hSiah2 is a new vav binding protein which inhibits Vav-mediated signaling pathways

MOLECULAR AND CELLULAR BIOLOGY, 0270-7306/99/$04.0010May 1999, p. 3798–3807 Vol. 19, No. 5 Copy igh © 1999, Ame ican Socie y o Mic obiology. All Righ s Rese ed. hSiah2 Is a New Va Binding P o ein Which Inhibi s Va -Media ed Signaling Pa hways ANTONIA GERMANI,* FRANCISCO ROMERO,† MARTIN HOULARD, JACQUES CAMONIS,‡ SYLVIE GISSELBRECHT, SIEGMUND FISCHER, AND NADINE VARIN-BLANK Ins i u Cochin de Ge´ne´ ique Mole´culai e, U363 INSERM, Hoˆpi al Cochin, Uni e si e´ Pa is V, 75014 Pa is, F ance Recei ed 3 Sep embe 1998/Re u ned o modi ica ion 27 Oc obe 1998/Accep ed 25 Janua y 1999 The hema opoie ic p o o-oncogene a has been cha ac e ized as a Rac1-GDP/GTP exchange p o ein which egula es cy oskele al eo ganiza ion as well as signaling pa hways leading o he ac i a ion o s ess-ac i a ed p o ein kinases (SAPK/JNKs). Fu he mo e, a o e exp ession enhances basal and T-cell ecep o (TCR)- media ed s imula ion o he nuclea ac o o ac i a ed T cells (NFAT). We epo he e he in e ac ion be ween Va and hSiah2, a mammalian homolog o D osophila Se en in absen ia (Sina) ha has been implica ed in R7 pho o ecep o cell o ma ion du ing D osophila eye de elopmen ia he p o easome deg ada ion pa hway. Va and hSiah2 in e ac in i o and in i o and colocalize in he cy oplasm o hema opoie ic cells. The S c homology domain o Va and he C- e minal egion o hSiah2 a e equi ed o his in e ac ion. We p o ide e idence o a nega i e egula ion by hSiah2 o Va -induced basal and TCR-media ed NFAT-dependen ansc ip ion. O e exp ession o hSiah2 also inhibi s he onco-Va -induced JNK ac i a ion. Al hough he Va - in e ac ing domain is loca ed in he C- e minal po ion o hSiah2, he N- e minal egion o hSiah2 is necessa y o he inhibi o y ole ha seems o be independen o he p o easome deg ada ion. The a p o o-oncogene p oduc , p95 a , is exp essed p i- ma ily in cells o hema opoie ic o igin (40). The oncogenic o m a ises om he dele ion o 67 N- e minal amino acids o c-Va and induces umo s in nude mice (15, 41). The p ima y s uc u e o Va has se e al s uc u al mo i s ound in p o eins in ol ed in cell signaling, e.g., an N- e minal leucine- ich e- gion, an acidic domain, a Dbl homology (DH) domain, a pleck- s in homology domain, a cys eine- ich sequence and a p oline- ich sequence, wo S c homology (SH3) domains lanking a single SH2 domain, and, inally, wo pu a i e nuclea localiza- ion sequences (59). Indeed, i has been demons a ed ha Va in e ac s, ia i s SH3 domains, wi h di e en cy oplasmic p o- eins, such as G b2 (53, 70), he e ogeneous ibonucleop o ein (hnRNP) K (11, 31), he ocal adhesion p o ein zyxin (33), and Cbl-b (10). Mo eo e , we desc ibed he in e ac ions o he C-SH3 domain o Va wi h Ku-70 and hnRNP C, wo p edom- inan ly nuclea p o eins (58, 60). Despi e he nume ous pa ne s desc ibed, he p ecise unc- ion o Va in cell-signaling pa hways is unclea . Upon s imu- la ion ia immune ecep o s (T-cell ecep o [TCR], B-cell ecep o [BCR], and FcRs) o a ious cy okine o g ow h ac- o ecep o s (cKi , Epo, in e leukin-2 [IL-2], IL-3, in e e on, epide mal g ow h ac o , pla ele -de i ed g ow h ac o , and o he s), Va is apidly and ansien ly y osine phospho yla ed (46, 59). Bo h S c and Syk/Zap70 amily kinases ha e been implica ed in Va phospho yla ion (19, 42). Biochemical e i- dence has shown ha ac i a ed Va ca alyzes, ia i s DH do- main, he con e sion o Rac1 p o ein, a membe o he Rho amily o GTPases, o he ac i e GTP-bound s a e (18, 29, 51). Rac1 ac i a ion leads in u n o he s imula ion o he JNK pa hway (17). Howe e , ecen in i o s udies on a 2/2 mice cas doub on he ole o Va in JNK ac i a ion (24, 35). P e ious s udies on a 2/2 mice also indica ed impai ed T-cell de elopmen and a poo p oli e a ion o ma u e T cells wi h a educed esponse o s imula ion h ough he TCR (25, 67, 72). O e exp ession o a seems o coope a e wi h Syk (19) and SLP76 (69) o syne gis ically induce basal and TCR-ac i a ed ansc ip ion o ei he he IL-2 gene o epo e cons uc s con aining binding si es o nuclea ac o o ac i a ed T cells (NFAT) p esen in he IL-2 p omo e (68). Recen indings om a 2/2 -ac i a ed lymphoid cells also showed he absence o IL-2 ansc ip ion e en hough he JNK ac i i y was s ill no mal (24, 35). These expe imen s demons a ed ha Va is an e ec o molecule ha unc ions downs eam o a a ie y o hema opoie ic cell ecep o s. Finally, Va 2, a p o ein highly homologous o Va bu wi h a mo e ubiqui ous exp ession and poo ly exp essed in hema opoie ic cells, has been iden i ied. I has been p oposed ha his p o ein may ha e Va -like unc- ions in nonhema opoie ic cells (30, 64). To u he in es iga e he ole o Va , we a emp ed o iden i y p o eins ha bind o i s S c homology domains (SH3- SH2-SH3) by he yeas wo-hyb id sys em. We iden i ied hSiah2, a human homolog o D osophila Se en in absen ia (Sina) (12), a ing inge (C 3 HC 4 )-con aining p o ein ha is equi ed o he co ec in eg a ion o signal ansduc ion downs eam o he y osine kinase ecep o Se enless (se ) and he Ras/Ra mi ogen-ac i a ed p o ein kinase (MAPK) pa