion that This showing nucleus. study the to localise ciain sjde yrdcdTRidcdIP TCR-induced reduced by judged as activation, ial,w hwdta uain nteS2adSH3 and SH2 the in mutations that showed we Finally, uain nteS2adSH3 and SH2 the in Mutations u tde hwdta uaino igeaioai nthe in acid amino single a of mutation that showed studies Our B oan(80)cue ag rcino a1to Vav1 of fraction large a causes (W820K) domain a1lclsto n ucin5311 function and localisation Vav1 c oteLTSP6complex, LAT/SLP76 the to 1 B B assVv omv othe to move to Vav1 causes oan fVv ul or fully Vav1 of domains B oanmybn to bind may domain c n t subsequent its and 1 B oan ic this since domain, 3 second a , c 2F 1. B Journal of Cell Science etd fnlcnetain0.25 concentration (final peptide CD8 F5 isolate To culture Cell methods. standard using Vav1 construct in this Mutations into pMSCV-EGFP-Vav1. an introduced generate into were to the cloned vector was between retroviral frame Ser-Thr- linker pMSCV reading 5 acids the open a amino the stop fusion with the EGFP-Vav1 encoding the to codon, This AGTACTGGA, Gly. stop without sequence joined the a EGFP and of including consisting of (Clontech) two cDNA frame Vav1 pEGFP-C1 reading mouse from complete open (EGFP) taken protein complete was fluorescent the codon green Vav1, enhanced between mouse protein and fusion a generate To constructs DNA SLP76 or C57BL/6J from cells marrow chimeras Bone marrow bone of Generation disrupting mutation a carrying Mice Mice Methods and Materials we and GEF-dependent its functions. localisation both we for -independent SLP76, this essential and is via for recruitment microclusters this required that to show and domains IS identified the have to recruited is Vav1 Vav1 for of PLC of domains function activation several adaptor the these to this which contribute by N- to mechanism other contribute The the possibly Vav1. and addition, may domain In domains CH 2011). the terminal al., that et identified (Sylvain study same pathway this in domains SH3 the by enhanced is that binding a SLP76, 5312 ylsswt C ufr(.5MNH M (0.15 buffer ACK with lysis by preparations splenocyte from removed were erythrocytes and disrupted, mechanically L7,wr ecie rvosy( previously the 1997). described in al., were et mutation Turner Y112F,Y128F SLP76, 2009; al., the et carrying Saveliev 2005; Mice al., et (Prisco previously described ln)adat-D8(75 ln)atbde tcnetain1.5 concentration at antibodies clone) (37.51 anti-CD28 and clone) SH3 and SH2 the implicated recent a also data, cells PLC our T to with of Vav1 Jurkat agreement activation of In in binding TCR-induced flux. study the the calcium that for to propose leading critical we this, is of SLP76 calcium view TCR-induced normal In a for flux. required are that domains two rma from reconstitution. niois uiywscekdb lwctmtyadwstpclyi h ag of range the in typically was and cytometry 2 flow 80–90%. by checked was anti-Mac1 Purity and antibodies. anti-B220 anti-CD8, using (Dynabeads) selection negative by purified hc hywr sdfrcnuaefraineprmns O11 CD4 DO11.10 experiments. formation conjugate for used were they which ta. 92.Mc ern h 5adD1. 