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p 2 1 a n d C a l c i n eu r i n S y n e r g i z e to R e g u l a t e t h eN u c l e a r F a c t or o f A c t i v a t e d T C e l lsBy M. W oodrow , N eil A. Clipstone,* and D. CantrellFrom the Lym phocyte Activation Laboratory, Imperial Cancer Research Fund , London, W C 2A3PX , England and the Beckm an Center or M olecular and Genetic Medicine, How ard HughesM edical Institut6 Stanford University Schoo l of M edicine, Stanford, California 94305

S u n l m a r yIn T lymphocytes, trigge ring of the T cell receptor (T CR ) induces several signaling cascadeswh ich ultim ately synergize to induce the activity o f the nuclear factor of activated T cells (NFAT ),a D N A binding co mplex crit ical to th e inducibi l i ty and T cel l specifici ty of the T cel l grow thfactor interleukin 2. On e imm ediate consequence of T cell activation via the TC R is an increasein cytosolic calcium. Calc ium signals are imp orta nt for NFA T inductio n, an d recent studies haveidentified calcineurin, a calcium-calm odulindepende nt serine-threonine phosphatase, as a prominentcom ponen t of the calcium signaling pathway in T ceils . A second important molecule in T CRsignal transduction is the guanine n ucleotide binding protein, p21 r~, w hic h is coupled to theTC R by a protein tyrosine kinase dependent mechanism. Th e experiments presented here showthat expression by transfection of m utation ally activated calcineurin or activated p21 r~ alone isinsuff ident for NFAT transactivation. However, coexpression of the activated calcineurin w ithactivated p21 r~ could m im ic TC R signals in NFA T induc tion. These da ta identify calcineurinand p21 ~as as cooperative partners in T cell activation.

T lymph ocyte grow th is control led by the cytokine in ter-leukin 2 (IL-2). Quiescent T cel ls do n ot express theIL-2 gene (1). Afte r T cell activation via the T cell recepto rfor antigen (TC R), there is transcriptional indu ction of IL-2gene expression (2). T he target for TC R-derived s ignals inthe IL-2 g ene is a T cell-specific transcriptional enhan cer lo-cated in a region 275 bp 5 of the gene (3). The cooperativeactivity of at least eigh t proteins th at interact w ith this en-hancer appears to contr ol IL-2 gene transcription (4). Severalof these proteins, such as AP-1, NFxB , and O ct-1 are ubiqui-tous and re gulate gene transcription in m any cells. However,a protein complex terme d the n ude ar factor of act ivated Tcells (NFAT ) 1 has been identified tha t seem s to determ inebot h th e T cell specificity and induc ibility of IL-2 gen e regn-lation (5). In particular, NFA T is a specific arget for the TC R-derived signals that control IL-2 gene expression. NFAT isthou ght to be composed of tw o proteins: one is T cell-specific,cytoplasmic, and translocates to the nucleus upon T cell acti-vation; the other is apparent ly a mem ber of the A Pl familyof transcriptional factors (i.e., Fos/Jun), p redo min antly nu-clear, and present only in activated ce lls (6-10).The expression of a funct ional NFAT complex in T cel ls

Abbrev ia t ions used in th i s paper : CAT, ch loramp henico l ace ty l t ransferase ;C N A , w i l d t y p e c a l c in e u r i n A su b u n i t ; C N B , w i l d t y p e ca l ci n eu r in Bsubuni t ; C N M , ca lc ineurin A m utan t ; NFAT , nuclear fac to r o f ac t ivatedT ce l ls ; PKC, p ro te in k inase C; I r I 'K, p ro te in ty rosine k inase .

is controlled by synergistic signaling pathways initiated bythe T C R (11). Th e immediate signaling consequenceso f T C Rtrigge ring include activation of protein tyrosines kinases(PTKs), wh ich facilitates the rapid elevation o f intracellularcalcium concentration and activation of protein kinase C (PKC)(12). Calciu m and PK C are imp ortan t signals in T cell acti-vation since pharmacological agents such as calcium iono-phores and phorb ol esters, that elevate intracellular calciumand activate PKC, respectively, can bypass the requ ireme ntsfor TC R triggering and synergize o induce a functional NFATcomplex (1 3, 14).A third signaling pathway tha t involves he guanine nucleo-tide bindin g proteins p21 ~s also originates from the T C R(15-18). The Ras proteins rapidly accumulate in an activatedGT P-boun d s tate upon T C R triggering and also in responseto s t imulation of PKC. However, al though the T C R inducesPKC act ivat ion, P KC does not appear to mediate TC R regu-lation of p21 r~s (19). Instea d, the T C R couples to p21 r~s viaa PKC-independent mechanism requiring the in tegri ty ofTCR-regulated PTKs.In recent studies, it has been shown that Ras function isessential for TC R signal transduction as udg ed by th e demon-strat ion that expression of a dominant inhibi tory mu tant ofp21 ra~, N17 ras, prevents T C R induc tion o f NFAT and theIL-2 gene (20, 2 1). Moreover, expression of a constitutivelyactivated Ras protein, p21 v H ... , has a dom inan t positiveeffect on NFAT induction and is able to generate a signal

1517 J. Exp . Med. 9 The Rockefe l ler Univ ersi ty Press 9 0022-1 007/93 /1 1 /1517/0 6 $2 .00Volume 178 Nov em ber 199 3 1517-1522

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that synergizes wi th a calcium signal to induce a level of NFA Tthat mim ics the TC R response. Th e calcium-regnlated signalsthat cooperate w ith p21 ra~ to in duce N FAT have not y et beeniden ti f ied . However , a com p onent o f the ca lc ium s igna l tha ti s es sen ti al fo r T C R induc t ion of NF AT has recen t ly beeniden t if i ed . The ca lc ium -ca lm od ul in dependent phospha tasecalc ineurin (22-25), which is the major cel lular target forthe im m unosuppres s ive d rugs cyc lospor in A and F K 506 invivo, is activated in response to T C R tr igg ering o r s t imula-t ion of cel ls with calc ium ionophore (26). The express ionof a t runca ted con s t i tu t ive ly act ive C a2+-indePend ent cata-lyt ic subu nit ofc alc ine urin generates a s ignal that can subst i-tute for calc ium s ignals and synergize w ith pho rbol es tersto induce the IL -2 gene (22) . Overexpres s ion of wi ld typecalcineurin can a lso aug men t TCR- indu ced NFA T-dependenttranscript ion (23).