hway du ing D osophila R7 pho o ecep o de elopmen (9, 13, 22, 26, 43). Recen ly i was shown ha Sina ac s oge he wi h Phyllopod (PHYL), induced by he Ras pa hway, o a ge he ep esso o cell a e de e mina ion T am ack (TTK) o deg ada ion by he p o easome pa hway (45, 66). Th ee highly conse ed mu- ine sina homologs (Siah1A,Siah1B, and Siah2) and wo hu- man homologs (hSiah1 and hSiah2) ha e been desc ibed (1, 20, 34, 37). Al hough he unc ion o he mammalian Siah p o eins has no been elucida ed, ecen s udies sugges ed ha hey migh be in ol ed in ubiqui in-media ed p o eolysis o se e al * Co esponding au ho . Mailing add ess: Ins i u Cochin de Ge´ne´- ique Mole´culai e, U363 INSERM, Hoˆpi al Cochin, 27 ue du Fau- bou g Sain Jacques, 75014 Pa is, F ance. Phone: 33(1)40469332. Fax: 33(1)46339297. E-mail: [email p o ec ed]. † P esen add ess: Depa amen o de Mic obiologı´a, Facul ad de Biologı´a, Uni e sidad de Se illa, 41080 Se ille, Spain. ‡ P esen add ess: Ins i u Cu ie, U248 INSERM, 75005 Pa is, F ance. 3798 on Ma ch 6, 2018 by USE/BCTA.GEN UNIVERSITARIAh p://mcb.asm.o g/Downloaded om p o eins (38, 71), as well as in g ow h a es and p53-induced apop osis (2, 47, 49). In his s udy, we showed ha Va and hSiah2 in e ac bo h in i o and in coimmunop ecipi a ion ex i o expe imen s. We de e mined ha he C- e minal hal o hSiah2 and he en i e SH3-SH2-SH3 egion o Va a e c i ical o binding. By im- muno luo escence s udies, we showed ha he wo p o eins colocalize in he cy oplasm o hema opoie ic cells. We u he demons a ed ha pa hways s imula ed by Va , namely, induc- ion o NFAT-dependen ansc ip ion and onco-Va -media - ed ac i a ion o JNK, a e la gely impai ed in cells o e exp ess- ing hSiah2. This inhibi ion is modula ed by he N- e minal egion o hSiah2 and does no in ol e ubiqui in-media ed p o- eolysis, a unc ion p e iously sugges ed o hSiah2. MATERIALS AND METHODS Plasmids. SHVAV con aining SH3-SH2-SH3 domains ( esidues 623 o 837 o Va ) was PCR ampli ied om pKLS1 (p o ided by M. Ba bacid, Mad id, Spain, and X. R. Bus elo, New Yo k, N.Y.) and used o he DNA-binding domain o LexA in pVJL10 (58). pGEX- 240 and pGEX- 460 we e ob ained by cloning he 240 and 460 cDNA agmen s, espec i ely, om pGAD- 240 (amino acids [aa] 13 o 324) and pGAD- 460 (aa 105 o 324) in o he EcoRI-No I si es o pGEX-4T2 (Pha macia Bio ech Inc.). Cons uc s encoding a unca ed o m o hSiah2 (pGEX- 240Da and pGEX- 240Db) we e ob ained by PCR ampli ica ion om pGAD- 240 wi h app op ia e oligonucleo ides (139 o 1555 and 139 o 1475, espec i ely) ollowed by liga ion in o BamHI-SalI si es o pGEX-4T2. Exp ession plasmids o myc- agged hSiah2 cDNA agmen s we e gene a ed by cloning he co esponding EcoRI-No I agmen s om pGAD- 240 and pGAD- 460 in o a pcDNA3-de i ed plasmid, pCAN-M2, o gene a e pCAN- 240 and pCAN- 460, espec i ely. The ull-leng h hSiah2 was gene a ed by eplacing he SpeI-D aIII agmen o pBK-CMV 240 wi h a PCR agmen ampli ied om human genomic DNA by using a 59p ime in he noncoding sequence o mouse Siah2 ( his sequence was highly conse ed among mouse Siah and hSiah1) and a39in e nal p ime o he 240 clone (nucleo ides 361 o 383). pKES-Va is a pcDNA3-de i ed plasmid con aining a ull-leng h mouse cDNA unde he con- ol o he cy omegalo i us p omo e (1a). pEF-Myc- agged Va was kindly p o ided by A. Al man (San Diego, Cali .). pEF-Myc- agged onco-Va was ob ained by eplacing he Va EcoRI-Bs XI agmen wi h he co esponding sequence om pJC7 (15). The NFAT-luci e ase epo e cons uc (kindly p o- ided by O. Acu o, Pa is, F ance) was de i ed om he pUBT-luc plasmid (21) and con ained he luci e ase gene unde he con ol o he human IL-2 p omo e NFAT-binding si e (23). pSV-bgal ec o (P omega) con ained he b-galac osi- dase gene d i en by he simian i us 40 p omo e . HA-JNK and pGEX-cJun we e p o ided by P. C espo (San ande , Spain). pBK-CMV hSiah1 was p o ided by R. B. Amson (Pa is, F ance). Yeas wo-hyb id sys em. Saccha omyces ce e isiae L40 (MATa p1 leu2 his3 LYS::lexA-HIS3 URA3::lexA-lacZ) was g own a 30°C in YPD medium (1% yeas ex ac , 2% polypep one, 2% glucose) and sequen ially co ans o med by he li hium ace a e me hod (65) wi h pVJL10-SHVAV and a cDNA lib a y om Ju ka cells used o he GAL4 ac i a ion domain in pGAD1318 (58). Double ans o man s we e pla ed on yeas d opou medium lacking T p, Leu, His, Lys, and U a (65). A e 5 days a 30°C, colonies we e pa ched on he same medium and eplica pla ed on Wha man 40 il e s o es o b-galac osidase ac i i y (8). Posi i e clones we e escued and es ed o speci ici y by e o ans o ma ion in o L40 ei he wi h pVJL10-SHVAV o wi h ex aneous a ge s (pLexA-Ras 12 o pLex-Lamin). Sequences o cDNA inse s om posi i e clones o he wo-hyb id sc een we e ob ained o bo h s ands wi h an au oma ic sequence (Applied Biosys ems model 373A) by he dideoxy- e mina ion me hod o Sange e al. (62). Sequence compa isons we e done wi h he FASTA p og am. Cell cul u e, ans ec ion, and an ibodies. Ju ka cells o simian i us 40 T-an igen (T-Ag)- ans ec ed Ju ka cells (p o ided by G. Baie , Innsb uck, Aus ia) we e g own in RPMI 1640 medium (Gibco) supplemen