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JACoP of laserarea and nm ImageJ 561 using performed mW Analysis (http://spider.science.strath.ac.uk 50 was ). software a Winfluor with and using synchronised UK), capture was Hamble illumination image Source, and (Point USA), laser Nevada, nm CrystaLasers, provided 488 (GLC-050-561, was mW light Excitation 20 2003). a al., by a et (Mashanov system as (Knipe TIRF described Invitrogen) based previously objective (A10042, UPLSAPO as Olympus an Ig out using 2010), carried anti-rabbit al., et was donkey imaging AF568 TIRF antibody. sc- with secondary antibody, polyclonal Cruz) (rabbit anti-SLP76 Santa an 9062, using detected was secondary SLP76 a antibody. A10262, as Systems) R&D IgY, (NL018, (chicken NL493 IgY anti-chicken anti-GFP goat using gelatin with Invitrogen) detected 0.25% was and EGFP-Vav1 S4521) G7765)]. (Sigma, in performed (Sigma, saponin was antibodies 0.1% secondary containing and primary [PBS of solution Binding blocking min. X- 2 Triton for 0.3% PBS with permeabilised in and 100 PBS, with twice washed temperature, min room at 20 paraformaldehyde for 3% with fixed were cells stimulation, Following control. 10 at (both antibodies anti-CD3 eeclue (0.7–1 cultured were uentnswr meitl sdt nettre el rfoe n trdat stored and frozen or cells target infect to 2 used immediately were supernatants el sda Psi ojgt omto xeiet,wr sltdfo h spleens the 129S8- from isolated of were experiments, formation conjugate in APCs as used cells ET,1 M a de otemedium. the to added was glycol-bis(2-aminoethylether)- mM) plasma 10 the across To (EGTA, ethylene influx 2002). without al., stores et intracellular membrane, (Reynolds from described release calcium previously measure as Biosciences) (BD (2 cytometers am Indo-1 and ml), niC4 n anti-TCR and anti-CD43 2h, 4 rsneo iu o 8h. 48 for virus of presence enatvtdfr3dy ihat-D0(0.5 anti-CD40 with days had 3 that mice for C57BL/6 10 from activated cells with been B pulsed with conjugated APCs were cells lymphoma T cell P14 peptide. T B agonist 5C.C7 CH27 2009). carried with al., conjugated was et were (Singleton EGFP-Vav1 described cells previously of as accumulation processing image of using patterns out the of classification subsequent uentn t20 p o ,weesCD8 whereas viral concentrated h, 1 the with for cells rpm the 2000 of at centrifugation by supernatant ‘spinfected’ were cells abt(okad 1-3-2) inl eedtce iha dse Infrared Odyssey an with detected were anti- Signals IRDye800 611-132-122). and (Rockland; A21058) (Invitrogen; rabbit anti-mouse Biotechnology), conjugated Upstate Fluor-680 (4G10; Signaling, Alexa (sc-7383, anti-phosphotyrosine (Cell Erk2 Biotechnology), anti-phospho anti-Vav1 Cruz Biotechnology), Santa Cruz immunoblotting: The Santa for Cruz). (sc-154, anti-Erk2 Santa used 2502), (C-14, were Vav1 against antibodies Vav1 2002). antibody following al., an et using (Reynolds immunoprecipitated earlier was described as performed were immunoblotting 37 peptide. agonist 80 oiaemcolse omto,atvtdTclswr tmltdfr3mnat min 3 for stimulated were cells T activated formation, microcluster image To ˚ 2+ ncvrlp Mte oprto;P5-.-0C rvosycae with coated previously P35G-1.5-10-C) Corporation; (MatTek coverslips on C ˚ e m o ae s.Tclswr rndcdwt ervrlvcos2 F CD8 (F5 h 24 vectors retroviral with transduced were cells T use. later for C lxi el aeldwt eC-ojgtdat-D,anti-CD3 anti-CD4, PerCP-conjugated with labelled cells T in flux (10 itr ormv eldbi,cnetae ycnrfgto (15,000 centrifugation by concentrated debris, cell remove to filters m ˚ )adrsseddi /0ho h rgnlvlm.Concentrated volume. original the of + 1/10th in resuspended and C) m e ABcTcls r4 otatvto D1.0CD4 (DO11.10 activation post h 48 or cells) T BALB/c Vav1 /lfor g/ml m 21 ln)adi oecssas ihat-D8(75 clone) (37.51 anti-CD28 with also cases some in and clone) (2C11 /l n D8(10 CD28 and g/ml) 2 / 2 rBALB/c- or Vav1 6 m 2+ 10 /l.Pl--yiecae oesiswr sda negative a as used were coverslips coated Poly-L-lysine g/ml). m 2 b 6 flux /l oeua rbs a esrdwt SI flow LSRII with measured was Probes) Molecular