The EMBO Journal vol.15 no.17 pp.4515-4525, 1996

Unique signal transduction of Eyk: constitutivestimulation of the JAK-STAT pathway by anoncogenic receptor-type tyrosine kinase

Chen Zong, Riqiang Yan1, Avery August,James E.Darnell,Jrl andHidesaburo Hanafusa2Laboratory of Molecular Oncology and 'Laboratory of Molecular CellBiology, The Rockefeller University, 1230 York Avenue, New York,NY 10021-6399, USA

2Corresponding author

The proto-oncogene c-eyk, the cellular counterpart ofa transforming oncogene, v-eyk, encodes a receptorprotein tyrosine kinase with a distinctive extracellularregion. We now demonstrate that c-Eyk can be con-stitutively activated through dimerization, and that theactive Eyk displays a unique signaling pattern. Whenthe kinase domain of c-Eyk was fused to the extracellu-lar and transmembrane domains of CD8, the resultingchimera showed elevated kinase activity and causedcellular transformation. We found that the activatedEyk kinases, both v- and c-Eyk, constitutively stimulatethe JAK-STAT pathway, while exerting little effect onother signaling routes such as the Ras-MAP kinaseand the JNK pathways. The activated Eyk kinasesspecifically stimulate tyrosine phosphorylation ofSTAT1, STAT3 and JAK1. These downstream mole-cules also co-immunoprecipitate with the constitutivelydimerized form of Eyk. The Eyk kinase activity isrequired for STAT1 stimulation. We found that theactivation of STAT1 but not STAT3 correlates wellwith cellular transformation. In constitutively stimulat-ing the JAK-STAT pathway, particularly STAT1, Eykis unique in its downstream signaling and may bedependent on this pathway for cellular transformation.Keywords: Eyk/JAK-STAT/receptor tyrosine kinase/signal transduction/transformation

IntroductionThe oncogene v-eyk (East Lansing tyrosine kinase) wasfirst isolated from an acute avian retrovirus, RPL30 (Jiaet al., 1992). It encodes a transmembrane receptor-typetyrosine kinase (TK), p69gp37-Eyk, in which the intracellular(IC) region of a putative receptor tyrosine kinase (RTK)is fused to the viral gp37 glycoprotein (Jia et al., 1992).The proto-oncogene c-evk, from which v-eyk was derived,codes for an RTK with a distinctive extracellular (EC)region, containing two immunoglobulin-like and twofibronectin III-like motifs (Jia and Hanafusa, 1994). Thecharacteristics of c-Eyk's EC structure and kinase domainsequence put it in the same subfamily of TKs as Axl/Ufo(O'Bryan et al., 1991). Other known members in thissubfamily include Sky/Rse/Tif/Brt/Tyro 3 (Dai et al.,1994; Fujimoto and Yamamoto, 1994; Lai et al., 1994;

Mark et al., 1994; Ohashi et al., 1994) and Mer (Grahamet al., 1994). Recently, Gas6 and Protein S, two factorsinvolved in blood coagulation, have been shown to beligands for Axl and Sky/Tyro 3, respectively (Stitt et al.,1995; Varnum et al., 1995). Comparison between thesequences of v-eyk and c-eyk revealed that v-evk wasprobably transduced from the nucleotides 1606-2987 ofc-evk, a portion encoding the kinase domain andC-terminus. At the amino acid level, there are onlytwo point mutations in v-Eyk, as compared with thecorresponding region of c-Eyk, one in the kinase domain,the other in the C-terminus. Aside from these mutations,v-Eyk has an identical sequence to c-Eyk in their ICdomains. However, unlike the stringently regulated c-Eyk,which is inactive and not transforming in chicken cells(C.Zong and H.Hanafusa, unpublished data), v-Eyk kinaseis constitutively active. It significantly increases cellulartyrosine phosphorylation and causes transformation ofchicken embryo fibroblasts (CEFs) (Jia et al., 1992).Consequently, RPL30 infection in vivo induces chickenerythroblastosis, fibrosarcomas, endotheliomas, viscerallymphomatosis and hemorrhage (Fredrickson et al., 1964).