Th e p otent ia l fo r p21TM to co operate w ith calc ineurin hasnot yet been explored, but act ivat ion of ras is a goo d candi-date for the physiological partne r for calc ineurin in NFA Tind uc tio n fo r at least tw o reasons. First, p21 ~'~, like cal-c ineurin, has an essentia l funct ion in TC K regulat ion of NFAT(21). Sec ond, p21 ras can replace some requ irem ents .forphorb ol es ter act ivat ion of PK C in NF AT ind uct ion. Accord-ing ly , the ob jec t o f the p resen t s tudy was to exam ine thepoten tial for p21 ~ and calcineurin to synergize du rin g T cellac t iva t ion and subs t i tu te fo r the TCR in the induc t ion ofNFAT. The present data show that in two T cel l l ines , thehum an leukem ia Jurka t and the m ur ine thym om a EL4, ex-press ion of e i ther const i tut iv ely act ive p21 ~'~ or a co nst i tu-tively active calcineurin is no t alon e sufficient for NFA T tran-script ion. H owever, in com binat io n, act ivated p21 ~s andca lc ineur in can syne rg ize to g ive induc t ion of NF AT tha tm im ics the re sponse induced by TCR. These da ta fu r the rdefine TC K s ignal t ransdu ct ion as involving cooperat ive in-teractions b etw een signals by calcine urin and p21 ~'~ con-trolled signaling pathways.

aterials and ethod sReagents Ionomycin (calcium salt) and phorbol-12, 13-di-butyrate w ere from Calbiochem Corp. (UK). U CH T1 (reactivefor human CD 3) was used at 10 ~g /ml in culture (27). [uC]-Acetyl coenzyme A (at 50-60 m Ci/m mo l) was from AmershamInternational (Buckinghamshire, LrK).Cells and Transfections The human T leukemia,Jurkat, an d themurine cells EL4 were maintained in R PM I 1640 supplementedwith 10% heat-inactivated FCS. Gro wth was at 37~ and hu-midified in 5% CO2/95% air. Cells were transfected via dec-troporation (Gene Pulser; Bio-Rad Laboratories, UK ), according

to the manu facturer's instructions. Briefly, u rkat cells were pulsed(2 x 107/ml) in media + serum, at 960 ~F and 310 V. EL4 cells(supplemented with 10 ~M B-m e for growth), at 108/ml, werepulsed at 250 V and 960/~F. Cultures were transfected and im-mediately stimulated, as necessary, for 24 h.Phsmids I1 .2 chloramphenicol acetyl transferase (CAT) (11),and N FAT CAT (5) have been previously described. IL-2 CAT con-tains regulatory sequences upstream o f the I1,2 gene (from - 326to +47), that include the I1,-2 minimal promoter and enhancer,directing transcription o f the reporter gene. NFA T CAT containsthree copies of the sequence 5 ' G GAGGA AAAAC TGTTT-

CATACAGAAGC~GT3' (corresponding to the sequence from po-sition -28 4 to -25 8 relative to the start of transcription of the11.2 gene) upstream of the I1.2 minimal promoter driving thereporter gene CAT. This sequence has been identifiedas the bindingsite for NFAT, the A RR E-2 site of the human I1.,2 enhancer (3).I1,2 mpCA T is a deletion mutant o f NFAT CAT in wh ich thebinding sites for NFA T havebeen deleted, leaving he I1,2 minimalpromoter to drive CAT activity. The sequence of the I1,2 minimalpromo ter w as confirmed by ds sequencing (Sequenase, version 2.0;United States Biochem . Corp., C leveland, OH ). The construct usedfor the expression of activated Ras was created by inserting the0.7-kb BamHI fragment of CM Vras (21) into the BamHI site ofpEF BOS (2 8). Orientation was determined by restriction analysisand activity was assessed n a focus forming assay. CN A and C NBhave bee n described (22), and are expressed under the co ntrol o fthe SRc~ promoter o f HTLV-1 (23). CNM is a truncation ofCNAo~4, in w hich a sto p codon has been introduced at aa397,resulting in a constitutively active, Ca2+-ind ependent phospha-tase (29).C A T A s s a ys Assayswere performed according to the m ethodof Sleigh et al. (30), w ith modifications. Briefly, cells were lysedin 100 #1 of 0.65% NP-40, 10 mM iris, pH 8.0, 1 mM EI ~A ,150 mM NaC1, for 10 min on ice. Cellular debris was pelleted,and the lysate heat was treated at 68~ for 10 min before use. Assayconditions were 150 mMTris, pH 8.0, 0.05/~Ci [14C]acetylcoen-zyme A , and 2 m M chloramphenicol. Chloramphenicols were ex-tracted wit h ethyl acetate, and the amoun t o f radioactivity in theacetylated products and nonacetylated substrate of each reactionwas determined by liquid scintillation counting of organic andaqueous phases, respectively.R e s u l t s

A Cons t i tu t ive ly Ac t i ve M utan t o fCa k ineur in Synerg i zes w i thPhorbo t Es te rs to Regu la te NF AT To m on i to r N F AT ac t iv ity ,we em ployed t rans ien t t rans fect ion and quan t i t a t ion o f theexpression of a CA T reporter gene wh ose ac t ivi ty is regu-la ted by a t r im er o f the NF AT b in d ing s it e, loca ted ups treamof the IL -2 m in im a l p rom ote r . T he s igna ling requ i rem entsfor expression of this transfected NFA T reporter ge ne in Jur katcells have been described (3, 6, 13). Ionom ycin, wh ich e le-vates intraceUular calc ium, and Pdbu, wh ich activates PK C,synerg ize fo r m axim a l NF A T ind uc t ion (F ig . 1 a ) .To examine th e effect of calc ineurin o n NFAT, Jurk at cellswere co t rans fec ted w i th NF AT CAT p lus one o r a com bina -t ion o f calc ineurin express ion constructs : CNAo~ 4, the w ildtype ca ta ly t ic subuni t o f ca lc ineurin (CN A); a wi ld type ca l-c ineurin B subu nit (CN B), or the const i tu t ively active cal-c ineur in A m utan t (CN M). The l a t t e r is a de le t ion m u tan tof the calc ineurin cata lyt ic subunit , ca lc ineurin A~x4, thatl acks func t iona l ca lm odul in b ind ing an d au to inh ib i to ry do-m a ins , and has CaZ+- independent cons t i tu t ive phospha taseac t iv i ty in v i t ro (29). The da ta show th a t expres s ion of wi ldt y p e C N A a n d C N B e i th e r a lo n e o r i n c o m b i n a t i o n d oe sno t induce a m arked ac t iva t ion o f NF A T CAT. A s desc r ibedpreviously, iono my cin a lone has a s l ight s t imu latory effecttha t i s enhanced in ce ll s t rans fected wi th the co m bina t iono f C N A a n d C N B ( 23 ).IMBu alone has no apparent s t imulatory effect, but the m osts tr iking observation was the s t rong syn ergy between the C N Mand P dbu tha t re su l t ed in a level o f NF AT indu c t ion com -