g/ml, / el/l ihI- 1 gm)fr9 .Vv-eiin B Vav1-deficient h. 96 for ng/ml) (15 IL-2 with cells/ml) 2 niois uiywstpcly85–95%. typically was Purity antibodies. el o 2hi elmdu.Sbeunl,cells Subsequently, medium. cell T in h 72 for cells T Vav1 m + 2 /l n usqetimnpeiiainand immunoprecipitation subsequent and g/ml) rP4CD8 P14 or / 2 ieb eaieslcin(yaed)using (Dynabeads) selection negative by mice 6 0 .0Aojcieadacso build custom a and objective 1.40NA 100 m + /l n usdwt 10 with pulsed and g/ml) el xrsigEF-a1and EGFP-Vav1 expressing cells T + el eeclue nthe in cultured were cells T N,N,N 9 ,N 9 ttactcacid -tetraacetic + m el) CD4 cells). T /l.CD4 g/ml). m e m MMCC Mgp33 (10 m + g/ g T + + , Journal of Cell Science ** etgoo . acii,M,Vlna . aael,E n aiai S. Capitani, and E. Caramelli, S., Volinia, M., Marchisio, V., Bertagnolo, eobnn rget fmueVv eeepesdin expressed were Vav1 mouse of fragments Recombinant isothermal and (ITC) proteins calorimetry recombinant titration of was purification signal and mean Expression Each generate to to proteins. set combined was the total which were of min, experiments of 1 intensity multiple at the those EGFP-Vav1 and of of 100% to signal ratio phosphorylation the proteins a to software. as normalised phosphorylated determined manufacturer’s of was the phosphorylation bands with of analysed degree and The Biotechnology) (Li-Cor Imager el .M,Aiev,N,Vra . aeo,T . aiie-hms . Norris, G., Vasiliver-Shamis, O., T. A., Cameron, R., Balbo, Varma, N., O., Anikeeva, M., A. Perl, Beal, N., Hassan, H., and M. A. Pauker, V. N., Barr, Shirasu, C., M., S. Barda-Saad, Bunnell, R., Titerence, A., and Braiman, N. M., Hogg, Barda-Saad, T., Norton, N., S. Pagakis, A., Mathiot, M., Bracke, L., Ardouin, K. M. Rosen, and Y. X. Huang, E., W. Lowry, B., Aghazadeh, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.105148/-/DC1 online available material Supplementary release. immediate Deposited for 089185]. number PMC fellowship. [grant in Trust R.K. Wellcome U117573808]; programme Curie by and and Council supported was T.C. Research U117565398 Marie Medical U117527252, K.R., UK numbers J.R., EU the [grant A.S., by a funded O.K., were 025449). V.T. and EIF (fellowship number fellowship 508-2005) long-term (fellowship EMBO ALTF an of number recipient a Edina was M.F. assistance, Daniel Funding advice. expert and for Koretzky mice for Gary chimeric and of Smerdon Zachacz generation Steve in Billadeau, help Agnieszka for Schweighoffer thank * We figures: the Acknowledgements on indicated are two-tailed two-tailed differences nonparametric unpaired the significant an using Statistically out test, carried were Mann–Whitney comparisons statistical All analysis Statistical 16 at expressed was (669–771) domain utatdfo h a T aapirt nlsswt h otae ir Origin Micro software, the model. with binding analysis single-site to a prior assuming data 7.0, version ITC raw the from subtracted eune:p12 DpEPD Y2:DDYSN and DGDpYESPNE pY128: were following EDDpYESPND, the Peptides contained 20 nM. and at peptide 280 performed Bristol were and mM pY112: experiments at 1 of Protein ITC 2pY:EDDpYESPNDDDPDGEDDGDpYESPNE. EDTA, University absorbance peptides. mM Mawby, 1 the sequences: by W. NaCl, re-suspend by mM determined to 150 synthesised used 7.5, were pH also Hepes concentrations was mM carried (20 which was filtration buffer TCEP), gel All The ITC filtration. protein. with in gel the overnight by out of homogeneity incubated to (Pierce), C-terminus purified beads the and streptavidin-coated at protease biotinylation to site a bound Protease containing was PreScission vector