Since several oncogenic TKs constitutively activate theRas-MAP kinase pathway, we tested whether v-Eyk orc-Eyk does the same. Our results indicated that the Ras-MAP kinase pathway is only modestly activated in v-Eyk-transformed cells, as measured by the activities of theMAP kinases and the MAP kinase kinase (MEK) (C.Zonget al., unpublished data). We also examined the JNK(c-Jun N-terminal kinase) pathway, which is involved insignaling in response to environmental stresses (Smealet al., 1991, 1992; Devary et al., 1991, 1992; Hibi et al.,1993; Galcheva-Gargova et al., 1994; Sluss et al., 1994).JNK kinases were not activated in the v-Eyk-transformedcells (C.Zong et al., unpublished data), as measured byboth the GST-c-Jun in-gel kinase assay and the c-Jun N'binding kinase assay (Pulverer et al., 1991; Smeal et al.,1991, 1992; Devary et al., 1992; Minden et al., 1994).These data will be described in detail in a separate paper(C.Zong et al., in preparation).

Since neither the Ras-MAP kinase nor the JNK pathwaywas activated, we examined another signaling route, theJAK-STAT pathway that also transduces signals from thecell surface to the nucleus in response to cytokine andgrowth factor treatment of cells (reviewed by Damellet al., 1994; Ihle et al., 1994). Many cytokines, some ofwhich stimulate growth, and growth factors, includingthose whose receptors have intrinsic tyrosine kinaseactivity, have been shown to utilize the JAK-STAT path-way, with different combinations of JAKs and STATsbeing activated by different polypeptide ligands (Schindlerand Damell, 1995). These findings prompted us to lookinto the possible influence exerted by v-Eyk and theactivated c-Eyk tyrosine kinases on the JAK-STATpathway.

© Oxford University Press 4515

C.Zong et aL

Here we have utilized a novel CD8-based fusion system(A.August and H.Hanafusa, unpublished data). By makinga CD8-c-Eyk chimera, we constitutively stimulated thec-Eyk kinase activity and studied the effects on the JAK-STAT pathway of both v-Eyk and the activated c-Eyk.We present evidence that the activated Eyk kinases con-stitutively activate the JAK-STAT pathway, specificallyJAKI, STATI and STAT3, and that Eyk kinase activity isessential for STAT1 activation, which correlates well withcellular transformation. Overexpression of STAT 1, anaturally occurring dominant negative form of STAT1, inthe Eyk-transformed cells significantly suppressed theanchorage-independent growth of these cells. In addition,we demonstrate that Eyk stimulates the JAK-STAT path-way in a manner distinct from Src, which only activatesSTAT3.

ResultsSTAT1 is constitutively activated in theRPL30-transformed CEFsSince Eyk kinase does not seem to activate the Ras-MAPkinase and JNK pathways significantly (data not shown),we tested the possible involvement of the JAK-STATpathway in Eyk-induced signal transduction. In vivo, theSTATs are phosphorylated rapidly on tyrosine residuesupon stimulation by cytokines such as interferon (IFN)-cxor -y. The phosphorylated STATs translocate immediatelyinto the nucleus and bind to specific DNA sequences suchas ISRE (interferon-stimulated response element) or GAS(IFN-y activation site) to activate specific gene expression(Darnell et al., 1994; Ihle et al., 1994). A simple test forSTAT activation is a DNA binding assay analyzing theretarded mobility of the oligonucleotides to which activ-ated STATs bind. In our initial studies, a 32P-labeled M67oligonucleotide containing a consensus binding site formany known STATs was used as a probe to analyzethe nuclear extracts from v-eyk-transformed CEFs (seeMaterials and methods). In normal CEFs (Figure 1, lanes4-6) that had been serum starved for 48 h, there was aweak but constitutive activation of STAT3, as indicatedby the SIF A band (c-sis inducible factor), which corre-sponds to a STAT3/3 homodimer (Sadowski and Gilman,1993; Zhong et al., 1994). The intensity of this band wasenhanced by serum stimulation of the quiescent cells(lanes 1-3). In contrast, in v-eyk-transformed CEFs, inaddition to much stronger STAT3 activation, STAT1 wassignificantly activated, as indicated by the SIF C band,which contains the STATI/I homodimer (Sadowski andGilman, 1993; Zhong et al., 1994) (lanes 7-10). Antibodiesagainst human STAT1 (Schindler et al., 1992) and mouseSTAT3 (Zhong et al., 1994), which had been tested cross-reactive with chicken STATI and 3, were used to co-incubate with the reaction mixtures. SIF A (lanes 3, 6 and9) and SIF C (lane 8) bands were abolished in thiscase by specific anti-STAT3 and anti-STAT1 antisera,demonstrating that these DNA-protein complexes didcontain chicken STAT3 and STAT1, respectively. Theseresults in chicken cells suggested that transformation byv-Eyk was accompanied by constitutive activation ofSTAT molecules and, more specifically, STAT1.

