Grb2 and Its Apoptotic Isoform Grb3-3 Associate with Heterogeneous Nuclear Ribonucleoprotein C, and These Interactions Are Modulated by Poly(U) RNA

G b2 and I s Apop o ic Iso o m G b3-3 Associa e wi h He e ogeneous Nuclea Ribonucleop o ein C, and These In e ac ions A e Modula ed by Poly(U) RNA* (Recei ed o publica ion, May 29, 1997, and in e ised o m, Janua y 9, 1998) F ancisco Rome o‡§, F ancisco Ramos-Mo ales¶, A ica Domı´nguez¶, Rosa Ma ı´a Rios¶, Fabien Schweigho e i, B uno Tocque´i, Jose´ An onio Pin o -To o**, Siegmund Fische ‡, and Ma ı´a To ole o¶ F om he ‡Ins i u Cochin de Ge´ne´ ique Mole´culai e, U363 INSERM, Hoˆpi al Cochin, 27 ue du aubou g Sain Jacques, 75014 Pa is, F ance, ¶Depa amen o de Mic obiologı´a, Facul ad de Biologı´a, Uni e sidad de Se illa, Apdo. 1095, 41080 Se illa, Spain, iRhoˆne-Poulenc-Ro e , Cen e de Reche che de Vi y-Al o ille, 13 quai Jules Guesde-BP14, 94403 Vi y su Seine Cedex, F ance, and **Ins i u o de Recu sos Na u ales y Ag obiologı´a, Consejo Supe io de In es igaciones Cien ı´ icas, Apdo. 1052, 41080 Se illa, Spain G b2 is an adap o molecule comp ising one S c ho- mology (SH) 2 and wo SH3 domains. This p o ein has a na u al iso o m named G b3-3 wi h a dele ion wi hin he SH2 domain. Nume ous e idence poin s o a unc ional connec ion be ween SH2- and SH3-con aining p o eins and molecules implica ed in RNA biogenesis. In his con- ex , we ha e examined he binding o G b2 and G b3-3 o he e ogeneous nuclea ibonucleop o ein (hnRNP) C. By he use o an in i o gene ic app oach and h ough in i o expe imen s, we u nish e idence ha bo h G b2 and G b3-3 in e ac wi h hnRNP C p o eins. Subcellula ac iona ion s udies clea ly show ha G b2 is pa ially localized in he nucleus. In addi ion, coimmunop ecipi- a ion expe imen s demons a e ha G b2zhnRNP C complexes exis in in ac hema opoie ic cells. The ca - boxyl- e minal SH3 domains o G b2 and G b3-3 a e p i- ma ily esponsible o he associa ion wi h hnRNP C. Howe e , al hough he p oline- ich mo i o hnRNP C is in ol ed in he in e ac ion wi h G b2, i is no in he binding o G b3-3. Fu he mo e, poly(U) RNA inhibi s he associa ion o G b2 wi h hnRNP C, whe eas i en- hances he in e ac ion be ween G b3-3 and hnRNP C. These indings sugges ha he G b2/G b3-3-hnRNP C in e ac ions migh ul ill di e en biological unc ions. G b2/Ash is he mammalian homolog o Sem-5 and D k in Caeno habdi is elegans and D osophila melanogas e , espec- i ely. G b2 is a ubiqui ous 25-kDa p o ein composed o one S c homology 1 (SH) 2 and wo SH3 domains. This adap o molecule plays an essen ial ole in cell g ow h and di e en ia ion and, in addi ion o o he unc ions, connec s y osine kinase ecep o s o ac i a ion o he Ras pa hway. I in e ac s ia i s SH3 domains wi h he p oline- ich egions o he mammalian Sos exchange ac o s. This G b2zSos complex exis s in he cy osol o quiescen cells and upon g ow h ac o s imula ion is ec ui ed in an SH2-dependen manne o he plasma memb ane, whe e Sos s imula es nucleo ide exchange on Ras (see Re . 1 o e- iew). G b2 has also been shown o in e ac wi h he ubiqui ous Ras exchange ac o C3G (2) and he hema opoie ic guanine nucleo ide exchange ac o Va (3). Fu he mo e, in NRK cells, G b2 has been shown o be in ol ed in g ow h ac o con ol o cy oskele al s uc u e (4). The SH2 domain o G b2 can also bind o ocal adhesion kinase (5), o he ecep o p o ein phos- pha ase a (6) and o he chime ic Bc /Abl p oduc s in human leukemias (7). Finally, se e al o he p o eins, such as Abl, Cbl, dynamin, synapsin, o 5-lipoxygenase, can bind o he SH3 domains o G b2, sugges ing ha his adap o molecule may play a ole in many cellula ac i i ies (1). Se e al iso o ms o Ash/G b2 ha e been desc ibed. Ash-m and Ash-s a e wo a iso o ms gene a ed om a single gene by unusual al e na i e splicing e en s. Mic oinjec ion o Ash-m o Ash-s in o Balb/c 3T3 cells inhibi ed DNA syn hesis induced by pla ele -de i ed g ow