1518 Ras and Calcineurin Regulate NFAT

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effec t on the P KC induc ib i l i ty of an AP-1 reporte r const ruc t(Fig. 1 b).v Ha ms Synergizes with Cakium onophore o RegulateNFATCAT inJurkat Cells. p21 v-H .... is m uta ted at c od on 12 and59 (31). These mutations render the Ras proteins insensitiveto the Ras-G TPase ac t iva t ing pro te ins such tha t the Ras ac-cumulates in cells in an active GT P- bo un d state (32). W he nexpressed in the murine thymoma EL4, s igna ls genera ted byp21 v-H . . . synergized wi th e i ther the ca lc ium ionopho reionom ycin or the PKC ac t ivator Pdbu to induce NFA T (21).The Jurka t cel ls used in the present s tu dy d i ffe r f rom EL 4cells in their activation response to Pdbu (33). Hence in EL4cells, PKC activation alone is sufficient for NFAT activity,whereas in Jurkat cells there is an apparent ob ligator y require-ment for ca lc ium signa ls for NFAT induc t ion (11). T he da tain Fig . 2 a show the characte r is t ics of NFAT regula t ion inJurk at cells co transfect ed wi th NF AT CA T and p21 ~-H ... . .The mutationally activated Ras alone had a stimulatory effecto n N FA T th a t w a s n o t fu r th e r e n h a n ce d b y Pd Bu t r e a tme n tof the cells. Howev er, p21 ~H ... generated a stron g sy nerg ywith ionom ycin to induce levels of NFAT comparable to tha tseen in ionomycin and PdBu-treated cells. The fact thatp21 r~s alone o nly elicits a weak stim ulation of NF AT CA T

Fig ure 1. Constitutively activated calcineurin synergizes with phorbolester to regulate NFAT ranscriptional activity. (a)Jurkat cells were trans-fected with NFAT CAT and calcineurin expression vectors either alone,or in combination was described in Materials and Methods. lonom ycinwas used at 0.5/~g/ml and PdBu at 50 ng/ml, as indicated. Here, 10 #gof reporter construct was used in concert with 10/~g of each calcineurinconstruct. Cells were electroporated and stimulated immediatelyafter trans-fection for 24 h before cytosolic extracts were made and reporter geneactivity was assessed. Percent conversion represents the ratio of radioac-tivity extracted in the o rganic phase vs. total radioactivity n organic plusaqueous phases. ([7) control - NFAT CAT alone; ([] ) CN A - NFATCAT + 10/*g wt CNA ; ([~) CNB - NFAT CAT + 10 #g wt CNB;(~ ) CN A + CNB - NFAT CAT + 10 #*g wt CNA + 10/~g wt CNB;( . ) CNM - NFAT CAT + 10/*g CNM. T he data shown are represen-tative of more than five experiments. Percent conversion n control un-stimulated samples ranged from 2.4 to 7.7%. (b) Jurkat cells were trans-fected with 2/~g of AP-1 CAT and 20/~g of CNM or not (control). ([])= control unstimulated cells; ( I ) = Pdbu 50 ng/ml. Fold induction isthe ratio o f activity (% conversion) n stimulated cells vs. control unstimu-lated cell (no added CNM ). The data show n are representative of threeexperiments. Percent conversion n control unstim ulated cells ranged from1.0 to 5.9%.

parable to tha t observed in response to iono mycin p lus Pdbu(Fig. 1 a ) . These da ta indica te tha t the C N M but not wi ldtype CN A or C NB can subst i tu te for ionomycin- induced ca l -c ium signa ls in the regula t ion o f NFAT. The e ffec t of theC N M on N FAT ac t iv i ty was se lec tive as jud ged by two in-dependent observa t ions. Fi rs t , the CN M did not enhance ex-pression of a CA T reporte r gene dr iven by the IL-2 minimalp ro mo te r a lo ne (d at a n o t sh o w n ) . Se c o nd , t h e CN M h a d n o

15 19 Woodrow et al.

Fig ure 2. v-Ha-ras regulates NFAT CAT and AP-1 CAT inJurkat cells.(a) Jurkat cells were transfected with 10 t~g NFAT CAT , and 40 k~g ofEF empty vector (control) or EF v-H a-ras (v-Ha-ras). Stimulation was asin Fig. 1: (I-l) unstimulated, (1~) iono, (~) Pdbu, @) iono + Pdbu.Activity was as in Fig. 1. The data sho wn are representative of three ex-periments. Percent conversion n control unstimulated samples as per Fig.1 a. (b) Jurkat cells were transfected with 2/z g of AP-1 CAT and 20 ~gof EF em pty (control) or 20/~g EF v-Ha-ras (v-Ha-ras). Stimulation, (D )control, unstimulated or (m) PdBu treated, and activity are as describedin Fig. 1. The data shown are representative of four experiments. Percentconversion in control unstimulated samples ranged from 1.2 to 5.9% .

