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. A.G. was
he ecipien o a Pos e Ve om INSERM (F ance).
REFERENCES
1. Adams, M. D., M. Dubnick, A. R. Ke la age, R. Mo eno, J. M. Kelley, T. R.
U e back, J. W. Nagle, C. Fields, and J. C. Ven e . 1992. Sequence iden i-
ica ion o 2,375 human b ain genes. Na u e 355:632–634.
1a.Al man, M. Pe sonal communica ion.
2. Amson, R. B., M. Nemani, J. P. Rope ch, D. Is aeli, L. Bouguele e , I. Le
Gall, M. Medhioub, G. Lina es-C uz, F. Le h osne, P. Pas u aud, L. Piou -
e, S. P ieu , L. Susini, V. Al a o, P. Millasseau, C. Guidicelli, H. Bui, C.
Massa , L. Cazes, F. Du ou , H. B uzzoni-Gio anelli, H. Owadi, C. Hen-
nion, G. Cha pak, A. Tele man, e al. 1996. Isola ion o 10 di e en ially
exp essed cDNAs in p53-induced apop osis: ac i a ion o he e eb a e
homologue o he D osophila se en in absen ia gene. P oc. Na l. Acad. Sci.
USA 93:3953–3957.
3. Angel, P., E. A. Alleg e o, S. T. Okino, K. Ha o i, W. J. Boyle, T. Hun e ,
and M. Ka in. 1988. Oncogene jun encodes a sequence-speci ic ans-ac i-
a o simila o AP-1. Na u e 332:166–171.
VOL. May 1999 INHIBITION OF Va PATHWAYS BY hSiah2 3805
on Ma ch 6, 2018 by USE/BCTA.GEN UNIVERSITARIAh p://mcb.asm.o g/Downloaded om
4. Biggs, W. H. D., and S. L. Zipu sky. 1992. P ima y s uc u e, exp ession, and
signal-dependen y osine phospho yla ion o a D osophila homolog o ex-
acellula signal- egula ed kinase. P oc. Na l. Acad. Sci. USA 89:6295–6299.
(E a um, 90:6377, 1993.)
5. Boguski, M. S., and F. McCo mick. 1993. P o eins egula ing Ras and i s
ela i es. Na u e 366:643–654.
6. Bohmann, D., T. J. Bos, A. Admon, T. Nishimu a, P. K. Vog , and R. Tjian.
1987. Human p o o-oncogene c-jun encodes a DNA binding p o ein wi h
s uc u al and unc ional p ope ies o ansc ip ion ac o AP-1. Science
238:1386–1392.
7. Bo den, K. L., and P. S. F eemon . 1996. The RING inge domain: a ecen
example o a sequence-s uc u e amily. Cu . Opin. S uc . Biol. 6:395–401.
8. B eeden, L., and K. Nasmy h. 1985. Regula ion o he yeas HO gene. Cold
Sp ing Ha bo Symp. Quan . Biol. 50:643–650.
9. B unne , D., N. Oelle s, J. Szabad, W. H. R. Biggs, S. L. Zipu sky, and E.
Ha en. 1994. A gain-o - unc ion mu a ion in D osophila MAP kinase ac i-
a es mul iple ecep o y osine kinase signaling pa hways. Cell 76:875–888.
10. Bus elo, X. R., P. C espo, M. Lopez-Ba ahona, J. S. Gu kind, and M. Ba -
bacid. 1997. Cbl-b, a membe o he Sli-1/c-Cbl p o ein amily, inhibi s
Va -media ed c-Jun N- e minal kinase ac i a ion. Oncogene 15:2511–2520.
11. Bus elo, X. R., K. L. Suen, W. M. Michael, G. D ey uss, and M. Ba bacid.
1995. Associa ion o he a p o o-oncogene p oduc wi h poly( C)-speci ic
RNA-binding p o eins. Mol. Cell. Biol. 15:1324–1332.
12. Ca hew, R. W., and G. M. Rubin. 1990. se en in absen ia, a gene equi ed
o speci ica ion o R7 cell a e in he D osophila eye. Cell 63:561–577.