h ac o (8). G b3-3 is a human iso o m o G b2 hough o a ise by al e na i e splicing, ca ying a de- le ed non- unc ional SH2 domain bu e aining unc ional SH3 domains (9). The esidues dele ed in he SH2 domain ( esidues 60–100 in G b2) pa icipa e in he binding o phospho y osine- con aining p o eins. Indeed, G b3-3 did no bind o phospho- yla ed epide mal g ow h ac o ecep o and inhibi ed epide - mal g ow h ac o -induced ansac i a ion o a Ras- esponsi e elemen . This inhibi ion was o e come by G b2, sugges ing ha depending on he a io o G b2/G b3-3, G b3-3 migh se e as a supp esso o G b2 unc ions. Se e al e idences implica e G b3-3 in apop osis (9, 10), bu he mechanism emains un- known. In ligh o he di ec in e ac ion o G b3-3 wi h hSos1, i could be a gued ha he apop o ic e ec o G b3-3 migh be a consequence o he down- egula ion o Ras GTP loading, bu his alone migh no be su icien . Thus, G b3-3 and G b2 may ha e common pa ne s besides Sos, and G b3-3 may compe e wi h G b2 o i al Ras-independen pa hways. In he las se e al yea s, an inc easing numbe o SH2- and SH3-con aining p o eins ha in e ac wi h molecules impli- ca ed in RNA biogenesis ha e been desc ibed (11–16). In his con ex , we conside ed i o g ea in e es o analyze he beha - io o an exclusi ely nuclea RNP, hnRNP C, in ega d o i s abili y o in e ac wi h an adap o molecule, G b2 (and i s iso o m G b3-3), in ol ed in ansducing signals om ecep- o s. hnRNP C p o eins (C1, M 41,000; C2, M 43,000) a e among he mos abundan p e-mRNA binding p o eins. C2 is iden ical o C1, excep o a 13- esidue inse ion due o a * This wo k was suppo ed in pa by Comisio´n In e minis e ial de Ciencia y Tecnologı´a (Minis e io de Educacio´n y Ciencia, Espan˜a) G an SAF96–0275 and by Associa ion pou la Reche che su le Cance . The cos s o publica ion o his a icle we e de ayed in pa by he paymen o page cha ges. This a icle mus he e o e be he eby ma ked “ad e - isemen ” in acco dance wi h 18 U.S.C. Sec ion 1734 solely o indica e his ac . § Suppo ed by pos doc o al ellowships om he EEC and Fonda ion pou la Reche che Me´dicale. To whom co espondence should be ad- d essed. Tel.: 33-140469332; Fax: 33-146339297; E-mail: ome o@icgm. cochin.inse m. . 1 The abb e ia ions used a e: SH, S c homology; AD, ac i a ion do- main; GST, glu a hione S- ans e ase; hnRNP, he e ogeneous nuclea ibonucleop o ein; PAGE, polyac ylamide gel elec opho esis. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 13, Issue o Ma ch 27, pp. 7776–7781, 1998 © 1998 by The Ame ican Socie y o Biochemis y and Molecula Biology, Inc. P in ed in U.S.A. This pape is a ailable on line a h p://www.jbc.o g7776 This is an Open Access a icle unde he CC BY license. 39-nucleo ide inse in he co esponding mRNA, p obably de- i ed om a common p e-mRNA by al e na i e splicing. C p o eins a e ound in he nucleus du ing in e phase, bu du ing mi osis hey a e dispe sed h oughou he cell. An ibody inhi- bi ion and immunodeple ion expe imen s implica ed hnRNP C in p e-mRNA splicing (see Re . 17 o e iew). Ou esul s demons a e ha (i) G b2 and G b3-3 in e ac wi h hnRNP C, (ii) bo h iso o ms pa ially localize in he nucleus, and (iii) while he G b3.3-hnRNP C associa ion is s imula ed by poly(U) RNA, G b2-hnRNP C in e ac ion is comple ely inhibi ed. The implica ions o hese esul s a e discussed. EXPERIMENTAL PROCEDURES Cloning and Si e-di ec ed Mu agenesis—The wild ypes g b2 and g b3-3 (9) and he mu an s g b2 P49L,g b2 G203R,g b3-3 P49L and g b3-3 G162R (9, 18) we e cloned in ame wi h gal4-DB (DNA-binding) in pGBT10 (19) o yield pGBT10-G b2, pGBT10-G b3-3, pGBT10-G b2 P49L, pGBT10-G b2 G203R, pGBT10-G b3-3 P49L and pGBT10- G b3-3 G162R, espec i ely. The mu an s G b2 (P49L-G203R),G b3-3 (P49L-G162R) and 90 (5PA) we e made in he yeas wo-hyb id ec o s pGBT10-G b2 G203R, pGBT10-G b3-3 G162R, and pGAD- 90 ( esi- dues 120–290 o hnRNP C1), espec i ely, using he T ans o me si e- di ec ed mu agenesis ki om CLONTECH. The mu agenic p ime s we e 59-GGA AAA GAC GGC TTC ATT TTA AAG AAC TAC ATA GAA ATG-39, o P49L mu a ions, and 59-CCA GCA CGT GTA GCT GCT GCA GCT GCT ATT GCT CGG GC-39, o 5PA mu a ion. The mu a ed bases a e unde lined. 