ed wi h 10% hea -inac i a ed e al cal se um (Boeh inge Mannheim), 2 mM L-glu amine, penicillin, and s ep omycin in a 5% CO 2 humidi ied a mosphe e a 37°C. Fo T-Ag Ju ka cells, he medium was supplemen ed wi h 2 mg o Gene icin pe ml (Gibco). Ra basophilic leukemia (RBL) and COS-7 cells we e cul u ed in Dul- becco’s modi ied Eagle’s medium supplemen ed wi h 10% e al cal se um, 2 mM L-glu amine, penicillin, and s ep omycin. COS-7 cells we e ans ec ed by he DEAE-dex an echnique (16). T-Ag Ju ka cells (10 6 ) we e elec opo a ed (a 260V and 960 mF) in 0.5 ml o RPMI 1640 supplemen ed wi h 20% e al cal se um in a Gene Pulse cu e e (Bio- Rad). The an i-Va monoclonal an ibody (MAb) was pu chased om Ups a e Bio- echnology. MAbs agains myc and hemagglu inin (HA) epi opes we e pu - chased om he Ame ican Type Cul u e Collec ion (9E10-CRL 1725) and Babco (12CA5), espec i ely. The an i-CD3 MAb (UCHT1) was p o ided by G. Bismu h (Pa is, F ance), and an i-CD28 was p o ided by D. Oli e (Ma seille, F ance). Rabbi polyclonal an i-hSiah2 an ibodies we e gene a ed agains a pep ide co esponding o aa 150 o 162 o hSiah2 (Syn em). Immuno luo escence s aining. RBL o Ju ka cells we e ixed in phospha e- bu e ed saline (PBS) plus 3% pa a o maldehyde and pe meabilized in PBS plus 0.1% T i on X-100. The co e slips we e insed and blocked o 10 min in PBS plus 0.2% bo ine se um albumin p io o incuba ion wi h an ibodies. Fixed cells we e incuba ed simul aneously wi h bo h p ima y an ibodies (an i-Va MAb and an i-hSiah2 se a, 1:500) o 30 min and hen incuba ed wi h donkey an i-mouse an ibody coupled o Texas ed (1:250) and wi h donkey an i- abbi an ibody coupled o luo escein iso hiocyana e (1:100). Fo pep ide-blocking expe imen s, he an i-hSiah2 an ise um was deple ed by incuba ion o 1ha 4°Cwi h a ni ocellulose memb ane sa u a ed wi h 1 mg o he immunizing pep ide p io o immuno luo escence s aining. The co e slips we e moun ed in Mowiol wi h Dabco an i ading (Hoechs , F ank u , Ge many). The s aining pa e n was analyzed by con ocal lase -scanning mic oscopy (Bio-Rad) a wo di e en emis- sion wa eleng hs ( luo escein iso hiocyana e, 522/535; Texas ed, 605/632), and colocaliza ion was pe o med by u he analysis o supe posed images ha we e ob ained as TIFF iles. In i o binding and immunop ecipi a ion expe imen s. Glu a hione S- ans- e ase (GST) usion p o eins we e induced and pu i ied as p e iously desc ibed (58). Lysa es om lysis o 10 7 Ju ka cells in Nonide P-40 (NP-40) bu e (10 mM T is-HCl [pH 7.4], 150 mM NaCl, 10% glyce ol, 1% NP-40, 1 mM di hio- h ei ol, 1% ap o inin, 1 mM phenylme hylsul onyl luo ide [PMSF], 1 mg o peps a in pe ml, 1 mg o leupep in pe ml) we e incuba ed o 2 h wi h usion p o ein bound o glu a hione-coupled aga ose beads (Pha macia). The beads we e washed i e imes in NP-40 lysis bu e , esuspended in Laemmli bu e and ac iona ed by sodium dodecyl sul a e-polyac ylamide gel elec opho esis (SDS- PAGE) (12% polyac ylamide). The p o eins we e elec o ans e ed o ni ocel- lulose memb anes and p obed wi h p ima y an ibody (MAb an i-Va dilu ed 1:1,000 in T is-bu e ed saline [pH 7.6]–0.05% Tween 20) ollowed by seconda y an ibody conjuga ed o ho se adish pe oxidase and hen e ealed wi h he ECL ki (Ame sham). Fo coimmunop ecipi a ion expe imen s, COS-7 and T-Ag Ju ka cells we e lysed in NP-40 bu e app oxima ely 48 h a e ans ec ion and lysa es we e incuba ed a 4°C wi h an i-myc ag MAb o 4 h ollowed by p o ein A-Sepha ose (Pha macia) o 1 h. The p ecipi a es we e analyzed as desc ibed below. Kinase assays. Subcon luen COS-7 o T-Ag Ju ka cells we e ans ec ed as desc ibed p e iously (16). A e ans ec ion, he cells we e cul u ed o 48 h, washed wi h PBS, and lysed a 4°C in a bu e con aining 20 mM T is-HCl (pH 7.5), 137 mM NaCl, 2 mM EDTA, 1% T i on X-100, 25 mM b-glyce ophospha e, 1 mM sodium anada e, 1 mM PMSF, 20 mg o ap o inin pe ml, and 20 mgo leupep in pe ml. Clea lysa es we e immunop ecipi a ed by incuba ion wi h an i-HA an ibodies (12CA5) o 2ha 4°C. Immunocomplexes we e eco e ed wi h Gamma bind Sepha ose beads (Pha macia) and washed wice in lysis bu e and once mo e in kinase bu e (25 mM HEPES [pH 7.5], 25 mM MgCl 2 ,25mM b-glyce ophospha e, 1 mM sodium anada e, 1 mM PMSF, 20 mg o ap o inin pe ml, 20 mg o leupep in pe ml). JNK ac i i y was de e mined a e esuspen- sion o he immunocomplexes in 50 ml o kinase bu e con aining 10 mCi o [g- 32 P]ATP (3,000 Ci/mmol) pe eac ion and 50 mM unlabeled ATP, wi h 1 mg o GST–c-Jun usion p o ein as a subs a e (16). A e 30 min a 30°C, he eac ions we e s opped by he addi ion o 50 ml o sample bu e , and he p oduc s we e boiled a 95°C o 5 min and esol ed by SDS-PAGE (12% polyac ylamide). Au o adiog aphy was pe o med wi h he aid o an in ensi ying sc een and quan i a ed wi h a Phospho Image (Molecula Dynamics). Luci e ase assays. T-Ag Ju ka cells (10 7 ) we e elec opo a ed wi h 5 mgo he epo e plasmid pNFAT-Luc oge he wi h 20 mg o pEF-Va -Myc wi h o wi hou 20 mg o pCAN- 240/pCAN- 460. Simila amoun s o emp y ec o we e used as a con ol. A 24 h a e ans ec ion, 10 6 cells we e ei he le uns