13. Chang, H. C., N. M. Solomon, D. A. Wassa man, F. D. Ka im, M. The ien,
G. M. Rubin, and T. Wol . 1995. phyllopod unc ions in he a e de e mi-
na ion o a subse o pho o ecep o s in D osophila. Cell 80:463–472.
14. Cle enge , C. V., W. Ngo, D. L. Sokol, S. M. Luge , and A. M. Gewi z. 1995.
Va is necessa y o p olac in-s imula ed p oli e a ion and is ansloca ed
in o he nucleus o a T-cell line. J. Biol. Chem. 270:13246–13253.
15. Coppola, J., S. B yan , T. Koda, D. Conway, and M. Ba bacid. 1991. Mech-
anism o ac i a ion o he a p o ooncogene. Cell G ow h Di e . 2:95–105.
16. Coso, O. A., M. Chia iello, J. C. Yu, H. Te amo o, P. C espo, N. Xu, T. Miki,
and J. S. Gu kind. 1995. The small GTP-binding p o eins Rac1 and Cdc42
egula e he ac i i y o he JNK/SAPK signaling pa hway. Cell 81:1137–1146.
17. C espo, P., X. R. Bus elo, D. S. Aa onson, O. A. Coso, M. Lopez-Ba ahona,
M. Ba bacid, and J. S. Gu kind. 1996. Rac-1 dependen s imula ion o he
JNK/SAPK signaling pa hway by Va . Oncogene 13:455–460.
18. C espo, P., K. E. Schuebel, A. A. Os om, J. S. Gu kind, and X. R. Bus elo.
1997. Phospho y osine-dependen ac i a ion o Rac-1 GDP/GTP exchange
by he a p o o-oncogene p oduc . Na u e 385:169–172.
19. Decke , M., S. Ta a e-Decke , C. Cou u e, T. Mus elin, and A. Al man.
1996. Func ional and physical in e ac ions o Syk amily kinases wi h he Va
p o o-oncogene p oduc . Immuni y 5:591–604.
20. Della, N. G., P. V. Senio , and D. D. Bow ell. 1993. Isola ion and cha ac e i-
sa ion o mu ine homologues o he D osophila se en in absen ia gene
(sina). De elopmen 117:1333–1343.
21. de Ma in, R., J. S asswimme , and L. Philipson. 1993. A new luci e ase
p omo e inse ion ec o o he analysis o weak ansc ip ional ac i i ies.
Gene 124:137–138.
22. Dickson, B. 1995. Nuclea ac o s in se enless signalling. T ends Gene 11:
106–111.
23. Emmel, E. A., C. L. Ve weij, D. B. Du and, K. M. Higgins, E. Lacy, and G. R.
C ab ee. 1989. Cyclospo in A speci ically inhibi s unc ion o nuclea p o-
eins in ol ed in T cell ac i a ion. Science 246:1617–1620.
24. Fische , K. D., Y. Y. Kong, H. Nishina, K. Ted o d, L. E. Ma enge e, I.
Kozie adzki, T. Sasaki, M. S a , G. Chan, S. Ga dene , M. P. Nghiem, D.
Boucha d, M. Ba bacid, A. Be ns ein, and J. M. Penninge . 1998. Va is a
egula o o cy oskele al eo ganiza ion media ed by he T-cell ecep o .
Cu . Biol. 8:554–562.
25. Fische , K. D., A. Zmuldzinas, S. Ga dne , M. Ba bacid, A. Be ns ein, and
C. Guidos. 1995. De ec i e T-cell ecep o signalling and posi i e selec ion o
Va -de icien CD4
1
CD8
1
hymocy es. Na u e 374:474–477.
26. Fo ini, M. E., M. A. Simon, and G. M. Rubin. 1992. Signalling by he se enless
p o ein y osine kinase is mimicked by Ras1 ac i a ion. Na u e 355:559–561.
27. Fuchs, S. Y., B. Xie, V. Adle , V. A. F ied, R. J. Da is, and Z. Ronai. 1997.
c-Jun NH
2
- e minal kinases a ge he ubiqui ina ion o hei associa ed
ansc ip ion ac o s. J. Biol. Chem. 272:32163–32168.
28. Gale, N. W., S. Kaplan, E. J. Lowens ein, J. Schlessinge , and D. Ba -Sagi.
1993. G b2 media es he EGF-dependen ac i a ion o guanine nucleo ide
exchange on Ras. Na u e 363:88–92.