90.1 ( esidues 120–167 o hnRNP C1), 90.2 ( esidues 168–290 o hnRNP C1), 90.3 ( esidues 120–209 o hnRNP C1), 90.4 ( esidues 148–187 o hnRNP C1), 90.5 ( esidues 148–209 o hnRNP C1), and 90.6 ( esidues 137–187 o hnRNP C1) we e polyme - ase chain eac ion-ampli ied om pGAD- 90 and cloned in ame wi h gal4-AD (ac i a ion domain) in pGAD1318 (20). Sequencing o polym- e ase chain eac ion agmen s and poin mu a ions was pe o med on bo h s ands wi h an au oma ic sequence (Ame sham Pha macia Bio- ech) using he Sange dideoxy- e mina ion me hod (21). Yeas Two-hyb id Me hods—Saccha omyces ce e isiae s ain H 7c was co ans o med wi h he indica ed plasmids by he li hium ace a e me hod (22). Double ans o man s we e pla ed on yeas d op-ou me- dium lacking T p and Leu (22). They we e g own o 3 days a 30 °C, and hen 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 (23) and on yeas d op-ou medium lacking T p, Leu, and His, and supplemen ed wi h 5 mM3-amino-1,2,4- iazole (22) ( he Gal4-G b3-3 usion p o ein had a weak ansc ip ional ac i i y i yeas s g ew only in selec i e medium). Plasmids pGBT-SNF1 and pGAD-SNF4 (24), ca ying un e- la ed p o eins, and pGAD-hSos1 ( esidues 1131–1333) (19), a known pa ne o G b2, we e used as con ols. Cell Cul u e and Subcellula F ac iona ion—Ju ka T cells (clone J77.6.8) we e g own in RPMI 1640 medium (Li e Technologies, Inc.) supplemen ed wi h 10% hea -inac i a ed e al cal se um (Boeh inge Mannheim), 2 mML-glu amine, penicillin, and s ep omycin, in a 5% CO 2 humidi ied a mosphe e a 37 °C. UT7-S Epo cells (a g ow h ac o - dependen human megaka yoblas ic cell line) (25) we e main ained in a -medium (Li e Technologies, Inc.) wi h 10% e al cal se um and e y h- opoie in a 2 uni s/ml. Ju ka and UT7-S Epo cell cy osol and nuclea ac ions we e p epa ed essen ially as desc ibed (26). In Vi o Binding S udies—The Esche ichia coli BL21 s ain was ans o med wi h pGEX-de i ed plasmids and bac e ia incuba ed wi h 1m Misop opyl-1- hio- b -D-galac opy anoside. GST (glu a hione S- ans e ase) usion p o eins we e eco e ed and pu i ied by a ini y ch oma og aphy wi h glu a hione-aga ose beads (Sigma). Nuclea ex ac s om 10 7 Ju ka cells (150 m g o p o ein) we e dilu ed o 150 mMNaCl and supplemen ed wi h 1% Nonide P40, and he supe na an incuba ed o 2ha 4°Cwi h usion p o eins (0.2–3 m g) bound o glu a hione-coupled aga ose beads. The washed beads we e dissol ed in sodium dodecyl sul a e (SDS)-sample bu e , sub- jec ed o SDS-polyac ylamide gel elec opho esis (PAGE), elec o- blo ed, and p obed wi h di e en an ibodies. Fo he RNA compe i ion expe imen s, nuclea ex ac s we e p ein- cuba ed wi h poly(U) RNA (Ame sham Pha macia Bio ech) a 50 m g/ml o 30 min a 4 °C be o e adding GST usion p o eins. Coimmunop ecipi a ion Expe imen s—Nuclea ex ac s om 3 310 7 Ju ka cells (450 m g o p o ein) we e dilu ed o 150 mMNaCl and supplemen ed wi h 0.05% B ij 96 (polyoxyle hylene-10-oleyle he ); a - e cen i uga ion, supe na an s we e incuba ed wi h p eimmune se- um o 30 min and hen wi h p o ein A-Sepha ose beads (Ame sham Pha macia Bio ech) o 1ha 4°C.A e cen i uga ion, he supe na- an s we e incuba ed o 12–14 h wi h polyclonal an i-G b2 (San a C uz Bio echnology, Inc., San a C uz, CA) an ibodies, monoclonal an i- hnRNP C (4F4) an ibodies (p o ided by D . G. D ey uss, Uni e si y o Pennsyl ania) (27) o p eimmune se um, and hen wi h p o ein A- Sepha ose beads o 1 h and cen i uged. The beads we e washed, dissol ed in SDS-sample bu e , and subjec ed o SDS-PAGE. Fil e s we e de eloped wi h di e en an ibodies. Fo blocking expe imen s, he an igenic pep ide ( esidues 195–217) o an i-G b2 (San a C uz Bio echnology, Inc.) was