Fig. 1. STATI is specifically activated in v-eyk-transformed CEFs.One ,ul of nuclear extract each from v-eyk-transformed (lanes 7-10),normal (lanes 4-6) and serum-stimulated (lanes 1-3) CEFs wasincubated with the 32P-labeled M67 probe in the gel mobility shiftassay (see Materials and methods). The positions of SIF A and SIF Care marked with arrows. Addition of anti-STATs antisera abolished thecorresponding SIF A (lanes 3, 6 and 9) and C (lane 8) bands. Themiddle band between SIF A and C is a non-specific band,co-migrating with SIF B.

c-Eyk tyrosine kinase can be activated viadimerizationThe first experiment linking Eyk and the JAK-STATpathway was carried out in chicken cells where STATshave been less well characterized than in mammaliancells. To better analyze the downstream effects of Eyk,we expressed and studied Eyk kinase in mammalian cells.Unlike v-Eyk, which is constitutively active, c-Eyk is notnormally tyrosine phosphorylated and shows no kinaseactivity in chickens cells. Since its natural ligand isunknown, it is impossible to stimulate c-Eyk with itsligand and study its downstream effects. To circumvent thisproblem, we constructed chimeric receptor-like tyrosinekinases between CD8 and Eyk to promote receptor dimer-ization. CDc-Eyk was made by fusing the transmembrane(TM) and IC domains of c-Eyk to the EC region of CD8(Figure 2A). We also made a CDv-Eyk, consisting of theCD8 EC and TM domains and the v-Eyk IC domain(Figure 2A), a fusion similar to that between v-Eyk andgp37 in p69gp37-Eyk. The wild-type (wt) chicken c-eykwas also put into the same vector pMEX as a control(Figure 2A).The CD8 surface molecules are normally expressed as

disulfide-linked homodimers and multimers of a 32 kDaprotein on peripheral T cells (Snow and Terhorst, 1983;Ledbetter et al., 1985; Snow et al., 1985). Since theextracellular Cys residues of CD8 can form intermoleculardisulfide bonds, the chimeric molecules will most likelybe constitutively dimerized after expression, bringingthe two kinase domains together and stimulating trans-

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and H.Hanafusa, unpublished data).

Forty eight hours after CDc-eyk, CDv-eyk, c-eyk and

pMEX were transfected separately into COS7 cells, the

expression and tyrosine phosphorylation of the Eyk kinases

were examined. Anti-phosphotyrosine (PY) Western

blotting of the anti-Eyk immunoprecipitates showed that

CDc-Eyk and CDv-Eyk were phosphorylated equally on

tyrosine (Figure 2B, lanes 3 and 4). In vitro kinase assays

also proved that CDc-Eyk was constitutively activated to

a level comparable with CDv-Eyk (data not shown).

Consistent with the kinase activities, CDc-Eyk and CDv-

Eyk significantly increased the level of cellular tyrosine

phosphorylation (lanes 7 and 8). These data clearly demon-

strated that c-Eyk kinase is activated to the same degree

as v-Eyk in the chimera with CD8, which promotesdimerization. The wt c-Eyk also seemed to be partially

Fig. 2. c-Eyk tyrosine kinase is activated through dimerization.(A) Schematic protein structures of CDc-Eyk, CDv-Eyk and wt c-Eyk.Their cDNAs were all constructed into vector pMEX: the CD8 portionis hatched and the Eyk portion is blank. The functional domains ofc-Eyk are marked: Ig, immunoglobulin-like domain; FNIII, fibronectinIII-like domain. (B) Anti-phosphotyrosine Western blot of anti-Eykimmunoprecipitates from 200 p.g lysates (lanes 1-4) and 40 .tg totallysates (lanes 5-8) of COS cells transiently transfected with CDc-eyk,CDv-eyk, c-eyk or pMEX. The positions of the Eyk kinases areindicated with arrows, as seen on the anti-Eyk Western blot (data notshown). (C) A colony formation assay of 3Y1 cells stably transfectedwith CDc-eyk (CE2 and CE9), CDv-eyk (CV6 and CVIO), c-eyk (El)or pMEX.

activated in COS cells, a phenomenon not observed inchicken cells and probably due to the extremely highexpression in COS cells, which may result in clusteringand spontaneous activation. Its activation was reflected inits own tyrosine phosphorylation (lane 2) and in theincreased total tyrosine phosphorylation in the cells(lane 6).