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a n d a l s o t h a t R a s d o e s n o t s y n e r g i z e w i t h P d B u w a s n o td u e t o i n s u f f i c i e n t e x p r e s s i o n o f R a s . T h u s , t h e l e v e l o fp 2 1 v ' H ' ~ e x p r e s s e d i n t h e s e e x p e r i m e n t s w a s a l o n e s u ff ic i en tt o s t i m u l a t e m a x i m a l l ev e ls o f A P - 1 a c t i v i t y , i. e. , c o m p a r a b l et o t h e l e v el o f A P - 1 a c t i v i t y i n d u c e d i n r e s p o n s e t o P K C s t i m -u l a t i o n w i t h p h o r b o l e s t e r ( F i g . 2 b ) .Cakineurin and p21 ~ Synergize to Regulate NF AT p2 1 v-m'~sa n d C N M c a n b o t h g e n e r a te s i gn a ls t h a t r e g u la t e N F A T , b u tt h e s e s i g n a l s a p p e a r t o d i ff er . C N M s y n e r g i z e s w i t h P K C ,b u t n o t c a l c i u m c o n t r o l l e d s i gn a ls t o r e g u l a t e N F A T , w h e r e a sp 2 1 ~m - '~ h a s t h e c o n v e r s e a b i l it y t o s y n e r g i z e w i t h c a l c i u m ,b u t n o t P K C - c o n t r o l l e d p a t h w a y s . T o e x a m i n e t h e p o t e n -t i a l f o r p 2 1 r.~ a n d c a l c i n e u r i n t o s y n e r g i z e i n N F A T i n d u c -t i o n , J u r k a t c e l l s w e r e c o t r a n s f e c t e d w i t h N F A T C A T a n dp 2 1 ~ - H ' ~ a n d C N M , e i t h e r a lo n e o r i n c o m b i n a t i o n . D a t aaveraged f rom fou r expe r imen ts (F ig . 3 ) s how tha t p21 ~ 'H . . .i n c o m b i n a t io n w i t h C N M i n d u c ed a s t ro n g e x p r es s io n o fN F A T c o m p a r a b l e t o t h a t s e en i n r e s p o n s e t o e i t h e r p 2 1 ~ - H '~p l u s io n o m y c i n o r C N M p l u s P d b u . B o t h p 2 1 ~ a n d c al -c i n e u ri n a r e a c t iv a t e d in r e s p o n s e t o t r ig g e r i n g o f t h e T C R( 15 , 2 4 , 2 5 ) . T h e d a t a s h o w t h a t p 2 1 v - H ' ~ p l u s C N M a l soi n d u c e d N F A T C A T t o a l ev el g r e at e r t h a n t h a t i n d u c e d i nr e sp o n se to T C R t ri g g er i ng w i t h t h e C D 3 a n t i b o dy U C H T 1(Fig. 3) .T h e r e g u l a t o r y e f f e c t o f p 2 1 ~-H . .. . o n N F A T i s n o tc o n f i n e d t o J u r k a t c e ll s b u t a l s o o c c u r s i n a m u r i n e T c e l lE L 4 . T h e e f fe c t o f c a lc i n e u ri n m u t a n t s o n N F A T e x p r e ss i o ni n c e l l s o t h e r t h a n J u r k a t h a s n o t b e e n e x p l o r e d i n p r e v i o u ss t u d i e s . T h e d a t a i n F i g . 4 s h o w t h a t i n E L 4 c e l l s e x p r e s s i o no f C N M b u t n o t w i l d t y p e C N A o r C N B c an s yn e rg iz e w i t hP d b u o r p 2 1 ~-H . ... t o i n d u c e N F A T . E x p r e s s i o n o f C N Ma l o n e in d u c e d o n l y a w e a k N F A T r e s p o n s e, b u t i n c o m b i n a -t i o n w i t h P d b u o r w i t h t h e c o n s t i t u t i v e l y a c t i v e R a s , t h e

i

s

100

8 0

60

40

2 0

0

F ig ur e 3. Calcineurin synergizes wi th v-Ha-ras to regulate NFAT CA Tin Jurkat cells. Jurkat cells were transfected with 10/~g NFAT CAT plus10/~g of v-Ha-ras or C N M or both , as indicated. Stim uli are as indicatedin Fig. 1; in addition, UCHT1 (10 I~g/ml in culture) was added as indi-cated. Percent maximum response represents the ratio of activity seen insamples vs. that in iono + Pdbu-treated cultures w itho ut added v-Ha-rasor CN M (mean iono + pdbu = 84 .5 _+ 0 .6%). D ata are the mean fromfour experiments, error bars represent SEM. P ercent conversion in controlunstimulated samples as per Fig. 1 a.

30 [] control[ ] C N A[] CNB

2 0z

~ l o

0 c o n t ro l p d b u v -H a - ra ss t i m u l i

Fig ur e 4 . Calc ineurin synerg izes wi th v-Ha-ras to regula te NFAT CATin EL4 cell s . EL4 cel l s were t ransfec ted w i th 20 /~g of NFA T CA T and5 0 / ~ g o f e it h e r (V ]) EF emp t y, ( [ ] ) C N A , (~ A ) C N B , o r ( l l ) C N M ,as indicated. Stimuli were applied and data are represented as in Fig. 1.The figure is representative of two experiments.ac t ive calc ineur in m utan t synerg ized to indu ce a leve l o f NFA Ta c t i v i t y s i m i l a r t o t h a t i n d u c e d i n r e s p o n s e t o c e l l a c t i v a t i o nb y i o n o m y c i n i n c o m b i n a t i o n w i t h P d b u ( F i g . 4 ) .

i s c u s s i o nI n T l y m p h o c y t e s , p 2 1T M i s s t i m u l a t e d v i a t h e T C R i n amech anism invo lv ing TC K inhib i t ion o f ras GTPase-ac tiva ting