imu- la ed o s imula ed in g ow h medium con aining an i-CD3 MAb (10 mg/ml) plus an i-CD28 (10 mg/ml). A e 8ha 37°C, he cells we e lysed in 200 ml o bu e con aining 100 mM KPO 4 (pH 7.8), 1 mM di hio h ei ol, and 0.5% T i on X-100. Lysa e (20 ml) was mixed wi h 100 ml o assay bu e (200 mM KPO 4 [pH 7.8], 10 mM ATP, 20 mM MgCl 2 ) ollowed by 100 ml o 1 mM luci e in. Luci e ase ac i i y was de e mined in iplica e and exp essed as a bi a y uni s (AU) a e no maliza ion o he b-galac osidase alues o co ec o a ia ion in ans ec- ion e iciency. RESULTS hSiah2, a new Va -in e ac ing p o ein. To iden i y p o- eins implica ed in Va signaling pa hways, we used he yeas wo-hyb id sys em wi h he SH2 and SH3 domains o Va (SHVAV, aa 623 o 837) used o he DNA-binding domain o LexA as a bai o sc een a Ju ka T-cell cDNA lib a y. O app oxima ely 4 310 6 clones sc eened, 450 posi i e clones we e analyzed o hei speci ic in e ac ion wi h SHVAV. O hese, six clones om h ee independen LexA usions VOL. May 1999 INHIBITION OF Va PATHWAYS BY hSiah2 3799 on Ma ch 6, 2018 by USE/BCTA.GEN UNIVERSITARIAh p://mcb.asm.o g/Downloaded om s ongly in e ac ed wi h SHVAV. Sequence analysis o he yeas plasmids showed ha hey we e iden ical o hSiah2 (37). The p e iously desc ibed Sina/Siah p o eins con ain an N- e minal cys eine- ich egion (C 3 HC 4 ) called he ing in- ge domain and, in he C- e minal egion, wo basic clus e s close o a bipa i e nuclea localiza ion sequence (12, 20) (Fig. 1A). The wo la ges clones isola ed, 240 and 472 (aa 13 o 324), con ained almos he en i e coding sequence o hSiah2, whe eas he sho es one, 460 (aa 105 o 324), main ained only he C- e minal 11 aa o he ing inge do- main o hSiah2. A s ong in e ac ion was also de ec ed when 240 was exp essed as a usion o he LexA DNA-binding domain and SHVAV was exp essed as a usion o he Gal4 ac i a ion domain. Full-leng h hSiah2 was isola ed and, as expec ed, in e ac ed wi h SHVAV. As indica ed in Fig. 1B, no ansac i a ion was obse ed when di e en hSiah2 clones we e coexp essed wi h un ela ed usion plasmids (pLexA-Ras 12 o pLexA-lamin). When hSiah2 clones we e es ed wi h G b2, which, like Va , has closely spaced SH3- SH2-SH3 domains (63), no epo e gene ac i i y was de- ec ed (Fig. 1B), sugges ing ha he hSiah2-SHVAV in e - ac ion equi es a he speci ic SH3-SH2-SH3 sequences. Finally, when 240 was cloned in bo h pLexA and pGAD, a s ong sel -in e ac ion was obse ed in he yeas ap assay, sugges ing a possible dime iza ion p ocess o hSiah2 (Fig. 1B). Taking accoun o hSiah1/hSiah2 homology, he expec ed in e ac ion be ween SHVAV and hSiah1 was also obse ed (Fig. 1B). hSiah2 in e ac s wi h Va in i o, and he p o eins coim- munop ecipi a e om COS-7 and Ju ka T cells. The in e ac- ion be ween Va and hSiah2 was hen con i med by an in i o binding assay. Di e en hSiah2 egions used o GST (Fig. 2A) we e exp essed in Esche ichia coli, and he a ini y-pu i ied p o eins we e es ed o hei abili y o bind endogenous Va om Ju ka T-cell lysa es. Consis en wi h he yeas wo-hyb id assay, bo h hSiah2 usion p o eins, con aining an almos ull- leng h hSiah2 (GST- 240) o only he C- e minal po ion o hSiah2 (GST- 460), we e able o in e ac wi h Va (Fig. 2B). To delinea e he egion o hSiah2 esponsible o his in e ac- FIG. 1. Va in e ac s wi h hSiah2 in he yeas wo-hyb id sys em. (A) Sche- ma ic ep esen a ion o hSiah2 and he clones ob ained om he wo-hyb id sc eening. (B) P o ein in e ac ion in he wo-hyb id sys em. The L40 epo e s ain was co ans o med wi h 1 mg o he indica ed pLex- and pGAD-de i ed plasmids, and in e ac ions we e de ec ed as b-galac osidase ac i i y. FIG. 2. In i o binding be ween hSiah2 and Va . (A) Schema ic ep esen- a ion o GST-hSiah2 usion p o eins. (B) Binding o Va o GST-hSiah2 usion p o eins. To al-cell lysa es om 10 7 Ju ka T cells we e incuba ed a 4°C o 2 h wi h 1 mg o he usion p o eins o GST alone. (C) (Top) Lysa es om uns imu- la ed (0 min) and CD3-plus-CD28-s imula ed (1 min and 30 min) Ju ka T cells (10 7 ) we e incuba ed wi h 1 mg o GST- 240. The esul ing complexes we e esol ed by SDS-PAGE, and he Wes e n blo (Wb) was de eloped wi h an i-Va MAb. The le -hand lane con ains o al lysa e om 2 310 5 cells. (Bo om) Wes e n blo analysis wi h an iphospho y osine an ibody (an i-PTy ) o he o al- cell ex ac s used in he op panel. 3800 GERMANI ET AL. MOL.CELL.BIOL. on Ma ch 6, 2018 by USE/BCTA.GEN UNIVERSITARIAh p://mcb.asm.o g/Downloaded om ion, we p oduced C- e minal dele ions o 38 aa (GST- 240Da) o 164 aa (GST- 240Db) o clone 240. Wi h he la e dele- ion, he in e ac ion wi h Va could no longe be de ec ed (Fig. 2B). A simila esul was also ob ained in he yeas wo-hyb id assays ( esul s no shown). This indica es ha he domain o hSiah2 esponsible o he in e ac ion wi h Va lies be ween aa 160 and 286 o hSiah2 and does no in ol e he ing inge domain, which is known o be a p o ein-p o ein in e ac ion domain (7). We used he yeas wo-hyb id assay o delinea e he hSiah2-binding si e in he Va molecule. None o he indi idual SH3 o SH2 domains no subdomains such as N- e - minus–SH3-SH2 o SH2-SH3–C- e minus