Both CDv-Eyk and CDc-Eyk, but not the wt c-Eyk,transform rat 3Y1 cellsTo test whether the constitutively activated c-Eyk andv-Eyk can cause transformation, anchorage-independentgrowth of 3Y1 cells overexpressing comparable levels ofCDc-Eyk, CDv-Eyk or wt c-Eyk was examined (Figure2C). 3YI/CDv-Eyk (CV6 and CVIO) and 3YI/CDc-Eyk(CE2 and CE9) formed colonies soon after suspension(3-4 days), while 3YlI/c-Eyk (El) and 3YI/pMEX didnot grow even after 2 weeks. The non-expressing, butdrug-resistant cell lines selected during CDc-eyk and CDv-eyk transfection also failed to grow in soft agar (data not

shown). 3YI/CDv-Eyk cells displayed evident morpho-

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Fig. 3. STATI is constitutively activated in the CDc-eyk- and CDv-eyk-transfected COS cells. (A) STATI is constitutively phosphorylated ontyrosine in CDv-Eyk- and CDc-Eyk-transfected COS cells, and the dimerized Eyk kinases co-immunoprecipitate with STAT1. An epitope (FLAG)-tagged STATI was transiently co-expressed with CDv-eyk, CDc-eyk, wt c-eyk or pMEX in COS cells. The tagged STATI was immunoprecipitatedfrom 200 p.g of total cell lysate with an anti-FLAG M2 monoclonal antibody and analyzed by anti-phosphotyrosine Western blotting (upper panel).The same membrane was stripped and reblotted with anti-STATI antiserum (lower panel). The co-precipitated CDv-Eyk and CDc-Eyk (indicated byhollow arrows on the right) were confirmed by anti-Eyk Western blotting (data not shown). (B) Activation of STATI DNA binding activity in COScells transfected with CDv-Eyk or CDc-Eyk. The nuclear extracts from COS cells transfected with CDv-eyk (lanes 10-14), CDc-eyk (lanes 7-9),c eyk (lanes 4-6) or pMEX (lanes 1-3) were subjected to gel mobility shift analysis using 32P-labeled M67 probe. Anti-STATs antisera causedspecific supershifts of the corresponding SIF A (lanes 6, 9 and 12) and SIF C (lanes 8 and 11) bands as marked by hollow arrows. In thecompetition reaction, 100-fold excess cold M67 probe was mixed with the labeled probe (lane 13). Pre-immune serum was used as a control forsupershifting (lane 14).

logical changes with increased refractility and had longprocesses that formed a mesh-like local structure (datanot shown). The changes in 3Y1/CDc-Eyk cells weremore subtle, with only slightly higher refractility andslightly more rounding of the cells (data not shown). 3Y1/c-Eyk cells did not show any discernible phenotypicchanges under the microscope (data not shown).

In transformed 3Y1 cells, both CDv-Eyk and CDc-Eykhad high specific kinase activity, while c-Eyk activity wasbarely detectable, as measured by both in vitro kinaseassays and the level of cellular tyrosine phosphorylation(data not shown). These data corresponded well with thecolony formation analysis, indicating that the constitutivelyactivated v-Eyk and c-Eyk cause transformation. Theundetectable kinase activity of the wt c-Eyk in 3Y1 cellswas probably due to a significantly lower expression in3Y1 cells than in COS cells.

STAT1 is constitutively activated in the CDv-eyk-and CDc-eyk-transfected COS cellsTo characterize further the direct effects of the Eyk kinaseon the JAK-STAT pathway, transient transfections ofvarious Eyk-expressing constructs (Figure 2A) werecarried out in COS cells.

Tyrosine phosphorylation of STAT1 in the presence ofthe Eyk kinases was examined. An epitope (FLAG)-taggedSTATI was co-expressed in COS cells with CDc-Eyk,

CDv-Eyk or wt c-Eyk. The exogenous STAT 1 wasimmunoprecipitated using an anti-FLAG M2 monoclonalantibody and subjected to SDS-PAGE and anti-PYWestern analysis (Figure 3A). The band at the top in allfour lanes is a non-specific protein binding to the M2antibody. The band immediately beneath this non-specificband is the FLAG-tagged STAT 1. Only in the cellstransfected with CDc-eyk or CDv-eyk was STATI phos-phorylated on tyrosine (Figure 3A, lanes 1 and 2). Reprob-ing of the same filter with anti-STAT1 antiserum showedcomparable amounts of STAT1 in all four lanes (lowerpanel). Two bands detected by the anti-PY antibody(marked by hollow arrows, lanes 1 and 2) were lateridentified as CDv-Eyk and CDc-Eyk on anti-Eyk Westernblot, respectively (data not shown). The wt c-Eyk was notco-precipitated with STATI (lane 3), though it was alsoexpressed at a fairly high level (data not shown).The DNA binding activity of STATI was analyzed.