p r o t e i n s (r a s- G A P s ) ( 1 5 , 1 8 ). O n e i m p o r t a n t r o l e f o r t h e T C Ris to con t ro l the express ion and fun c t ion of t ranscr ip t ion fac torss u c h a s N F A T , t h a t c o n t r i b u t e t o t h e c o n t r o l o f IL - 2 g e n et r a n s c r i p t i o n ( 6 ) . E x p r e s s i o n o f a m u t a t e d c o n s t i t u t i v e l y a c-t i v a t e d p 2 1 ~ i n d u c e s a r e s p o n s e i n T c e ll s t h a t c a n s y n e r -g i z e w i t h a c a l c i u m s i g n a l t o m o d u l a t e T c e l l a c t i v a t i o n a sj u d g e d b y N F A T i n d u c t i o n ( 2 1 , 3 4 ) . S i m il ar ly , o v e r e x p r e s -s i o n o f t w o s r c - f a m i l y k i n a s e s , p 5 6 1o k a n d p 5 9 f y , a p p e a r t ob e a b l e t o p a r t i a l l y r e p l a c e t h e c a l c i u m r e q u i r e m e n t f o r i n -d u c t i o n o f N F A T - d i r e c te d t r a n s c r ip t i o n ( 3 5 , 3 6 ) . T h e c o m -p o n e n t s o f t h e c a l c i u m s i g n a l in g p a t h w a y a b l e to c o o p e r a t ew i t h p 2 1 ras i n T c e ll s t o r e g u l a t e N F A T h a v e n o t b e e n p r e -v i o u s l y d e t e r m i n e d . H o w e v e r , r e c e n t s t u d i e s h a v e e st a b l is h e dt h a t c a l c i n e u ri n , w h i c h i s r e g u l a t e d b y in c re a se s i n c y t o s o l i cc a l c i u m , i s e s se n ti al f o r T C R s i g n a l in g f u n c t i o n ( 2 2 , 2 3 , 3 7 ) .T h e d a t a p r e s e n te d h e r e s h o w t h a t e x p r e s s i o n o f a t ru n c a t e d ,c o n s t i t u t i v e ly a c t i ve c a lc i n e u ri n i n b o t h a h u m a n l e u k e m i cT c e ll l in e , J u r k a t , a n d i n t h e m u r i n e t h y m o m a , E L 4 , c a ns u b s t i t u t e f o r c a l c i u m s i g n a l s a n d , i n s y n e r g y w i t h a n a c t i -v a t e d p 2 1 r ~ , s ti m u l a t e T c e ll s t o i n d u c e N F A T - m e d i a t e dt ranscr ip t ion . Prev ious s tud ies have ind ica ted tha t ca lc ineur inc a n s y n e rg i z e w i t h p h o r b o l e s t er s t o i n d u c e N F A T a n d h e n c et h e I L - 2 g e n e ( 2 2 , 2 3 ) . T h e c u r r e n t s t u d y i d e n t if i e s p 2 1 r'~a s a n a l t e r n a t iv e p a r t n e r f o r c a l c i n e u r i n i n T c e l l a c ti v a t i o n .N F A T i s k n o w n t o h a v e a c r i ti c a l fu n c t i o n i n t h e r e g u l a -t i o n o f t h e I L - 2 g e n e ( 1 1 , 2 1 ). H o w e v e r , t h e e x a c t c o m p o s i -t i o n o f t h i s t r a n s c r i p t i o n a l f a c t o r is a s y e t u n r e s o l v e d . I t i sp r o p o s e d t h a t N F A T i s a c o m p l e x o f a t l e as t t w o c o m p o -n e n t s : a T c e ll -s p ec i fi c c y t o s o l i c c o m p o n e n t t h a t t r a n s l o c a te st o t h e n u c l e u s i n r e s p o n s e t o i n c r e a s e s i n c y t o s o l i c c a l c i u m ,a n d a n u c l e a r c o m p o n e n t , e x p r e s s e d o n l y i n a c t i v a t e d c e l l s ,

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t h a t a p p e ar s t o b e a m e m b e r o f t h e A P - 1 f a m i l y o f tr a n s cr i p-t iona l fac tors (6 , 7 , 9 , 10) . p21 ~ i s wel l es tab l i shed as an in -d u c e r a n d r e g u l a t o r o f A P - 1 i n fi b r o b l a s t s ( 3 8 ) . T h e p r e s e n td a t a c o n f i r m t h a t p 2 1 ~ i s a l so a b l e to i n d u c e A P - l - m e d i -a t e d t r a n s c r i p t i o n i n T c e l l s , w h e r e a s c a l c i n e u r i n h a s n o a p -p a r e n t r e g u l a t o r y e f f e c t . I t i s m o s t p r o b a b l e , t h e r e f o r e , t h a ti n t h e c o o p e r a t i o n b e t w e e n p 2 1 ras a n d c a l c i n e u r i n , R a sw o u l d c o n t r i b u t e t o N F A T i n d u c t i o n v i a c o n t ro l o f A P - 1p r o t e in s , w h i l e c a l c in e u r in w o u l d r e g u l a t e N F A T b y c o n t r o lof the subce l lu la r loca l iza t ion of the ca lcium-respons ive NF ATs u b u n i t . T h i s m o d e l f o r t h e c o o p e r a t i v e i n t e ra c t io n b e t w e e nc a l c in e u r i n a n d R a s i n N F A T i n d u c t i o n w a r r a n t s f u r t h e r e x -p l o r a t i o n . O n e c o m p l i c a t i o n l i e s i n t h e f a c t t h a t t h e A P - 1p r o t e i n s a r e a l a r g e h e t e r o g e n e o u s f a m i l y o f t r a n s c r i p t i o n a lf a c to r s , w h i c h c o m e t o g e t h e r i n v a r i o u s c o m b i n a t i o n s o f F o sa n d J u n h o m o - a n d h e t e r o d i m e r s t o f o r m f u n c t io n a l c o m -p l e x e s ( 3 9 ) . W h e n T c e l l s a r e a c t i v a t e d , t h e y e x p r e ss m u l -t i p l e F o s a n d J u n p r o t e i n s ( 4 0 ) , a n d t h e i d e n t i t y o f t h e p a r -t i c u la r p a r t n e rs p r e s e n t i n t h e A P - 1 c o m p l e x t h a t c o n t r i b u t et o N F A T i n d u c t i o n i s n o t y e t c l e a r ( 7 , 9 , 1 0 ) .T h e d o w n s t r e a m e f f e c t o r s o f b o t h p 21 TM a n d c a l c i n e u r i na r e n o t k n o w n . O n e p o t e n t i a l R a s r e g u l a t e d p a t h w a y t h a tc o u l d c o n t r i b u t e t o N F A T r e g u l a t i o n i s t h e M A P k i n as e s ig -n a l i n g c a s c a d e ( 4 1 ). p 2 1 r~ c a n r e g u l a t e t h e a c t i v i t y o f t h eM A P k i n a s e E r k 2 i n J u r k a t c e l ls ( 4 2 ). S u b s t r a t e s f o r E r k 2i n c l u d e t r a n s c r i p t io n a l f a c t o rs s u c h as t h e A P 1 m e m b e r c-jun,a n d E l k - l , p r o t e i n t h a t c o m p l e x e s w i t h s e r u m r e s p o n s e f a c to rt o t r a n s a c t i v a t e t h e c -f o s p r o m o t e r , w h o s e f u n c t i o n i s c o n -t r o l l e d b y p h o s p h o r y l a t i o n a t t a r g e t s i t e s f o r t h e M A P k i n a s ef a m i l y (4 3 - 4 5 ) . T h u s , t h e M A P k i n as e s c o u l d b e i n te g r a l t ot h e s i g n a l in g p a t h w a y s t h a t c o u p l e t h e T C R a n d p 2 1 ~ t othe ce l l nuc leus .