we e able o in e - ac wi h hSiah2 (clone 240), indica ing ha a con o ma ion p esen in he comple e SHVAV egion was equi ed o bind- ing o hSiah2 (da a no shown). The associa ion be ween Va and hSiah2 was no modula ed by cellula ac i a ion. Lysa es om uns imula ed o an i-CD3- plus an i-CD28-s imula ed Ju- ka cells exhibi ed he same amoun o Va p o eins bound o he GST- 240 usion p o ein (Fig. 2C, op), despi e an in- c eased Va y osine phospho yla ion a e ac i a ion (Fig. 2C, bo om). To u he analyze he associa ion be ween hSiah2 and Va , bo h COS-7 and T-Ag Ju ka cells we e ans ec ed wi h ex- p ession ec o s encoding Va alone, myc epi ope- agged hSiah2 alone ( 240 o 460), o myc epi ope- agged hSiah2 oge he wi h Va . This app oach was ende ed necessa y be- cause he an i-hSiah2 an ise um did no allow us o immuno- p ecipi a e he p o ein. The lysa es we e immunop ecipi a ed wi h an i-myc an ibody, and he immunocomplexes we e ana- lyzed o he p esence o Va and hSiah2 p o eins. As shown in Fig. 3, Va could be de ec ed in he immunocomplexes de i ed om bo h cell ypes co ans ec ed wi h Va and ei he he 240 o 460 clones. As expec ed, Va could no be de ec ed in an i-myc immunop ecipi a es om cells ans ec ed wi h ei he myc-hSiah2 ( 240 o 460) o Va cons uc s alone o om hSiah2-plus-Va ans ec an s immunop ecipi a ed wi h un- ela ed se a (Fig. 3A and B, op, and da a no shown). We ca ied ou ecip ocal immunop ecipi a ion expe imen s (IP an i-Va in Va and myc-hSiah2 ans ec ed cells), bu in spi e o he p esence o simila le els o he p o eins, he le el o myc- agged hSiah2 de ec ed was oo low o be meaning ul (da a no shown). This disc epancy migh be because he an i- Va an ibody in e e ed wi h hSiah2-Va in e ac ion. Colocaliza ion o Siah and Va . Fu he suppo o he in i o Va -hSiah2 in e ac ion was ob ained by s udying he sub- cellula localiza ion o endogenous Siah and Va by immuno- luo escence labeling. We used bo h Ju ka T cells and RBL cells, whose adhe ence p ope ies acili a e he isualiza ion o subcellula s uc u es. In bo h cell models, Siah and Va we e de ec ed mainly in he cy oplasm, al hough a weak signal was obse ed in he nucleus (Fig. 4C and E). No s aining was de ec ed wi h he p eimmune Siah an ise um ollowed by he seconda y an ibody (Fig. 4A) o when an i-Siah an ise um was deple ed wi h an excess o he immunizing pep ide p io o incuba ion wi h he cells (Fig. 4B). Al hough p e ious s udies indica ed ha D osophila Sina was a nuclea p o ein (12) and ha in ans ec ed COS-7 cells hSiahs we e dis ibu ed in dis- c e e cy oplasmic pa icles (38), we ound ha endogenous Siah was e enly dis ibu ed in he cy oplasm, wi h a p o- nounced pe inuclea localiza ion. In e es ingly, his egion is he majo si e o colocaliza ion o he wo p o eins (Fig. 4G). A e s imula ion o RBL cells ia agg ega ion o FcεRI, a pa ial nuclea ansloca ion o Va bu no Siah could be de ec ed (Fig. 4D and F), lea ing he majo colocaliza ion si e a ound he nucleus (Fig. 4F and H). These da a p o ide u - he e idence o he exis ence o a cy oplasmic in i o complex be ween Va and hSiah2 and ein o ce he coimmunop ecipi- a ion esul s showing ha he in e ac ions we e no induced du ing he expe imen al p ocedu e, al hough a speci ic con o - ma ion was equi ed o de ec his in e ac ion. hSiah2 inhibi s Va -media ed NFAT ac i a ion. I has been epo ed ha TCR s imula ion con ibu es o IL-2 p oduc ion h ough ac i a ion o di e en ansc ip ion ac o s, including NFAT and AP1 (55). O e exp ession o Va leads o an in- c eased basal NFAT ansc ip ional ac i i y, which is u he enhanced by TCR s imula ion (36, 68). We es ed whe he hSiah2 may also be in ol ed in Va -media ed NFAT ac i a- ion. To his end, we o e exp essed Va ei he alone o in combina ion wi h myc- agged hSiah2 (clone 240 o 460) in T-Ag Ju ka cells and examined hei ela i e e ec s on he ac i i y o a NFAT-luci e ase epo e cons uc (NFAT-luc) con aining a ime o an IL-2-de i ed cis-ac ing elemen cloned ups eam o he luci e ase epo e gene. The ac i a ion o his p omo e equi es coope a i e binding o NFAT p o- eins and AP1 ansc ip ion ac o s (55). As expec ed, Va o e exp ession led o a signi ican induc ion o he basal an- FIG. 3. hSiah2 in e ac s wi h Va in mammalian cells. COS-7 cells (A) o T-Ag Ju ka cells (B), ansien ly ans ec ed wi h 4 and 20 mg, espec i ely, o ei he pKES-Va (Va ), myc- agged pCAN- 240 (myc- 240), o pCAN- 460 (myc- 460) alone o wi h a combina ion o he ec o s (Va 1myc- 240; Va 1myc- 460), we e lysed in NP-40 bu e and immunop ecipi a ed wi h an i-myc MAb and p o ein G-Sepha ose beads. Immunop ecipi a es (IP) we e de ec ed wi h an i-Va MAb ( op). Exp ession o ans ec ed hSiah2 (clones 240 and 460) was e i ied by ep obing he ni ocellulose memb ane wi h an i-myc MAb (bo om). Wb, Wes e n blo . VOL. May 1999 INHIBITION OF Va PATHWAYS BY hSiah2 3801 on Ma ch 6, 2018 by USE/BCTA.GEN UNIVERSITARIAh p://mcb.asm.o g/Downloaded om sc ip ional ac i i y o he NFAT epo e cons uc ela i e o he con ol ec o , which was u he inc eased by CD3 plus CD28 s imula ion (Fig. 5A). Coexp ession o hSiah2 (clone 240) wi h Va inhibi ed he Va -induced NFAT ac i i y in uns imula ed cells, and a consis en educ ion was obse ed in CD3- plus CD28-s imula ed cells. On he o he hand, no inhi- bi ion was obse ed when Va was coexp essed wi h clone 460. Exp ession o ei he 240 o 460 alone did no signi i- can ly modi y basal and CD3- plus CD28-s imula ed NFAT ac i i y (Fig. 5A). A simila esul was also obse ed wi h he ull-leng h hSiah2. As epo ed in Fig. 6, hSiah2 inhibi ed Va -media ed NFAT induc ion in a dose-dependen manne . In e es ingly, hSiah1, which is highly homologous o hSiah2 apa om he N- e minal egion, did no exhibi any signi ican inhibi ion o Va -media ed NFAT ac i i y (Fig. 6). Since an in ac N- e mi- nal egion o hSiah2 is equi ed o he inhibi ion o Va - induced NFAT ac i i y, his inhibi o y e ec may e lec an associa ion o hSiah2 and Va in a complex in ol ing he N- e minal egion o hSiah2 wi h an as ye uncha ac e ized p o- FIG. 4. Immunolocaliza ion o Va and hSiah2 by con ocal immuno luo escence mic oscopy. RBL cells we e labeled wi h p eimmune Siah an ise um (A), Siah an ise um deple ed o he immunizing pep ide (B), an i-Va MAb (C and D), and an i-hSiah2 abbi polyclonal an ibody (E and F) as desc ibed in Ma e ials and Me hods. Colocaliza ion o ed luo escence om Va and g een luo escence om hSiah2 p oduced a yellow signal, indica ing an o e lap in he dis ibu ion o he wo p o eins (G and H). In panels D, F, and H, cells we e s imula ed (S im.) by FcεRI c oss-linking. Panels A and B we e ob ained wi h a much highe ansmission a e in o de o he signal o be de ec able. 3802 GERMANI ET AL. MOL.CELL.BIOL. on Ma ch 6, 2018 by USE/BCTA.GEN UNIVERSITARIAh p://mcb.asm.o g/Downloaded om ein. The N- e minal egion o hSiah1, which showed only 53% homology o i s coun e pa in hSiah2, could no media e such an in e ac ion and hence could no ha e any inhibi o y ole. hSiah2 inhibi s onco-Va -media ed JNK ac i a ion. Recen e idence showed ha ac i a ed Va ca alyzes GDP-GTP ex- change on Rac1 and ha Rac1-GTP s imula es he kinase ac i i y o JNK (17, 18). The inding ha hSiah2 in e ac ed wi h Va led us o in es iga e whe he hSiah2 may modula e Va -induced JNK ac i i y. To his end an HA- agged JNK and he oncogenic o m o Va (onco-Va ), which cons i u i ely ac i a es JNK, we e co ans ec ed in COS-7 o T-Ag Ju ka cells, alone o oge he wi h myc epi ope- agged hSiah2 ( 240 o 460 clones). As shown in Fig. 7A and C, coexp ession o 240 wi h onco-Va and HA-JNK ab oga ed almos com- ple ely he onco-Va -induced JNK ac i a ion ( h ee- o ou - old), which e u ned o le els ound in cells ans ec ed only wi h JNK. In con as , co ans ec ion o he N- e minally de- le ed hSiah2 ( 460), which s ill binds o Va , did no modi y he ac i a ion o JNK by onco-Va despi e simila le els o exp es- sion o he di e en p o eins (Fig. 7B and D). These esul s a o an inhibi o y ole o hSiah2 in he onco-Va -induced JNK ac i a ion and ag ee wi h he inhibi o y e ec obse ed wi h clone 240 bu no 460 in Va -induced NFAT ac i i y. hSiah2 does no dec ease he s abili y o Va p o ein. Bo h Sina and mu ine Siah2 ha e been implica ed in egula ing he p o easomal deg ada ion o di e se p o eins o which hey bind (38, 45, 66). This unc ion seems o be associa ed wi h he amino- e minal 114 aa o he Sina and Siah p o eins, including he ing inge domain, which in e ac s in a yeas ap assay wi h ubiqui in-conjuga ing enzymes and Ubc9 homologs (38, 45, 66). To in es iga e whe he hSiah2 exp ession a ec s Va p o ein s abili y, we pe o med a pulse-chase expe imen (Fig. 8). COS-7 cells we e ansien ly ans ec ed wi h myc epi ope- agged Va alone o oge he wi h myc epi ope- agged hSiah2 ( 240 o 460). Co ans ec ion o Va wi h ei he 240 o 460 did no accele a e Va deg ada ion (Fig. 8). We also obse ed ha he hal -li e o Siah was somehow much sho e han ha o Va . Taken oge he , hese indings exclude he no ion ha hSiah2 could al e Va s abili y. The e idence ha he N- e minal dele ion mu an o hSiah2 ails o inhibi Va -induced NFAT and JNK ac i i y p omp ed us o ask whe he he in- hibi o y mechanism o hSiah2 could be media ed by he deg- ada ion o one o mo e componen s o he signal ansduc ion pa hways induced by Va . JNK assays wi h COS-7 cells co- ans ec ed wi h hSiah2 and Va showed ha JNK inhibi ion did no occu ia he deg ada ion o ei he JNK, Rac1, o c-Jun (da a no shown). Addi ionally, ea men o he cells wi h he p o easome inhibi o LLnL (N-ace yl-Leu-Leu-no leucinal) o lac acys in a 50 mM did no modi y he inhibi o y e ec o hSiah2 in NFAT o JNK assays (da a no shown). Toge he , hese da a demons a e ha he inhibi o y mechanism o hSiah2 migh no in ol e a p o easome deg ada ion pa hway. DISCUSSION We epo he e he iden i ica ion o a new Va -associa ed p o ein, hSiah2, a human homolog o D osophila Se en in absen ia (Sina). Sina was ini ially desc ibed as a nuclea p o- ein implica ed in he speci ica ion o he R7 pho o ecep o cell h ough he Ras1-MAPK signaling cascade (4, 52), com- p ising he y osine kinase Se enless, G b2, and Ra (26, 28, 44, 50, 61). Sina in e ac s wi h ubiqui in-conjuga ing enzymes ha FIG. 5. Va -media ed NFAT ac i a ion is inhibi