Figure 3B shows a gel mobility shift analysis of thenuclear extracts from COS cells transfected with CDc-eyk, CDv-eyk, c-eyk or pMEX. In CDc-eyk- and CDv-eyk-transfected cells, STAT1 was strongly and selectivelyactivated (SIF C) (lanes 7 and 10). In both cases, the SIFC band could be supershifted by incubating the nuclearextracts with anti-STAT1 serum (lanes 8 and 11). The wtc-Eyk did not activate STATI (lanes 4-6), but activatedSTAT3 equally to CDc-Eyk and CDv-Eyk (SIF A) (lanes

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Fig. 4. STAT3 is constitutively activated in all CDc-eyk-, CDv-eyk- and wt c-eyk-transfected COS cells. (A) STAT3 is constitutively phosphorylatedon tyrosine in COS cells expressing all three Eyk kinases (Figure 2A), but only the dimerized Eyk kinases co-immunoprecipitate with STAT3.Immunoprecipitates of STAT3 from cells co-expressing STAT3 and the Eyk kinases (Figure 2A) were subjected to anti-phosphotyrosine Westernblotting (upper panel). The two hollow arrows on the left side below STAT3 indicate the positions of the co-precipitated CDc-Eyk and CDv-Eyk.The amount of STAT3 being precipitated was shown by anti-STAT3 Western blotting (lower panel) after stripping the same membrane. (B) TheDNA binding activity of STAT3 is constitutively activated in COS cells expressing all three Eyk kinases (Figure 2A). The nuclear extracts fromCOS cells co-transfected with CDv-eyk (lanes 13-16), CDc-eyk (lanes 9-12), wt c-eyk (lanes 6-8) or pMEX (lanes 3-5), together with a STAT3expression vector, were subjected to gel mobility shift analysis, using 32P-labeled M67 probe. The nuclear extract from COS cells treated with IL-6was used as a positive control (lane 1). The anti-STAT3 (lanes 4, 7, 10 and 14) and anti-STATI C-terminus (lanes 5, 8, 11 and 15) and N-terminus(lanes 12 and 16) antisera were used to confirm the identity of the components in SIF A and C bands. The supershifted bands are indicated withhollow arrows. The anti-STAT1 N' antiserum did not cause supershift to the SIF C band because dimerization of STATI prevented the epitope frombeing recognized.

4, 7 and 10). The anti-STAT3 antiserum decreased theSIF A mobility (lanes 6, 9 and 12). Excess cold M67competed away the binding of STATs to the labeled probe(lane 13), while the pre-immune serum had no effect oneither STATI or STAT3 binding to M67 (lane 14). Con-sistent results were also obtained from 3Y1 cells stablytransfected with CDc-eyk, CDv-eyk or wt c-eyk. In 3Y1cells transformed by CDc-Eyk and CDv-Eyk (Figure 2C),both STATI and STAT3 were constitutively activated,while no STAT1 activation was detected in the untrans-formed 3Y1/c-Eyk cells (data not shown).

STAT3 is constitutively activated in all CDc-eyk-,CDv-eyk- and wt c-eyk-transfected COS cellsFrom Figure 3B, activation of the endogenous STAT3seems relatively weak, compared with that of STAT1. Itseemed possible that the low STAT3 activation wasgenerally due to a lower endogenous expression in COScells. Therefore, we tested the activation of STAT3 incells where STAT3 was overexpressed.A STAT3 expression vector was co-transfected into

COS cells with CDv-eyk, CDc-eyk, c-eyk or pMEX. Theanti-PY Western blot of the immunoprecipitated STAT3demonstrated strong tyrosine phosphorylation of STAT3in all CDv-eyk-, CDc-eyk- and c-eyk-transfected cells

(Figure 4A, upper panel). Reblotting of the same filterwith anti-STAT3 antiserum showed comparable amountof STAT3 in each lane (lower panel). Bands indicated bytwo hollow arrows below STAT3 were later identifiedon anti-Eyk Western blot as CDc-Eyk and CDv-Eyk,respectively (data not shown). The gel mobility shiftanalysis showed that all three kinases, including CDc-Eyk, CDv-Eyk and wt c-Eyk, strongly stimulated theDNA binding activity of STAT3 (SIF A) (Figure 4B, lanes6, 9 and 13). The anti-STAT3 antiserum caused supershiftof SIF A (lanes 7, 10 and 14).