protein SH3-SH2-SH3B a pET22b by protocols. modified preceded a standard site in to expressed was according (596–845) chromatography filtration gel itain HAS2(9–7)adS2S3 6985 eeepesdat expressed were gel (669–845) by SH2-SH3B the homogeneity to of and purified removal 37 (596–771) was After protein SH3A-SH2 chromatography. the filtration. Protease affinity PreScission GST by by GST-tag purified Sciences), Life P ˚ ucinadcl oaiaina h muooia synapse. immunological the at polarization cell and J. function L. V. Tybulewicz, hshioiie3kns uiggauoyi ifrnito fH-0cells. HL-60 of differentiation and Lett. granulocytic C-gamma1 during phospholipase domain. 3-kinase to secretory Vav phosphoinositide tyrosine-phosphorylated the of association to Nuclear delivery granule lytic Y. of Sykulev, pathway and Immunity the L. regulate M. signaling Dustin, J., P. polymerization. actin for critical al. are et 2315-2328. complex P. SLP-76 Schuck, the E., at Appella, interactions C., J. Houtman, H., Yamaguchi, cytoskeleton. actin the to receptor E. L. Samelson, 790-797. o eifo uoniiino h b oooydmi fpooocgn a by Vav proto-oncogene of domain homology Dbl the phosphorylation. of tyrosine autoinhibition of relief for rmpT8 Nvgn ihaHis a with (Novagen) pET28a from C , ˚ sn nIC20clrmtr(ELf cecs.Haso iuinwere dilution of Heats Sciences). Life (GE calorimeter ITC-200 an using C .1 *** 0.01, 441 480-484. , 31 632-642. , P , 0.001. 20) yai oeua neatoslnigteTcl antigen cell T the linking interactions molecular Dynamic (2005). 20) a1tasue C inl eurdfrLFA-1 for required signals TCR transduces Vav1 (2003). Cell 102 625-633. , a.Immunol. Nat. ˚ saGTfso rmpE-P (GE pGEX-6P1 from fusion GST a as C 6 20) ieiso al elreceptor cell T early of Kinetics (2009). tg n uiidb iklafnt and affinity nickel by purified and -tag, 6 t ts,o ihrseattest. exact Fisher’s or -test, 80-89. , 20) tutrlbasis Structural (2000). u.J Immunol. J. Eur. 21) Cooperative (2010). .coli E. 6 SEM. MOJ. EMBO h SH2 The . P , (1998). FEBS 0.05, 29 33 , , Jordan,M.S.,Smith,J.E.,Burns,J.C.,Austin,J.E.,Nichols,K.E., ekn,M . sn . tnhob,J .adGifts .M. G. Griffiths, and C. J. Stinchcombe, A., Tsun, R., M. Jenkins, S., Gisselbrecht, A., Germani, F., Regnier-Ricard, R., Arudchandran, M., Houlard, osne,L . re,I . wt . h,T,Buit,D . aisn . Lewis, L., Davidson, M., D. Bautista, T., Chi, W., Swat, A., I. Graef, J., L. Holsinger, a,J,Lb-hls . a,B,Su . i,Y,Mselr .D,Kiha .M., U. Krishna, D., R. Mosteller, Y., Xia, X., Shu, B., Das, K., Luby-Phelps, J., Han, S. Katzav, and A. Alcover, I., Hornstein, M., Groysman, S. A. Shaw, and K. A. Chakraborty, L., M. Dustin, heck .E,Bou,A,Zag . ahw,I . ua .L,Fse,S A., S. Foster, L., G. Hura, I., I. Mathews, H., Zhang, A., Brooun, E., J. Chrencik, L. M. Dustin, and R. Varma, G., Campi, rku,A,Boly .K,Smn . ai,M . hw .S,Aln .M and M. P. Allen, S., A. Shaw, M., M. Davis, C., Sumen, K., S. Bromley, A., Marenge Grakoui, K., Tedford, H., Nishina, Y.-Y., Kong, K.-D., Fischer, A. G. Koretzky, and N. Fang, N., Desai, S., Bromley, J., Li, D., A. Holdorf, W., M. Olszowy, L., M. Dustin, unl,S . igr .L,Hn,D . aqe .H,Jra,M . Seminario, S., M. Jordan, H., B. Jacque, I., D. Hong, L., A. Singer, C., S. Bunnell, obok,D,Huhln,R . an,C . rct . aeoo . Caplen, A., Takesono, P., Precht, M., C. Labno, A., R. Houghtling, D., Dombroski, unl,S . og .I,Kro,J . aaai . cld,C . ar .A. V. Barr, J., C. McGlade, T., Yamazaki, R., J. E. Kardon, I., L. D. Samelson, Hong, C., and S. W. Bunnell, Zhang, P., R. Trible, V., Kapoor, C., S. Bunnell, otlo .S,Wles .E,Me .J,Tre,M,Ryod,L . rso A., Prisco, F., L. Reynolds, M., Turner, J., P. Mee, E., M. A. A. Walters, Gewirtz, S., and