used a 25 m g/ml. RESULTS In e ac ion o G b2 and G b3-3 wi h hnRNP C P o eins in he Two-hyb id Sys em—Func ional connec ions be ween SH2- and SH3-con aining p o eins and molecules implica ed in RNA biogenesis ha e been desc ibed. The e o e, i seemed o in e es o s udy he po en ial in e ac ions o G b2 and G b3-3 (Fig. 1A), adap o molecules in ol ed in signaling e en s, wi h hnRNP C, an exclusi ely nuclea RNP. We ha e in es iga ed he binding o hese p o eins o 90, a hnRNP C subclone FIG.1. In e ac ion o G b2 and G b3-3 wi h hnRNP C in he wo-hy- b id sys em. A, diag am o he domains o G b2 and G b3-3. N, amino; C, ca - boxyl; DSH2, dele ed SH2 domain o G b3-3 lacking he i s 40 esidues o he SH2 domain o G b2. B, diag am o he domains o he hnRNP C p o eins and 90 polypep ide. RNP, consensus RNA-bind- ing domain; P o, p oline- ich mo i ; NLS, nuclea localiza ion si e; Acidic, acidic do- main; D, egion dele ed in hnRNP C1. C, in e ac ion o G b2 wi h 90 polypep ide. H 7c epo e s ain was co ans o med wi h he indica ed plasmids. G ow h in he absence o his idine and wi h 5 mM 3-amino-1,2,4- iazole indica es he in e - ac ion be ween hyb id p o eins. Co ans- o ma ions wi h pGAD-hSos1 ( esidues 1131–1333) and pGAD-SNF4 we e used as con ols. Each pa ch ep esen s an in- dependen ans o man , and he e a e ou pa ches o e e y s ain. D, in e ac- ion o G b3-3 wi h 90 polypep ide (see C o de ails). G b2 and G b3-3 In e ac wi h hnRNP C 7777 isola ed in ou labo a o y om a Ju ka T cell oligo(dT) cDNA lib a y cons uc ed in ame wi h gal4-AD. This clone spans esidues 120/133 o 290/303 o hnRNP C1/C2, espec i ely (Fig. 1B). We ha e used he yeas wo-hyb id sys em o analyze hese associa ions. Fo his pu pose, he ull-leng h g b2 and g b3-3 we e cloned in ame wi h gal4-DB in pGBT10 and hen co ans o med wi h pGAD- 90 in H 7c cells. I he wo hyb id p o eins in e ac , he epo e s ain is expec ed o g ow in he absence o his idine and o p oduce b -galac osidase (28). H 7c ca ying pGBT10-G b2 and pGAD-hSos1 ( esidues 1131–1333) (19) was used as a posi i e con ol. Fig. 1 (Cand D) shows ha pGBT10-G b2/pGAD- 90 and pGBT10-G b3-3/pGAD- 90 con- e ed on H 7c yeas cells he abili y o g ow in he absence o his idine. Fu he mo e, ull-leng h Csk, ano he p o ein wi h SH2 and SH3 domains, and lamin, an un ela ed p o ein, did no in e ac wi h he 90 polypep ide (da a no shown). The e- o e, bo h G b2 and G b3-3 speci ically bind o hnRNP C p o- eins in he wo-hyb id sys em. Localiza ion o G b2/G b3-3 and hnRNP C Binding Si es—We nex sough o iden i y he speci ic G b2 and G b3-3 sequences implica ed in he in e ac ion wi h hnRNP C p o- eins. Mu a ions in he amino- e minal SH3 (N-SH3) o in he ca boxyl- e minal SH3 (C-SH3) domains o G b2 and G b3-3 g ea ly dec ease he a ini y o binding o he p oline- ich e- gion o hSos1 (9, 19, 29–31). We es ed he e ec o poin mu an s in he N-SH3 (P49L) and in he C-SH3 (G203R and G162R, espec i ely) domains o G b2 and G b3-3 (9, 18) on he in e ac ion wi h 90 in he yeas wo-hyb id sys em. In addi- ion, we gene a ed double N- and C-SH3 mu an s o G b2 (P49L-G203R) and G b3-3 (P49L-G162R) and assessed hei abili y o associa e wi h hnRNP C p o eins. S ain H 7c was co ans o med wi h pGAD- 90 and pGBT10 con aining he g b2 and g b3-3 mu an s in ame wi h gal4-DB. Visual com- pa ison o g ow h in he absence o his idine indica es ha mu a ions in ei he o he SH3 domains o bo h G b2 and G b3-3 dec eased he in e ac ion wi h hnRNP C (Fig. 2, Aand B), as epo ed o G b2/hSos1 (19). Howe e , he P49L mu a- ions we e less e ec i e han G203R o G162R, and he double mu a ions (P49L-G203R o P49L-G162R) comple ely abolished he in e ac ion. Mo eo e , we con i med by Wes e n blo expe - imen s ha wild- ype and mu an p o eins we e p oduced in simila amoun s in H 7c (da a no shown). These esul s indi- ca e ha he associa ion o G b2 and G b3-3 wi h hnRNP C p o eins is p ima ily media ed by he C-SH3 