JAK1 is Tyr phosphorylated in the CDc-eyk-,CDv-eyk- and c-eyk-transfected COS7 cellsThe JAK family kinases have been known to play a crucialrole in the cytokine activation of STATs (reviewed byDarnell et al., 1994; Ihle et al., 1994). JAKI was selectedfirst to test for its activation by Eyk. Since the endogenouslevel of JAKI is extremely low, human JAK1 was transi-ently co-transfected into COS cells with CDc-eyk, CDv-eyk, c-eyk and pMEX. Anti-PY Western analysis ofthe immunoprecipitated JAKI showed that its tyrosinephosphorylation was significantly elevated in CDc-eyk-,CDv-eyk- and wt c-eyk co-transfected cells (Figure 5,upper panel, lanes 1-3). An equal amount of JAKI was

4519

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.. .... .; t . ... _ .R n i - L 'r! h............ .. a.r 5 p ; 8bt54f 1 ( s; > i f 1- | ! ;Fig. 5. JAK1 tyrosine phosphorylation is stimulated by all three Eykkinases, but only CDc-Eyk and CDv-Eyk co-immunoprecipitate withJAKI. A JAKI-expressing vector was co-transfected into COS cellswith the various eyk constructs (Figure 2A). JAKI wasimmunoprecipitated from total cell lysates and subjected to anti-phosphotyrosine Western blot (upper panel). The JAKI p130 andanother pl0 protein, a putative JAK1 degradation product, areindicated with arrows. The co-precipitated dimerized Eyk kinases arealso marked (hollow arrows). Their identities were confirmed byanti-Eyk Western blot (data not shown). The amount of JAKI beingprecipitated was comparable in each lane, as shown by anti-JAKIWestern blot (lower panel).

precipitated in each lane (lower panel). In lanes 1 and 2,two doublet bands recognized by anti-PY antibody werelater identified as CDc-Eyk and CDv-Eyk (data not shown).The wt c-Eyk was not co-precipitated by anti-JAKIantibody despite its fairly high expression. This resultconfirmed the previous studies that JAKI binding to thecytokine receptors is enhanced by receptor dimerization(Darnell et al., 1994; Ihle et al., 1994). We also testedtwo other members of the JAK family kinases, JAK2 andTyk2. The tyrosine phosphorylation of neither seemedaffected by Eyk.

Eyk kinase activity is required for the activation ofSTAT1, but not STAT3To study whether Eyk kinase activity is required for STATactivation, a kinase-negative mutant of CDc-Eyk, calledCDc-EykM, was constructed by mutating the ATP bindingLys to Met (K609M). Theoretically, CDc-EykM shouldlose kinase activity yet maintain its ability to dimerizein vivo. CDc-EykM and the original CDc-Eyk were equallywell expressed when transfected into COS cells (Figure6A, left panel), but the kinase-negative mutant was notphosphorylated on tyrosine (middle panel), nor did itpossess any kinase activity in vitro (right panel). Gelmobility shift analysis of the nuclear extracts from bothnormal CDc-eyk- and CDc-EykM-transfected COS cellsshowed that CDc-EykM did not activate STAT1 (Figure6B, lane 1), while CDc-Eyk strongly stimulated STATI(SIF C band, lane 2). However, the DNA binding activityof the endogenous STAT3 was not affected by the kinasedeficiency of CDc-EykM (SIF A band, lanes 1 and 2).

STAT1f3 overexpression suppresses transformationby constitutively activated EykThe specific activation of STAT1 in the Eyk-transformedcells, both CEFs and 3Y1 cells, and the indispensablerole of Eyk kinase in STAT1 tyrosine phosphorylation,indicated the possibility that STAT1 may be requiredfor Eyk-initiated cellular transformation. To confirm thishypothesis, we transfected a STAT1IB (p84) expressionplasmid into CV1O and CE2 cells, which had beentransformed by CDv-Eyk and CDc-Eyk, respectively.Compared with STATax (p91), which is activated by Eyk,STAT1,B lacks the transcription activation domain, andtherefore could serve as a competitive suppresser to theax form once overexpressed (Schindler et al., 1992; K.Shuaiand J.E.Darnell,Jr, unpublished observations). Cells withelevated STAT I10 expression were selected, and theiranchorage-independent growth was analyzed in soft agar(Figure 7). These cells only showed an increased expres-sion of STAT1 of 1.5- to 5-fold, as compared with theparental cells (data not shown). However, all of themdisplayed significant reduction in their colony formationefficiency (Figure 7). In addition, the size of the coloniesformed in soft agar by these cells was also smaller thanthat of their parental cells (data not shown).