P. M. Costello, S. Luger, L., D. Sokol, W., Ngo, V., C. Clevenger, unl,S . ih,M,Yfe .B,Fnel .R,Cnly .C n eg .J. L. Berg, and C. L. Cantley, R., P. Findell, B., M. Yaffe, M., Diehn, C., S. Bunnell, O. Acuto, and S. M. Hershfield, A., F. Letimier, A., Cardona, F., Blanchet, azv . uhrad . aka,G,Y,T n es,A. Weiss, and T. Yi, G., Packham, M., Sutherland, S., Katzav, rgoi . abrii . ai-ln,N,Pv,R,Mrhso . rsil,S., Grassilli, E. M., Marchisio, L. R., Piva, Samelson, N., Varin-Blank, and E., Lambertini, L. F., Brugnoli, C. Sommers, M., Barda-Saad, A., Braiman, shnrne,A .adKrtk,G A. G. Koretzky, and C. A. Aschenbrenner, tegho elrcpo inlcnrl h oaiaino yooi ahnr to machinery cytotoxic of polarization the controls synapse. signal immunological receptor the cell T of N. strength Varin-Blank, and complexes. J. active Rivera, transcriptionally U., Blank, eiin ieipiaea ci eurmn o ypoyesga transduction. signal lymphocyte for requirement actin Biol. an Curr. R. implicate G. mice Crabtree, and deficient W. F. Alt, S., R. I3kns nrgltn ciaino a-eae unsn rpopaae yVav. by triphosphatases guanosine Rac-related of activation Science D. regulating Broek, in and 3-kinase A. PI M. White, R., J. Falck, er,J . tef,M,Rmg,P,Wde,H tal. et H. Widmer, P., Ramage, M., Streiff, J., J. Perry, signaling. for scaffolds as microclusters receptor Med. cell T complex-dependent 7155-7166. w euaoso h Tae,fnto oprtvl ssga rndcr nTcell T in transducers signal as pathways. cooperatively receptor-induced function antigen GTPases, Rho of regulators two activation. cell T L. M. Dustin, receptor. T-cell the al. by et mediated S. 8 reorganization Gardener, cytoskeletal G., of Chan, regulator M., a Starr, T., Sasaki, I., Kozieradzki, activity. promoter 16206-16212. interleukin-2 augment to pathways overlapping synapse. immunological the of Biol. function and structure T- al. in et polarity M. cytoskeletal P. and Allen, patterning contacts. A., receptor cell P. orchestrates Merwe, protein der adaptor van novel F., Rosenberger, P., Widder, .C,Br,V . oezy .A n aesn .E. L. Samelson, and activation. T-cell A. drives G. clusters signaling Koretzky, stabilized A., cooperatively V. Barr, M.-C., 20) iaeidpnetfntosfrIki C-nue euaino a and L. P. Vav Schwartzberg, of and regulation K. TCR-induced cytoskeleton. J. in actin Burkhardt, Itk the L., for R. functions Wange, Kinase-independent D., (2005). D. Billadeau, J., N. yaial euae inln assemblies. signaling E. regulated dynamically L. Samelson, and 828-843. xhnefco a saciia rndcro elrcpo inl otecalcium, the to signals receptor cell T pathways. of transducer NF-kappaB J. critical and L. a ERK, V. is Vav Tybulewicz, factor and exchange R. Zamoyska, N., Sarner, yai ci oyeiaindie elrcpo-nue pedn:arl o the for role LAT. a spreading: adaptor receptor-induced transduction cell signal T receptor-initiated drives polymerization cell actin Dynamic T the with Itk cascade. integrating signaling interactions Biochemical (2000). yoiekns A-0cnascaewt h H oano proto-Vav. of domain SH2 the with associate can Chem. ZAP-70 kinase tyrosine unn uloieecag eitdb h -elesnilVav1. essential T-cell the by mediated exchange nucleotide guanine a sncsayfrpoatnsiuae rlfrto n stasoae nothe into translocated is and line. proliferation T-cell a prolactin-stimulated of for nucleus necessary is Vav h D1 rmtri P-eie rmeoye:rl fVv nmodulating in Vav1 of differentiation. role ATRA-induced promyelocytes: 316 during APL-derived complexes in PU.1-containing promoter V. CD11b Bertagnolo, and the S. Capitani, S., Miscia, cells. 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