domains. SH3 domains a e known o bind p o eins h ough a he speci ic p oline- ich sequences (32). Human hnRNP C p o eins ha e a egion ich in p olines (YPARVPPPPPIARAVVPS, es- idues 126/139 o 143/156 (hnRNP C1/C2, espec i ely)) (33), which is con ained in he 90 subclone. To asce ain whe he he binding be ween G b2/G b3-3 and hnRNP C p o eins in- ol es he in e ac ion be ween a SH3 domain and a p oline- ich mo i , we subs i u ed alanines o he unde lined i e p olines o 90 o yield 90 (5PA). S ain H 7c was co ans- o med wi h pGBT10-G b2 o pGBT10-G b3-3 and pGAD- 90 (5PA) and he in e ac ion es ed in he yeas sys em. Fig. 2C shows ha G b2 does no bind o 90 (5PA) whe eas he G b3- 3- 90 (5PA) in e ac ion is clea ly de ec ed. The e o e, he G b2- hnRNP C associa ion is a classical SH3-p oline- ich in e ac ion, which is no he case o he G b3-3-hnRNP C in e ac ion. To localize he G b3-3 binding si e in hnRNP C, we sub- cloned se e al 90 agmen s in pGAD1318 (Fig. 3A). We es ed hese cons uc ions wi h pGBT10-G b3-3 in he wo-hyb id sys em, and only 90.3 con inued o in e ac wi h G b3-3 (Fig. 3B). This limi s he in e ac ing egion o esidues 120–209 o hnRNP C1, al hough his po ion does no ha e any known binding mo i s. G b2 and G b3-3 In e ac wi h hnRNP C P o eins in Vi o—To con i m he esul s ob ained wi h he yeas wo- hyb id sys em, he in e ac ions o G b2 and G b3-3 wi h hnRNP C p o eins we e s udied by in i o binding expe i- men s. The cDNAs o g b2,g b3-3,g b2 P49L,g b2 G203R, g b3-3 P49L, and g b3-3 G162R subcloned in o pGEX-de i ed plasmids (9, 18) we e used. The chime ic usion p o eins we e pu i ied om bac e ial lysa es on glu a hione-aga ose beads (see “Expe imen al P ocedu es”). Since C1 and C2 hnRNP p o- eins a e con ined o he nucleus, we used nuclea ex ac s o ca y ou he binding expe imen s. Nuclea ex ac s om a T lymphoma cell line (Ju ka , J77 clone) we e incuba ed wi h GST usion p o eins bound o glu a hione-aga ose beads. P o- ein complexes we e esol ed by SDS-PAGE, and he p esence o hnRNP C was de e mined by using an i-hnRNP C (4F4) an ibodies. As shown in Fig. 4, he chime ic GST-G b2 and GST-G b3-3 bind he endogenous human hnRNP C p o eins and, al hough hese in e ac ions we e usually pe o med wi h 3 m g o GST usion p o eins, GST-G b2 and GST-G b3-3 a concen a ions as low as 0.2 m g we e s ill able o associa e wi h hnRNP C. Mo eo e , by sil e s aining and [ 35 S]me hionine labeling o nuclea p o eins om Ju ka and UT7-S Epo cells (a g ow h ac o -dependen human megaka yoblas ic cell line), we assessed ha hnRNP C p o eins a e among he ew p o eins ha bind o GST-G b2 and GST-G b3-3 (da a no shown). Fig. 4 also shows ha he P49L mu a ion o he N-SH3 domains o G b2 and G b3-3 did no modi y he in e ac ion wi h hnRNP C, whe eas no binding was obse ed wi h C-SH3 mu an s o G b2 and G b3-3. The in e ac ions we e also obse ed when nuclea ex ac s we e p e iously ea ed wi h RNase (da a no shown), FIG.2.Si es o binding o G b2, G b3-3 and hnRNP C p o eins in he wo-hyb id sys em. Aand B, in e ac ion o 90 polypep ide wi h poin mu an s o G b2 and G b3-3. hSos1 includes esidues 1131– 1333. C, in e ac ion o G b2 and G b3-3 wi h a 90 mu an ( 90 (5PA)). See he legend o Fig. 1C o de ails. The e a e wo pa ches o e e y s ain. DB, usion wi h he DNA-binding domain o Gal4; AD, usion o he ac i a ion domain o Gal4. G b2 and G b3-3 In e ac wi h hnRNP C7778 indica ing ha G b2/G b3-3-hnRNP C in e ac ions a e no me- dia ed by RNA s ands. Taken oge he , hese indings con i m he associa ion o G b2/G b3-3 wi h hnRNP C and localize he in e ac ion in he G b2/G b3-3 C-SH3 domains. In Vi o Binding o G b2 and hnRNP C P o eins—G b2 has been desc ibed o be localized mainly in memb ane u les and in he cy oplasm (1, 34), whe eas hnRNP C p o eins a e e- s ic ed o he nucleus (35). To e alua e he physiological el- e ance o he G b2/G b3-3-hnRNP C in e ac ions i was impo - an o de e mine whe he