29. Han, J., B. Das, W. Wei, L. Van Aels , R. D. Mos elle , R. Khos a i-Fa , J. K.
Wes wick, C. J. De , and D. B oek. 1997. Lck egula es Va ac i a ion o
membe s o he Rho amily o GTPases. Mol. Cell. Biol. 17:1346–1353.
30. Henske, E. P., M. P. Sho , S. Jozwiak, C. M. Bo ey, S. Ramlakhan, J. L.
Haines, and D. J. Kwia kowski. 1995. Iden i ica ion o VAV2 on 9q34 and i s
exclusion as he ube ous scle osis gene TSC1. Ann. Hum. Gene . 59:25–37.
31. Hobe , O., B. Jallal, J. Schlessinge , and A. Ull ich. 1994. No el signaling
pa hway sugges ed by SH3 domain-media ed p95 a /he e ogeneous ibonu-
cleop o ein K in e ac ion. J. Biol. Chem. 269:20225–20228.
32. Hobe , O., B. Jallal, and A. Ull ich. 1996. In e ac ion o Va wi h ENX-1,
a pu a i e ansc ip ional egula o o homeobox gene exp ession. Mol. Cell.
Biol. 16:3066–3073.
33. Hobe , O., J. W. Schilling, M. C. Becke le, A. Ull ich, and B. Jallal. 1996.
SH3 domain-dependen in e ac ion o he p o o-oncogene p oduc Va wi h
he ocal con ac p o ein zyxin. Oncogene 12:1577–1581.
34. Holloway, A. J., N. G. Della, C. F. Fle che , D. A. La gespada, N. G. Cope-
land, N. A. Jenkins, and D. D. Bow ell. 1997. Ch omosomal mapping o i e
highly conse ed mu ine homologues o he D osophila RING inge gene
se en-in-absen ia. Genomics 41:160–168.
35. Holsinge , L. J., I. A. G ae , W. Swa , T. Chi, D. M. Bau is a, L. Da idson,
R. S. Lewis, F. W. Al , and G. R. C ab ee. 1998. De ec s in ac in-cap
o ma ion in Va -de icien mice implica e an ac in equi emen o lympho-
cy e signal ansduc ion. Cu . Biol. 8:563–572.
36. Holsinge , L. J., D. M. Spence , D. J. Aus in, S. L. Sch eibe , and G. R.
C ab ee. 1995. Signal ansduc ion in T lymphocy es using a condi ional
allele o Sos. P oc. Na l. Acad. Sci. USA 92:9810–9814.
37. Hu, G., Y. L. Chung, T. Glo e , V. Valen ine, A. T. Look, and E. R. Fea on.
1997. Cha ac e iza ion o human homologs o he D osophila se en in ab-
sen ia (sina) gene. Genomics 46:103–111.
38. Hu, G., S. Zhang, M. Vidal, J. L. Bae , T. Xu, and E. R. Fea on. 1997.
Mammalian homologs o se en in absen ia egula e DCC ia he ubiqui in-
p o easome pa hway. Genes De . 11:2701–2714.
39. Jacin o, E., G. We len, and M. Ka in. 1998. Coope a ion be ween Syk and
Rac1 leads o syne gis ic JNK ac i a ion in T lymphocy es. Immuni y 8:31–
41.
40. Ka za , S., D. Ma in-Zanca, and M. Ba bacid. 1989. a , a no el human
oncogene de i ed om a locus ubiqui ously exp essed in hema opoie ic cells.
EMBO J. 8:2283–2290.
41. Ka za , S., G. Packham, M. Su he land, P. A oca, E. San os, and J. L.
Cle eland. 1995. Va and Ras induce ib oblas ans o ma ion by o e lap-
ping signaling pa hways which equi e c-Myc unc ion. Oncogene 11:1079–
1088.
42. Ka za , S., M. Su he land, G. Packham, T. Yi, and A. Weiss. 1994. The
p o ein y osine kinase ZAP-70 can associa e wi h he SH2 domain o p o o-
Va . J. Biol. Chem. 269:32579–32585.