ed by hSiah2. (A) T-Ag Ju ka cells (10 7 ) we e ans ec ed wi h NFAT and pSVb-galac osidase epo e plasmids (5 and 1 mg, espec i ely) and 20 mg o ei he an emp y ec o ( ec o ), pEF-Va (Va ), pCAN- 240 ( 240), pCAN- 460 ( 460), o a combina ion o he ec o s as indica ed. A o al o 10 6 cells we e ei he le uns imula ed o s imu- la ed a e 24 h wi h an i-CD3 plus an i-CD28 o 8 h. Luci e ase ac i i y was measu ed and co ec ed o b-galac osidase ac i i y, and he esul s we e ex- p essed as a e age old induc ion ela i e o uns imula ed cells ans ec ed wi h he emp y ec o . The da a a e ep esen a i e o ou independen expe imen s. The basal ac i i y and he maximum NFAT esponses we e app oxima ely 600 and 2 310 5 AU, espec i ely. (B) T-Ag Ju ka cell lysa es om panel A we e analyzed by immunoblo ing o exp ession o Va and hSiah2 ( 240 and 460). Wb, Wes e n blo . FIG. 6. O e exp ession o hSiah2 inhibi s Va -induced NFAT ac i i y in a dose-dependen manne . T-Ag Ju ka cells (10 7 ) we e ans ec ed wi h NFAT and pSVb-galac osidase epo e plasmids (5 and 1 mg, espec i ely), 10 mgo pEF-Va (Va ), and inc easing concen a ions o pBK-CMV hSiah2 o pBK- CMV hSiah1 as indica ed. Luci e ase ac i i y was de e mined and no malized o b-galac osidase ac i i y o co ec o ans ec ion e iciency. VOL. May 1999 INHIBITION OF Va PATHWAYS BY hSiah2 3803 on Ma ch 6, 2018 by USE/BCTA.GEN UNIVERSITARIAh p://mcb.asm.o g/Downloaded om a e able o a ge TTK, a ansc ip ional ep esso o neu onal cell a e, o he deg ada i e pa hway (45, 66). Recen ly, Siah p o eins we e ound o bind ei he o he cy oplasmic domain o he ecep o o ne in 1 (DCC, dele ed in colo ec al cance ) o o he nuclea ecep o co ep esso (N-CoR) and o medi- a e hei deg ada ion ia a p o easome-dependen mechanism (38, 72). Addi ionally, i has been epo ed ha he p53-induc- ible hSiah1 is a nega i e egula o o cell p oli e a ion and ha i s in e ac ion wi h BAG1, a ubiqui in-like Hsp70-Hsc70- eg- ula ing p o ein, ab oga ed his an ip oli e a i e ole (47). The FIG. 7. Inhibi o y e ec o hSiah2 on onco-Va -media ed JNK ac i a ion. COS-7 (A) o T-Ag Ju ka (C) cells we e ans ec ed wi h 1 mg (A) o 5 mg (C) o pcDNA3-HA-JNK oge he wi h 3 mg (A) o 15 mg (C) o exp ession ec o s con aining cDNA o he indica ed plasmids. The o al amoun o ans ec ed DNA was kep cons an by using emp y pcDNA3 ec o . COS-7 cells ea ed wi h 1 mg o anisomycin pe ml o 20 min we e used as a con ol. The kinase eac ion was pe o med in an i-HA immunop ecipi a es om he co esponding cellula lysa es wi h pu i ied GST–c-Jun as a subs a e (A and C, op panels). The le els o HA-JNK p o ein we e con i med by Wes e n blo (Wb) analysis wi h an i-HA an ibody (bo om panels). Values in he his og am ep esen he means and he s anda d e o s o ou independen expe imen s. COS-7 (B) and T-Ag Ju ka (D) cell lysa es om panels A and C we e analyzed by immunoblo ing o exp ession o Va and myc-epi ope- agged hSiah2 ( 240 and 460). The addi ional bands in hese blo s could be due o deg ada i e e en s caused by onco-Va o e exp ession. FIG. 8. hSiah2 does no dec ease he hal -li e o Va . COS-7 cells we e co ans ec ed wi h 4 mg o myc- agged pEF-Va (myc-Va ) and pCAN- 240 (myc- 240) o pCAN- 460 (myc- 460). A 48 h a e ans ec ion, he cells we e pulse-labeled o 1 h wi h [ 35 S]me hionine, chased wi h cold me hionine o he indica ed imes, and hen lysed as desc ibed in Ma e ials and Me hods. Va and hSiah2 p o eins we e immunop ecipi a ed (IP) wi h an i-myc an ibody and analyzed by SDS-PAGE and au o adiog aphy. 3804 GERMANI ET AL. MOL.CELL.BIOL. on Ma ch 6, 2018 by USE/BCTA.GEN UNIVERSITARIAh p://mcb.asm.o g/Downloaded om high le el o e olu iona y conse a ion o he Siah and Sina p o eins be ween humans, mice, and D osophila (76%) sug- ges s ha hese p o eins may play a c ucial ole in cellula p ocesses such as p oli e a ion, di e en ia ion, and su i al. Th ough a wo-hyb id sc een in yeas , we de ec ed an in e - ac ion be ween Va and hSiah2 (Fig. 1), which was con i med by using hSiah2 GST usion p o eins (Fig. 2) and also by pe o ming coimmunop ecipi a ion expe imen s wi h co ans- ec ed COS-7 and Ju ka T cells (Fig. 3). We also ound, by immuno luo escence mic oscopy, a colocaliza ion o Siah and Va in he cy oplasm o hema opoie ic cells, whe e Siah is p edominan ly obse ed (Fig. 4). Al hough he o iginal s udies epo ed a nuclea localiza ion signal o Sina, a somehow con o e sial dis ibu ion in cy oplasmic pa icles has been e- po ed o ans ec ed hSiah p o eins (12, 38). Bo h Va and hSiah2 con ain nuclea localiza ion sequences which could me- dia e he ansloca ion o one o bo h p o eins o he nucleus. Se e al Va -in e ac ing nuclea p o eins, such as Ku70 (58), ENX-1 (32) and hnRNP C (60), ha e been epo ed, and he p esence o Va in he nucleus has al eady been documen ed (14). In he p esen s udy, we showed some nuclea accumu- la ion o Va in RBL cells a e FcεRI s imula ion ha did no co ela e wi h a delocaliza ion o hSiah2, indica ing ha he unc ional ele ance o he Va -hSiah2 in e ac ion should be cy oplasmic a he han nucleoplasmic. On he o he hand, we epo ed ha y osine phospho yla ion