G b2/G b3-3 we e p esen in he nucleus. Fo his pu pose, he localiza ion o G b2/G b3-3 was in es iga ed by subcellula ac iona ion. Wes e n blo expe i- men s pe o med on cy osol and nuclea ac ions o Ju ka and UT7-S Epo cells showed ha G b2 is p esen in bo h ac ions, whe eas li le o no Ra 1 was ound in he nuclea ac ions and hnRNP C was absen om he cy osol ac ions (Fig. 5). In addi ion, a simila localiza ion o G b3-3 was ound on NIH3T3 cells s ably ans ec ed wi h g b3-3 (36) (da a no shown). Thus, by subcellula ac iona ion, we demons a e he p esence o G b2/G b3-3 in he nucleus. Nex , o in es iga e he in e ac ion o G b2 and hnRNP C p o eins in he con ex o a li ing cell, we pe o med coimmu- nop ecipi a ion expe imen s wi h nuclea ex ac s om Ju ka cells. An i-G b2 immunop ecipi a es we e esol ed by SDS- PAGE and he blo s de eloped wi h an i-hnRNP C. Fig. 6A shows ha hnRNP C p o eins we e de ec ed in he an i-G b2 immunop ecipi a es, whe eas immunop ecipi a es ob ained wi h a p eimmune se um con ained no de ec able hnRNP C. Fu he mo e, he an i-G b2 an igenic pep ide blocked immu- nop ecipi a ion o hnRNP C (Fig. 6B). The ecip ocal expe i- men s, namely an i-hnRNP C immunop ecipi a es immuno- blo ed wi h an i-G b2, also yielded posi i e esul s (Fig. 6C). Simila esul s we e ob ained wi h nuclea ex ac s om UT7-S Epo (da a no shown). These da a clea ly demons a e ha endogenous G b2 is able o in e ac wi h hnRNP C p o- eins in he nuclei o in ac hema opoie ic cells. Poly(U) RNA Modula es he Associa ion o G b2/G b3-3 wi h hnRNP C—hnRNP p o eins can bind in i o o di e en sin- gle-s anded ibo- and deoxy ibopolynucleo ides, sugges ing ha hnRNP p o eins bind o he e ogeneous nuclea RNA e- ga dless o he nucleo ide sequence (37–41). Ne e heless, FIG.3. 90 polypep ide binding si e o G b3-3 in he yeas wo-hyb id sys em. A, di e en subclones o 90. See he legend o Fig. 1B o abb e ia ions. B, in e ac ion o G b3-3 wi h a ious 90 subclones. The H 7c epo e s ain was co ans o med wi h he indi- ca ed plamids. The in e ac ion be ween he wo hyb id p o eins is indica ed by he induc ion o lacZ exp ession (da k g ay pa ches). As- socia ions wi h G b2 we e used as con ols. Each pa ch ep esen s an independen ans o man , and he e a e wo pa ches o e e y s ain. DB, usion wi h he DNA-binding domain o Gal4; AD, usion o he ac i a ion domain o Gal4. FIG.4. The in e ac ions o G b2 and G b3-3 wi h hnRNP C p o eins in i o a e media ed by he C-SH3 domains. Exp ession o he GST- usion p o eins was induced by addi ion o isop opyl-1- hio- b -D-galac opy anoside. The bac e ial lysa es we e pu i ied on GST- aga ose beads. GST- usion p o eins we e incuba ed wi h nuclea ex- ac s om Ju ka cells (10 7 ), and hei associa ions wi h hnRNP C p o eins we e de e mina ed by immunoblo ing wi h an i-hnRNP C (4F4) an ibody. NE, nuclea ex ac om 10 6 Ju ka cells. These in e - ac ions we e de ec ed wi h di e en amoun s o GST usion p o eins (be ween 0.2 and 3 m g). FIG.5.De ec ion o G b2 in cy osol and nuclea ac ions o UT7-S Epo cells. A, subcellula ac iona ions o 0.5, 0.75, 1, and 2 3 10 6 UT7-S Epo cells we e sepa a ed on SDS-PAGE, ans e ed o a ni ocellulose memb ane, and hen p obed wi h an i-G b2 polyclonal an ibody. Band C, he same il e was eblo ed wi h an i-Ra 1 poly- clonal an ibody and an i-hnRNP C monoclonal an ibody, espec i ely, o con ol he pu i y o he ac ions. Exposu es o 1 min (A) and 120 min (Band C) using epichemiluminescence Wes e n immunoblo ing sys em (ECL, Ame sham Pha macia Bio ech). G b2 and G b3-3 In e ac wi h hnRNP C 7779 mo e s ingen in i o assays demons a ed ha hnRNP p o- eins ha e p e e ences o speci ic sequences. Fo example, hnRNP C p o eins ha e high a idi y o poly(U) RNA (42). To de e mine whe he poly(U) RNA has a ole in he associa ion o G b2/G b3-3 wi h hnRNP C, we examined hei binding in he p esence o absence o poly(U) RNA. Nuclea ex ac s om Ju ka cells we e incuba ed wi