43. Lai, Z. C., and G. M. Rubin. 1992. Nega i e con ol o pho o ecep o de-
elopmen in D osophila by he p oduc o he yan gene, an ETS domain
p o ein. Cell 70:609–620.
44. Li, N., A. Ba ze , R. Daly, V. Yajnik, E. Skolnik, P. Cha din, D. Ba -Sagi, B.
Ma golis, and J. Schlessinge . 1993. Guanine-nucleo ide- eleasing ac o
hSos1 binds o G b2 and links ecep o y osine kinases o Ras signalling.
Na u e 363:85–88.
45. Li, S., Y. Li, R. W. Ca hew, and Z. C. Lai. 1997. Pho o ecep o cell di e -
en ia ion equi es egula ed p o eolysis o he ansc ip ional ep esso
T am ack. Cell 90:469–478.
46. Ma golis, B., P. Hu, S. Ka za , W. Li, J. M. Oli e , A. Ull ich, A. Weiss, and
J. Schlessinge . 1992. Ty osine phospho yla ion o a p o o-oncogene p od-
uc con aining SH2 domain and ansc ip ion ac o mo i s. Na u e 356:71–
74.
47. Ma suzawa, S., S. Takayama, B. A. F oesch, J. M. Zapa a, and J. C. Reed.
1998. p53-inducible human homologue o D osophila se en in absen ia
(Siah) inhibi s cell g ow h: supp ession by BAG-1. EMBO J. 17:2736–2747.
48. Mus i, A. M., M. T eie , and D. Bohmann. 1997. Reduced ubiqui in-depen-
den deg ada ion o c-Jun a e phospho yla ion by MAP kinases. Science
275:400–402.
49. Nemani, M., G. Lina es-C uz, H. B uzzoni-Gio anelli, J. P. Rope ch, M.
Tuynde , L. Bouguele e , D. Che i , M. Medhioub, P. Pas u aud, V. Al a o,
H. de Sa kissan, L. Cazes, D. Le Paslie , I. Le Gall, D. Is aeli, J. Dausse ,
F. Sigaux, I. Chumako , M. O en, F. Cal o, R. B. Amson, D. Cohen, and A.
Tele man. 1996. Ac i a ion o he human homologue o he D osophila sina
gene in apop osis and umo supp ession. P oc. Na l. Acad. Sci. USA 93:
9039–9042.
50. Oli ie , J. P., T. Raabe, M. Henkemeye , B. Dickson, G. Mbamalu, B.
Ma golis, J. Schlessinge , E. Ha en, and T. Pawson. 1993. A D osophila
SH2-SH3 adap o p o ein implica ed in coupling he se enless y osine ki-
nase o an ac i a o o Ras guanine nucleo ide exchange, Sos. Cell 73:179–
191.
51. Olson, M. F., N. G. Pas e is, J. L. Go ski, and A. Hall. 1996. Faciogeni al
dysplasia p o ein (FGD1) and Va , wo ela ed p o eins equi ed o no mal
emb yonic de elopmen , a e ups eam egula o s o Rho GTPases. Cu .
Biol. 6:1628–1633.
52. Pelech, S. L., and J. S. Sanghe a. 1992. MAP kinases: cha ing he egula o y
pa hways. Science 257:1355–1356.
53. Ramos-Mo ales, F., F. Rome o, F. Schweigho e , G. Bismu h, J. Camonis,
M. To ole o, and S. Fische . 1995. The p oline- ich egion o Va binds o
G b2 and G b3-3. Oncogene 11:1665–1669.
54. Rao, A. 1994. NF-ATp: a ansc ip ion ac o equi ed o he co-o dina e
induc ion o se e al cy okine genes. Immunol. Today 15:274–281.
55. Rao, A., C. Luo, and P. G. Hogan. 1997. T ansc ip ion ac o s o he NFAT
amily: egula ion and unc ion. Annu. Re . Immunol. 15:707–747.
56. Rei , K., and D. A. Can ell. 1998. Ne wo king Rho amily GTPases in
lymphocy es. Immuni y 8:395–401.
3806 GERMANI ET AL. MOL.CELL.BIOL.
on Ma ch 6, 2018 by USE/BCTA.GEN UNIVERSITARIAh p://mcb.asm.o g/Downloaded om