T h e p o t e n t i a l f o r c o o p e r a t i o n b e t w e e n p 2 1 ~a, a n d c a 1-

c i n e u r in h a s n o t b e e n o b s e r v e d i n p r e v i o u s s t u d i e s o f R a sf u n c t i o n i n o t h e r c e l l t y p e s . H o w e v e r , t h e i d e a th a t R a s c o n -t r o l l e d s i g n a l i n g p a t h w a y s a r e n o t a l o n e s u f f i c i e n t f o r c e l l a c -t i v a t i o n i s n o t n o v e l . F o r e x a m p l e , o n c o g e n i c t r a n s f o r m a t i o ni n f i b r o b l a s t s r e q u i r e s a c o o p e r a t i v e i n t e r a c t i o n b e t w e e n R a sa n d o t h e r o n c o g e n e s ( 4 6 ) . T h e d e m o n s t r a t i o n h e r e i n t h a tR a s a n d c a l c i n e u r i n c a n c o o p e r a t e t o a c t i v a t e T c e l l s r a i s e sthe poss ib i l i ty tha t ca lc ineur in cou ld be a pro toonco gene . Thu s ,m u t a t i o n s i n c al ci n eu r in o r m u t a t i o n s t h a t p e r t u r b e d t h e r e g u -l a t io n o f c a l ci n e u ri n c o u l d b e o n c o g e n i c i f th e y o c c u r r e d i nc e l l s e x p r e s s i n g o n c o g e n i c R a s .

D e f e c t s in N F A T r e g u l a t i o n h a v e p r o f o u n d f u n c t io n a l c o n -s e q u e n ce s f o r T c el ls , a s j u d g e d b y t h e i m m u n o s u p p r e s s i v ee f f e c ts o f t h e d r u g s c y c l o s p o r i n A a n d F K 5 0 6 t h a t i n h i b i tt h e a c t i o n o f c a l c i n e u r in o n N F A T i n d u c t i o n ( 3 3 ) . T h e r e al -i z a t i o n t h a t p 2 1 T i s a n i m p o r t a n t p a r t n e r w i t h c a l c i n e u r i ni n N F A T r e g u l a t i o n s u g g e s t s t h a t R a s c o u l d a l so b e an i m -p o r t a n t t a r g e t f o r i m m u n o s u p p r e s s i o n . A l t e r n at i ve l y , a n yd e f e ct s i n R a s r e g u l a t i o n w o u l d h a v e se v er e i m m u n e c o n s e -q u e n c e s. T h i s i s s u p p o r t e d b y a r e c e n t s t u d y i n n o n o b e s e d i a-b e t i c m i ce , i n w h i c h m a t u r e ( C D 4 + 8 - , C D 4 - 8 + ) T c e ll sw e r e n o t o n l y u n a b l e t o p r o l i f e ra t e in r e s p o n s e t o T C K c r o s s -l i n k i n g , b u t c o u l d n o t d e m o n s t r a t e a c t i v a t i o n o f p 2 1 ra~ o rp42maPk a f te r li g a t io n o f t h e T C K ( 4 7 ). D i r e c t s t i m u l a t io no f P K C w a s a b l e t o a c t i v a te p 2 1 r ~, a n d a l s o t o r e s t o r e t h ec e l ls a b i l i ty t o g r o w i n r e s p o n s e t o T C R s i g n a li n g . T h i s s u g -g e s t s t h a t a d e f e c t m u s t e x i s t b e t w e e n t h e T C K a n d t h ep 2 1 r ~ p r o t e i n s i n t h e s e a n e r g i c c e ll s, s u p p o r t i n g t h e h y p o t h -e s is t h a t p 2 1 r ~ m a y p l a y a n i m p o r t a n t r o l e i n t h e g e n e r a t i o no f a n i m m u n e r e sp o n s e . H o w e v e r , i t r em a i n s t o b e s e e n i fR a s d e f e c t s e x i s t i n o t h e r a n e r g i c T c e l l m o d e l s o r a u t o i m -mune d iseases .

The authors wo uld l ike to thank P. Parker, E. Ro zeng urt , S. Rayter, and G. Crabtree for helpful comm entson the manuscript .Address correspondence to Dr. Melissa A. W oodrow, L ymph ocyte Activation Labo ratory, Imperial CancerResearch Fund, Ro om 613, L incoln s Inn F ie lds, London, W C2 A 3PX, U K.Rece ived for pub l ica t ion 24 M ay 1993 and in rev i sed orm 2 A ugu s t 1993 .

R e f e r e n c e s1. Sm ith, K.A. 1988. Interleukin-2: inception, impact and im-plications. Science Wash. DC). 240:1169.2. Crabtree, G.R . 1989 . Co nting ent g enetic regulatory events

in T lymphocyte activation. Science Wash. DC). 243:355.3 . Duran d , D.B. , M .R. Bush , J .G. Morgan , A . Weiss , and G.R.Crabtree. 1 987. A 275 basepair fragment at the 5 end of theinterleukin 2 gene enhances expression from a heterologouspromoter in response to signals from the T cell antigen receptor.J . Ex t z Med . 165:395.4. Em mel, E.A. , C.L. Verweij , D.B . Du rand, K .M. Higgins, E.Lacy, and G .R. Crabtree. 1989. Cyclosporin A specifically n-hibits function of nuclear proteins involved in T cell activa-tion. Science Wash. DC). 246:1617.5. Verweij , C.L . , C. Guidos, and G.K. Crabtree. 1990. Cell type1 5 2 1 W o o d r o w t a l.