h o wi hou poly(U) RNA (50 m g/ml), ollowed by glu a hione-aga ose beads con aining GST- G b2 o GST-G b3-3 usion p o eins. The esul ing complexes we e analyzed by Wes e n blo s wi h an i-hnRNP C. As shown in Fig. 7, poly(U) RNA abolished he G b2-hnRNP C in e ac- ion, whe eas i inc eased he G b3-3-hnRNP C associa ion. This e ec was no obse ed wi h o he RNA homopolyme s (poly( C), poly( G), o poly( A) RNA), and combina ion o any o hese poly-RNAs wi h poly(U) RNA did no modi y he e ec induced by poly(U) RNA alone (da a no shown). These indings indica e ha he G b2/G b3-3-hnRNP C in e ac ions a e mod- ula ed by poly(U) RNA and sugges ha he na u e o hese associa ions is di e en o G b2 and o G b3-3. DISCUSSION In he las se e al yea s, he epe oi e o complexes be ween RNPs and SH2- and SH3-con aining p o eins has inc eased (11–16, 43). To in es iga e new pa hways in ol ing G b2 and G b3-3, we s udied hei in e ac ions wi h hnRNP p o eins using di e en bu complemen a y app oaches. By he wo- hyb id sys em, we demons a ed ha G b2 and G b3-3 in e ac wi h a subclone o hnRNP C. hnRNP C1 and C2 bind s ongly o sequences ele an o he p ocessing o p e-mRNA, including he polypy imidine s e ch o in ons (i u idine- ich) (44–46). The hnRNP p o eins can be di ided in o wo g oups acco ding o hei nucleocy oplasmic anspo p ope ies. One g oup is comple ely es ic ed o he nucleus in in e phase cells, whe eas he o he g oup shu les be ween he nucleus and he cy oplasm. The hnRNP C p o eins belong o he i s g oup. They a e es ic ed o he nucleus no because hey lack a nuclea expo signal bu because hey bea a nuclea e en ion sequence ha is capable o o e iding nuclea expo signals (35). To e alua e he physiological ele ance o G b2/G b3-3- hnRNP C in e ac ions, i was c i ical o de e mine he subcel- lula localiza ion o G b2 and G b3-3. Ou subcellula ac ion- a ion s udies clea ly demons a e he p esence o bo h p o eins in he nucleus and he cy oplasm. We es ima ed ha 20% o endogenous G b2 is ound in he nucleus o Ju ka and UT7-S Epo cells. Mos impo an ly, by coimmunop ecipi a ion expe - imen s, we showed he exis ence o endogenous G b2-hnRNP C complexes in nuclea ex ac s om hema opoie ic cells. P e i- ous wo k pe o med by mic oinjec ion o GST-G b2 usion p o- ein in REF-52 cells did no desc ibe i s p esence in he nuclei (34). The explana ion o his disc epancy may lie in he addi ion o he GST, which may al e he no mal beha io o he na i e p o ein and p e en i s nuclea localiza ion (47). Binding s udies wi h GST usion p o eins localized he do- main in ol ed in in e ac ion wi h he endogenous hnRNP C p o eins o he C-SH3 o G b2 and G b3-3 since hese associa- ions we e comple ely abolished by G203R and G162R mu- an s. Howe e , in he wo-hyb id sys em in e ac ions we e p e en ed only in he double mu an s (P49L-G203R and P49L- G162R), sugges ing ha he N-SH3 may pa icipa e in he binding. I should be no ed ha in he wo-hyb id sys em only a agmen o hnRNP C ( esidues 120–290 o hnRNP C) was es ed. The e o e, he cons ain s o he binding imposed by he s uc u e o he na i e p o ein may be o e idden in he 90 polypep ide. The e o e, he in e ac ions a e mo e likely medi- a ed by he C-SH3 domains in i o. Al hough G b2 and G b3-3 use he same domain o bind o hnRNP C p o eins, ou esul s indica e ha he na u e o he associa ion mus be di e en . As expec ed o a con en ional SH3-media ed in e ac ion, mu a ion o he only p oline- ich mo i in hnRNP C supp essed he binding o G b2, bu , su - p isingly, in e ac ion wi h G b3-3 emained unchanged. We localized he G b3-3 binding si e o a mo e ex ended egion ( esidues 120–209 o hnRNP C1) p obably in ol ing a con o - ma ional mo i , as judged by he inding ha o he subclones con aining he po en ial in e ac ion egion we e unable o as- socia e wi h G b3-3. These esul s sugges ha signi ican di e ences exis be ween G b2 and G b3-3 in hei in e ac ions wi h common pa ne