The effects of Eyk kinases on the JAK-STATpathway are different from those of SrcBased on the described data, the activated Eyk kinases,including v-Eyk, CDc-Eyk and CDv-Eyk, constitutivelyactivate STAT1 and STAT3, while normally STATs areactivated only transiently upon cytokine stimulation(Schindler et al., 1992; Shuai et al., 1992; Hou et al.,1994; Wakao et al., 1994; Zhong et al., 1994). Some otherTKs, such as the receptors for epidermal growth factor(EGF) and platelet-derived growth factor (PDGF), canstimulate STAT1 or STAT3 (reviewed by Schindler andDarnell, 1995), but their effects are also transient insteadof constitutive. To illustrate that the signaling pattern ofthe activated Eyk is different from those of other TKs,we compared the effects of the Eyk kinases and c-Src onthe JAK-STAT pathway.

CDc-Src is a chimeric molecule made up of the CD8EC and TM domains and the entire c-Src molecule fusedin-frame, which has been shown to be constitutively activewith a significantly higher specific kinase activity thanthe normal c-Src (A.August and H.Hanafusa, unpublisheddata). The architecture of CDc-Eyk and CDc-Src is there-fore very similar. We and others have found independentlythat v-Src, CDc-Src and CDv-Src (made up of CD8 andv-Src) can all weakly stimulate endogenous STAT3, butnot STATI (Yu et al., 1995; C.Zong et al., unpublisheddata). Analysis of the nuclear extracts from COS cellsoverexpressing STAT3 revealed that stimulation of STAT3DNA binding activity was only seen in the CDc-src co-transfected cells (Figure 8, lanes 5-8). However, therewas no activation of STATI by CDc-Src, as there was byCDc-Eyk (lane 1). The activated Eyk kinases thus causea different pattern of STAT activation from Src. Therefore,the activation of the JAK-STAT pathway by the activatedEyk kinases is not only constitutive, but also unique witha specificity toward STAT1.

4520

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Fig. 6. Eyk tyrosine kinase activity is required for STATI activation. (A) The kinase-negative mutant CDc-EykM is expressed normally but has nokinase activity. Left panel, an anti-Eyk Western blot of 40 ,ug of total lysate; middle panel, an anti-phosphotyrosine Western blot of 40 gg of totallysate; right panel, an in vitro kinase assay of CDc-EykM or wt CDc-Eyk immunoprecipitated from 200 ,ug of total cell lysates. (B) A gel mobilityshift assay of STAT activation in COS cells transiently transfected with the kinase-negative mutant CDc-eykM and wt CDc-eyk using 32P-labeledM67 probe. The positions of SIF A and C are marked with arrows.

DiscussionThe CD8-based chimeric system effectivelyactivates orphan receptor kinasesThere are many ways to activate an orphan RTK artificially.In our experiments, we used the characteristics of CD8surface molecule to constitutively dimerize the fused Eykkinase domains. This design not only mimics the ligandstimulation of RTKs in vivo (Ullrich and Schlessinger,1990), but also has several advantages for studying thelong-term effects of RTK activation. First, in the CD8chimera system, the steady-state level of the constitutivelyactive kinase dimers is less affected by the receptordown-regulation. Upon ligand treatment, normal receptorsdimerize and are immediately down-regulated, trans-mitting only a transient signal (Ullrich and Schlessinger,1990). In the CD8 chimeric system, internalized chimerason the plasma membrane are replenished constantly bynewly synthesized active dimer kinases. In addition,kinase-active chimeras might also be present on thecytosolic membranous organelles such as the endoplasmicreticulum and Golgi complex, where they may not besubject to the normal down-regulation. Therefore, theconstitutively activated chimeric kinases continuously sendstimulatory signals, causing a sustained activation of thedownstream signaling pathways. Second, the chimeric

dimers can provide a platform for studying the interactionbetween the receptors and the downstream signalingmolecules, which would otherwise be difficult to analyzewith the transiently activated and rapidly internalizednormal receptors. In our studies, we found STAT1, STAT3and JAKI were all constitutively activated by thedimerized CDc-Eyk and CDv-Eyk. Also, they all stablyassociated with CDc-Eyk and CDv-Eyk as detected in theco-immunoprecipitation experiments. The relevance of thephysical associations to the activation of these moleculesby Eyk kinases is not clear, since the activation of STAT3and JAKI seems independent of the stable associationwith the Eyk kinase dimers, as both can be activated bywt c-Eyk. No stable association with wt c-Eyk wasdetected despite the reasonable expression level and kinaseactivity of c-Eyk in COS cells.