o Va did no in lu- ence he Va -hSiah2 in e ac ion (Fig. 2). Ne e heless, he mechanism by which Va migh link ecep o -media ed signals o he nucleus is no ye es ablished, and he in e ac ion be- ween Siah and Va could be egula ed wi hin he cell by an as ye uncha ac e ized s imulus, o e ing o Siah he possibili y o modula ing some Va unc ions. The Va -hSiah2 in e ac ion equi es a speci ic con o ma- ional s uc u e o Va p esen in he comple e SH3-SH2-SH3 domain (Fig. 2). Residues 160 o 286 in he C- e minal egion o hSiah2 a e also in ol ed in binding o Va bu do no con ain a p oline- ich sequence expec ed o in e ac wi h Va SH3 domains. The e o e, he in e ac ion be ween Va and hSiah2 p obably co esponds o a di e en p o ein-p o ein in- e ac ion. In e es ingly, Ku70 p o ein does no use i s p oline- ich mo i in binding o he ca boxy- e minal SH3 domain o Va (58), and Cbl-b needs he comple e SH3-SH2-SH3 do- main o Va o co ec binding (10). We also ha e e idence, ob ained wi h he wo-hyb id sys em, indica ing ha hSiah2 could dime ize h ough a egion (aa 105 o 160) adjacen o he Va -in e ac ing domain (da a no shown). I will be in e es ing o in es iga e whe he he wo in e ac ions Va -hSiah2 and hSiah2-hSiah2 a e simul aneous o compe i i e. In his way, i is plausible ha he o ma ion o he complexes is con olled by subs a e a ailabili y. Al hough he ing inge domains a e gene ally in ol ed in p o ein-p o ein in e ac ions, nei he Va - hSiah2 no hSiah2-hSiah2 in e ac ions equi e his domain (Fig. 2 and da a no shown). The exp ession o hSiah2 impai ed he ansc ip ional ac i - i y o NFAT epo e gene exp ession induced by Va in Ju ka T cells (Fig. 5). NFAT is an impo an ac i a o o gene an- sc ip ion o cy okines, such as IL-2, IL-3, IL-4, and umo nec osis ac o alpha (55). This ac i a ion is media ed by co- ope a i e binding o NFAT p o eins wi h ansc ip ion ac o s o he AP-1 amily (Jun-Fos complex) (54). I has been shown ha Va ac i a es IL-2 gene exp ession and syne gizes wi h he TCR s imula ion in inducing NFAT-dependen ansc ip ion (36, 68). Ou esul s con i m hese indings and u he dem- ons a e ha coexp ession o hSiah2 and Va causes a signi - ican dec ease o NFAT epo e ac i i y ha is mo e p o- nounced in TCR-s imula ed cells. In e es ingly, nei he he N- e minal dele ion mu an o hSiah2 no a membe o he Siah amily, hSiah1, is able o inhibi Va -induced NFAT ac- i i y (Fig. 5 and 6). I is known ha hSiah1 and hSiah2 p o- eins exhibi s iking homology, di e ging signi ican ly only a hei N e mini. Toge he , hese esul s a o a model in which hSiah2 modula ion o Va ac i a ion migh be dependen on he o ma ion o a speci ic signaling complex wi h o he e ec- o s, p obably h ough he N- e minal egion o hSiah2, bu no o hSiah1, which in u n unc ions coope a i ely o in luence he downs eam e en s leading o IL-2 gene egula ion. We also obse ed ha hSiah2 was able o block he onco-Va - induced JNK s imula ion in co ans ec ed COS-7 and T-Ag Ju ka cells (Fig. 7). I has been epo ed ha onco-Va leads o he s imula ion o GDP-GTP exchange in Rac1, a p o ein implica ed in cell p oli e a ion and cy oskele al o ganiza ion (5, 56, 57) as well as in he ac i a ion o he JNK cascade (16). This inhibi ion could no be ob ained wi h he N- e minal dele ion mu an o hSiah2 (aa 105 o 324, clone 460). Al- hough he e a e con o e sial da a epo ing, on he one hand, onco-Va cons i u i e ac i a ion o Rac p o eins and sequen- ial JNK ac i a ion a e ans ec ion in T-cell lines (17, 18) and, on he o he hand, unimpai ed JNK ac i i y in T cells isola ed om a 2/2 animals (24, 35), he implica ion o Sina in he D osophila Ras-MAPK-induced signaling and he p es- ence o Va in bo h pa hways in ol ing G b2 (53, 70) and Rac1 (18, 29, 51) sugges ha hSiah2 in e ac ion wi h Va migh also impai di e se signal ansduc ion pa hways. This co ela es wi h ecen indings demons a ing ha se e al pa hways could be implica ed in JNK ac i a ion in T cells (39, 56). Addi ionally, he inhibi o y ac i i y o hSiah2 seems o be independen o he p o eoly ic mechanism p e iously de- sc ibed o Sina and mSiah2 (38, 45, 66, 71). Ou own expe - imen s showed ha hSiah2 did no modi y he hal -li e o Va , and he use o p o easomal blocking agen s showed ha he inhibi o y e ec o hSiah2 on NFAT ac i i y and JNK pa hways does no seem o be media ed ia ubiqui in-p o easome-de- penden deg ada ion (da a no shown). S udies o iden i y hSiah2-in e ac ing p o eins a e unde way and will be help ul o cla i y i s inhibi o y ole. ACKNOWLEDGMENTS We hank G. Baie o T-Ag Ju ka cells, M. Ba bacid and X. Bus elo o plasmid pKLS1, A. Al man o pEF-Myc- agged Va and pKES-Va plasmids, O. Acu o o he NFAT epo e cons uc , P. C espo o HA-JNK and pGEX-cJun plasmids, R. B. Amson o plas- mid pBK-hSiah1, G. Bismu h o an i-CD3 an ibody, D. Oli e o an i-CD28 an ibody, I. Bouchae o echnical assis ance in con ocal mic oscopy, and D. Li man and I. Dusan e o c i ical eading o he manusc ip . This wo k was suppo ed by he Ligue Na ionale Con e le Cance — Axe oncoge´ne`se and Fonda ion pou le Reche che Medicale. 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