s. This iew is suppo ed by he esul s o he wo-hyb id sys em in which poin mu a ions in ei he o he SH3 domains o G b3-3 comple ely dis up ed he associa ion FIG.6.Coimmunop ecipi a ion o G b2 and hnRNP C p o eins. A, nuclea ex ac s om Ju ka cells (3 310 7 ) we e incuba ed wi h an i-G b2 o p eimmune (PI) se um and, a e washing, esol ed by SDS-PAGE, ans e ed o ni ocellulose il e s, and incuba ed wi h an i-hnRNP C (4F4). NE, nuclea ex ac om 7 310 5 Ju ka cells. B, nuclea ex ac s we e incuba ed wi h an i-G b2 in absence (2) o p esence (1) o an igenic pep ide (25 m g/ml). Wes e n blo was de eloped wi h an i-hnRNP C. C, simila expe imen using an i-hnRNP C and p eimmune (PI) se um ollowed by immunoblo ing wi h an i-G b2 polyclonal an ibody. The band o abou 40 kDa seen in he NE lane is a nonspeci ic band ha appea s some imes when using an i-G b2 polyclonal an ibodies om San a C uz Bio echnology. FIG.7.Associa ion o G b2 and G b3-3 wi h hnRNP C p o eins is modula ed by poly(U) RNA. Nuclea ex ac s om Ju ka cells (10 7 ) wi h o wi hou 50 m g/ml poly(U) RNA we e incuba ed wi h glu a hione beads bound o ei he GST o GST- usion p o eins. The esul ing complexes we e analyzed by Wes e n blo s wi h an i-hnRNP C(4F4). NE, nuclea ex ac om 10 6 Ju ka cells. G b2 and G b3-3 In e ac wi h hnRNP C7780 wi h hSos1 ( esidues 1131–1333), whe eas in he case o G b2 his pheno ype was only ob ained wi h he double mu an . Ou esul s a e in ag eemen wi h p e ious obse a ions showing ha , al hough bo h G b2 and G b3-3 bound o hSos1 in an SH3-dependen manne , he e we e quali a i e di e ences in hei espec i e binding o he exchange ac o (10). The hnRNP complexes con ain a leas 20 majo p o eins wi h di e en RNA binding speci ici ies. hnRNP C p o eins ha e s iking a idi y o poly(U) RNA (42). The e o e, we ex- amined he ole o poly(U) RNA in G b2/G b3-3-hnRNP C in e ac ions. We clea ly show ha , whe eas G b2 only binds o hnRNP C in he absence o poly(U) RNA, G b3-3-hnRNP C in e ac ion is enhanced by he p esence o poly(U) RNA. O he RNA homopolyme s do no ha e his e ec . Fou G b3-3 pa - ne s ha e been iden i ied so a : hSos1 (10), Va (18), adenosine deaminase (36), and now hnRNP C p o eins. Howe e , we epo o he i s ime ha an in e ac ion o G b3-3 is egula ed. The physiological signi icance o hese in e ac ions emains o be de e mined. The localiza ion o G b2 in he nucleus opens new pe spec i es. Up o now, G b2 has been shown o be in ol ed in signal ansduc ion om y osine-phospho yla ed ecep o s o cy osolic pa ne s. Ou esul s sugges ha G b2 migh also be in ol ed in communica ing signals om he cy oplasm o he nucleus. Ano he possibili y is ha G b2 unc ions as an adap o p o ein in he nucleus. I would be in e es ing o iden i y y osine-phospho yla ed pa ne s o G b2 in he nucleus. Ve y ecen ly, Nck, ano he adap o mol- ecule wi hou a nuclea localiza ion signal, has been ound in he nuclei o NIH3T3 and A431 cells in associa ion wi h Sam68 (48). Speci ic binding pa ne s ha e also been iden i ied o SH2 and SH3 domains o he adap o p o ein c-C k in cy osolic and nuclea lysa es (49). These s udies, oge he wi h ou e- sul s, sugges ha he e migh be signal ansduc ion mecha- nisms in he nucleus. The ac ha G b2/G b3-3-hnRNP C in e ac ions a e egula ed by poly(U) RNA g ea ly enhances he physiological ele ance o hese associa ions. Nuclea e- en ion sequence-bea ing p o eins, like hnRNP C, ha e been sugges ed o e ain p e-mRNA in he nucleus (35). The emo al o hnRNP C om p e-mRNA/mRNA is likely o be in ol ed in mRNA expo om he nucleus. I is possible ha G b2 and G b3-3 pa icipa e in his unc ion. On he o he hand, i has been epo ed ha inc eased exp ession o G b3-3 coincides empo ally wi h ex ensi e cell dea h in some issues (9, 10). Howe e , u he expe imen s will be necessa y o in es iga e he hypo he ical in ol emen o G b3-3zhnRNP C complexes in apop osis. 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