6 .

7.

8

specificity and activation requirements for N FAT-1 (nuclearfactor of activated T-cells) transcriptional activity determinedby a new m ethod using transgenic mice to assay ranscriptionalactivity of an activated nuclear factor.J . Biol . Chem . 265:15788.Flanagan , W.M. , B. Cor th~sy , R. J . Bram, and G.R. Crab-tree. 1991 . Nuclear association o f a T-cell t ranscription factorblocked by FK -506 and cyclosporin A. Nature Lond.). 352:803.Jain, J . , P .G. McC affrey, V.E. Valge-Archer, and A . Rao. 1992.Nuclear factor of activated T cells contains Fos and Jun. Na-lure Lond.). 356:801.McC affrey, P.G. , B.A . Perrino, T.R. Soderl ing, and A. Rao.1993. NF-ATp, a T lymphocyte DNA-binding protein that isa target for calcineurin and immun osuppressive dru gs.J . Biol.Chem. 268:3747.

Published November 1, 1993

8/12/2019 J Exp Med-1993-Woodrow-1517-22

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9 . No r th ro p , J .P . , K.S . Ul lman , and G.K . Crab t ree . 1993 . Char -a c t e r iz a t i o n o f t h e n u c l e a r a n d c y t o p la s m i c c o m p o n e n t s o f t h elym pho id speci f ic nuc lea r fac to r o f ac t iva ted T ce ll s (NF-AT)c o m p l e x , j . B i o l . C h e m . 268:2917.10 . Bo ise , L .H. , B . Pe t ryn iak , X. Moo , C.H. June , C. -Y. Wang ,T. Linds ten , K . Bravo , K. Kovary , J .M . L e iden , and C.B.T h o m p s o n . 1 9 9 3 . T h e N F A T -1 D N A b i n d i ng c o m p l e x i n a c-t ivated T cells contains Fro-1 and J u n k Mo l . Cel l . Biol . 13:1911.11 . Du rand , D.B. , J . -P . Shaw, M .K . Bush , K.E . Rep log le , R . B e-lagaje , and G.R . Cra b t ree . 1988 . Chara c te r iza t ion o f an t igenrecep to r re sponse e lemen ts wi th in the in te r leuk in -2 enhance r.M ol . C e l l . B i o l . 8:1715.12 . Weiss , A. , and J .B. Im boden . 1987 . Ce l l su r face mo lecu les andea r ly even ts invo lved in hum an T lym phoc y te ac t iva t ion . A dv .Immunol . 41:1.13 . Hiv ro z -Burg aud , C. , N . Cl ip s tone , and D. Can treU. 1991 . S ig -na l ing requ i remen ts fo r the exp ress ion o f the t ransac t iva t ionf a c t o r N F - A T i n h u m a n T l y m p h o c y t e s . E u r . J . Immunol .21:2811.14 . Mat i ll a , P .S . , K.S . UUm an , S . F ie r ing , E .A. Em me l , M.M cCu tcheon , G.R . Crab tree , and L.A . Herzenberg . 1990 . Th eac t ions o f cyclospo r in A and FK 506 su gges t a nove l s tep inthe ac t iva t ion o fT lymphocytes . E M B O E u r . M o l . B i o l. O rg a n . )j . 9 :4425.15 . Do wn war d , J . , J .D. Graves , P .H. W arne , S . Ray te r , and D.A .Can t re l l . 1990 . S t imu la t ion o f p21 r'~ upo n T-ce ll ac t iva t ion .Nature Land.) . 346:719.16. Do wn wa rd, J . , J .D. G raves, and D.A. C antrell . 1992. T he regu-l a t i o n a n d f u n c t i o n o f p 2 Y s i n T l y m p h o c y t e s . Immunol .Today . 13:89.1 7 . D o w n w a r d , J . 1 9 92 . R e g u l a t i o n o f p 2 Y ~s by G APs and gua-n i n e n u c l e o t i d e e x ch a n g e p r o t ei n s i n n o r m a l a n d o n c o g e n i ccells. C u ~ O p i n . D e v . G e n e t . 2:13.18 . G raves , J .D. , J . Do wn wa rd , S . Ray te r , P . W arne , A.L. T u t t ,M . G l e n n i e , a n d D . A . C a n t r e l l. 1 9 91 . C D 2 a n t i g e n m e d i a t e dac t iva t ion o f the guan ine nuc leo t ide b ind ing p ro te in s p21F .' inh u m a n T l y m p h o c y t e s . J . I m m u n o L 146:3709.19 . Izqu ie rdo , M . , J . Dow nw ard , J .D. Graves, and D.A. Can t re l l .1992 . Ro le o f p ro te in k inase C in T-ce ll an t igen recep to r regu -la t ion o f p2P s : ev idence tha t two p21TM egu la to ry pa thwayscoex is t in T ce l l s . M ol . C e l l . B i o l . 12:3305.20 . Ray te r, S ., M. W oodrow , D. Can t re l l, andJ . D own ward . 1992 .p 2 1 S m e d ia te s c o n t r o l o f I L 2 g e n e p r o m o t e r f u n c t i o n i n Tcell activation. E M B O E u r . M o l . B i o l . O r g a n .) J . 11:4549.21 . W oodrow , M.A . , S. Ray te r, J . Dow nw ard , and D.A . Can t re l l .1993 . p2Y S func t ion i s imp or tan t fo r T ce l l an t igen recep to rand p ro te in k inase C regu la t ion o f nuc lea r fac to r o f act iva tedT cells. J . Immunol . 150:1.22 . O 'K eefe , S .J ., J . Tamura , K.L . Kinca id , M .J . Tocc i , and E.A.O 'N e i l l . 1992 . FK-506 and C s-A-sens it ive ac t iva t ion o f thein te r leuk in -2 p rom ote r b y ca lc ineu r in . Nature Land.) . 357:692.23 . C l ip s tone , N.A . , and G.R. C rab t ree . 1992 . Iden t i f ica t ion o fca lc ineu r in a s a key s igna ll ing enz ym e in T - lymph ocy te ac t iva -t ion . Nature Land.) . 357:695.24 . Mc Keon , F . 1991 . W he n wo r lds co l lide : imm unosup pressan tsmee t p ro te in phospha tases . C e l l . 66:823.25 . Sch reibe r, S .L. 1992 . Imm unoph i l in -sens i t ive p ro te in phospha-tase action in cell s ignall ing pathways. C e l l . 70:365.26. Frum an, D .A. , C.B. K lee, B.E. Bierer , and S.J. Burakoff . 1992.Phospha tase ac t iv i ty in T lymp hocy tes i s inh ib i ted by FK -506and cyc lospo r in A. P roc . N at l . A c ad . S c i . US A . 89:3686.27. Beverley, P .C.L. , and R.E. Callard . 1981. Distin ctive functio nalcha rac ter i st ic s o f hum an T lymp hocy tes de f ined by E rose t -

t ing o r m onoc lonal anti-T cell antibody. E ur .J . Immunol . 11:329.28 . Mizush ima , S . , and S . Naga ta . 1990 . pEF-BOS, a powerfu lmammal ian exp ress ion vec to r . Nucle ic Acids Res . 18:5322.29 . Cl ip s tone , N .A. , and G .R. C rab t ree . 1993 . Ca lc ineu r in i s akey s igna ll ing e nzym e in T lymphoc y te act iva t ion and the ta rge to f t h e i m m u n o s u p p r e s s i v e d r u g s C s A a n d F K 5 0 6 . A n n .N . Y . A c ad . Sc i . In press .30 . S le igh , M .J . 1986 . A no nchro ma tog rap h ic a ssay fo r exp res -sion of the chlo ram phen icol acetyltransferase gene in eucaryoticcells. A n a l B i o c h e m . 156:251.31. Cap on, E . , E. Che n, A. Levinson, P . Seeburg, and D. Goeddel.1983 . Com ple te nuc leo t ide sequence o f the T24 hum an b ladderca rcinoma oncogen e and i t s no rma l homo logue . Nature Land.) .302:33.32 . McCormick , F . 1989 . ra s GTPase ac t iva t ing p ro te in : s igna lt ransmi t te r and s igna l t e rmina to r . C e l l . 56:5.33 . Sch reibe r, S .L. , and G.K . Crab t ree . 1992 . Th e mechan ism o fac t ion o f cyc lospo r in A and FK506 . ImmunoL Today . 13:136.34 . Ba lda ri , C .T. , A. H eguy , and J .L . Te l ford . 19 93 . ra s p ro te inac t iv i ty i s e s sen t ia l fo r T-ce l l an t igen recep to r s igna l t ransduc-t ion . J . B i o l . C he m. 268:2693.35 . B a ldar i , C .T. , A. He guy , and J .L . Te l ford . 1993 . Ca lc ium-depen dent cyclosporin A-sensit ive activation o f the in terleukin-2p r o m o t e r b y p 5 9 r J . B i o l . C he m. 268:8406.36 . Ba lda ri , C .T. , A. Heg uy , and J .L . Te l ford . 1993 . Ca lc ium de -pend en t ac t iva t ion o f the NF-AT t ransc r ip t ion fac to r byp5 6 fyn. F E B S F e d . E u r . B i oc he m. Soa ) Le tt . 323:233.37. Liu, J . , J .D . Farm er, W.S. Lane, J . F riedm an, I . Weissman,and S .L. Schre ibe r . 1991 . Ca lc ineu r in i s a com m on ta rge t o fcyc loph i l in , cyc lospo rin A, and FK BP-FK 506 complexes. Cell .66:807.38. Smeal, T. , B. Binetruy, D.A . Mercola , M. Birrer , and M . Karin .1991. On cogen ic and t ransc r ip t iona l coopera t ion wi th Ha-R asrequ i re s phosp hory la t io n o fc -J un o n se r ines 63 and 73 . N at ureLand.) . 354:494.39 . Ange l , P . , and M. Kar in . 1991 . Th e ro le o fJ un , Fos , and theAP-1 comple x in cell-proliferation and transfo rma tion. Biochim.Biophys . Ac ta. 1072:129.

40 . Sh ibuya , H. , M . Yoneyama , J . Ninomiya-Tsu j i, K . M atsumoto ,and T. Tan ignch i . 1992 . Ib2 and EG F recep to rs s t imu la te thehem opo ietic cell cycle via different s ignall ing pathways: dem on-s t ra t ion o f a nove l ro le fo r c-myc . Cel l . 70:57.41 . Ahn , N.G . , K. Sege r , and E.G. Krebs . 1992 . Th e mi togen -activated pro tein kinase activator. C u~ Opi n . C e l l B i o l . 4:992.42 . Izqu ie rdo , M. , S . J . Leeve rs , C . J . M arsha l l, and D. Can t re l l .1993. p21 r~ couple s the T cell antige n re cep tor to extracel-lu la r s igna l - regu la ted k inase 2 in T lymp hocy tes . J . E x t x M e d .178:1199.43. Pulverer , B.J . , J .M . K yriakis, J . Av ruch , E. Nik olakak i, andJ . R . W o o d g e t t . 1 9 9 1 . P h o s p h o r y l a t i o n o f c- j u n m e d i a t e d b yMAP k inases . N at ure Land . ) . 353:670.44 . Hi l l , C .S . , K. Mara is , S. Joh n , J . Wy nne , S . Da l ton , and R .Tre isman . 1993 . Func t iona l ana lysi s o f a g row th fac to r re spon-s ive t ransc r ip t ion fac to r complex . C e l l . 73:395.45 . Mara is, K. , J . Wynn e , and R. Tre i sman . 1993 . Th e SRF ac -cesso ry p ro te in ELK-1 con ta in s a g ro wt h fac to r - regu la ted t ran -sc r ip t iona l ac t iva t ion domain . C e l l . 73:381.46 . Hun te r , T . 1991 . Coop era t ion be tween oncogenes. Cell . 64:249.47 . Rap opo r t , M.J . , A .H . Laza rus, A. Ja rami llo , E . Speck , andT . L . D e l o v i t c h . 1 9 9 3. T h y m i c T c e ll a n e r g y in a u t o i m m u n enonobese d iabe t ic mice i s med ia ted by de f ic ien t T ce ll r ecep to rregu la t ion o f the pa thw ay o f p21 ~s ac t iva t ion . J . E x l ~ M e d .177:1221.

1522 Ras and Calcineurin Regulate NFAT

Published November 1, 1993