The constitutive activation of STAT1 is specific tothe activated Eyk kinases and correlated withtransformationWe demonstrate by gel mobility shift assays with M67probes that both STAT1 and STAT3 are constitutivelystimulated by the active Eyk kinases. STAT4, which isactivated by interleukin-12 in T helper cells (Yamamotoet al., 1994; Jacobson et al., 1995), was not activated by

4521

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Fig. 7. The expression of STAT1IB suppresses the anchorage-independent growth of the Eyk-transformed 3Y1 cells. Two 3Y1 celllines, CV1O (transformed by CDv-Eyk) and CE2 (transformed byCDc-Eyk), were transfected with a STAT1f expression construct. Thecell lines with elevated expression (2- to 5-fold compared withendogenous level, data not shown) of STAT1,B were selected forcolony formation analysis. After trypsinization, 3x 105 cells wereresuspended in the soft agar and incubated for 2 weeks before thecolonies were counted. The numbers plotted represent the counting of1/10 of a 10 cm plate. CVIO and CE2, parental 3Y1 cells transformedby CDv-Eyk and CDc-Eyk, respectively; 10-2 is derived from CVIOwith -2-fold overexpression of STATlI ; C-2, -3, -5 and -6 and M-2,-3 and -6 (transfected with a pMNC-based STAT1IB expressionconstruct, promoted by the CMV promoter, a gift from Dr J.Darnell)are derived from CE2, with 2- to 5-fold STAT1I expression ascompared with the parental cells.

Eyk (data not shown). The possibility of STAT5 or 6activation (Hou et al., 1994; Wakao et al., 1994; Pallardet al., 1995) by Eyk was also ruled out (data not shown)by testing with a probe derived from a casein kinasepromoter (Wakao et al., 1994).

There was a good correlation between the constitutiveactivation of STATI in vivo and transformation by Eyk.The three active kinases, v-Eyk, CDc-Eyk and CDv-Eyk,all induced a significant increase in cellular tyrosinephosphorylation, caused transformation and led to theconstitutive activation of STAT1. On the contrary, wtc-Eyk and the kinase-negative CDc-EykM, which did notactivate STAT1, failed to transform 3Y1 cells. Overexpres-sion of STAT1 I, which suppresses the transcription activ-ation by STATIcx, significantly reduced the colonyformation of the Eyk-transformed 3Y1 cells in soft agar.These data suggest that STAT1 may be a direct effectoror substrate that is at least partially responsible for Eyksignaling, and that the constitutive activation of STAT1may be indispensable for Eyk transformation.

While the Eyk kinase plays an essential role in phos-phorylating STAT1, we do not have evidence that Eyk isdirectly phosphorylating STAT1. However, if there is asecondary kinase involved, it is probably not JAKI, asJAKI was activated by all three Eyks, including CDc-Eyk, CDv-Eyk and the wt c-Eyk, an activation patterndifferent from that of STAT1.

The activation of STAT3 does not always correlatewith transformationOn the other hand, we found that the activation of STAT3alone is not always associated with transformation. First, in

Fig. 8. The effects of Eyk on the JAK-STAT pathway differ fromthose of Src. The nuclear extracts prepared from COS cells transientlyco-transfected with CDc-src (lanes 5-9) or pMEX (lanes 2-4) togetherwith a STAT3 expression vector were subjected to gel mobility shiftanalysis using 32P-labeled M67 probe. The nuclear extract fromCDc-eyk-transfected cells without STAT3 co-transfection served as acontrol. The positions of SIF A and C are marked with arrows. Thesupershifted band caused by anti-STAT3 antiserum is marked by ahollow arrow (lane 7).

normal quiescent CEFs, there was a weak but constitutiveactivation of STAT3, which could be augmented by serumstimulation (Figure 1). Second, neither wt c-Eyk- nor thekinase-negative CDc-EykM-transfected 3Y1 cells weretransformed, yet, in those cells, a weak STAT3 activationcould still sometimes be detected. An additional suggestionthat STAT3 activation may not be sufficient for cellulartransformation comes from the finding that CDc-Srcconstitutively activated STAT3 in 3Y1 cells (C.Zong et al.,unpublished data), yet it failed to stimulate anchorage-independent growth of these cells in soft agar (A.Augustand H.Hanafusa, unpublished data). Therefore, the sig-nificance of STAT3 activation in cellular transformationneeds further investigation.

It seems that STAT3 phosphorylation is mediated by atyrosine kinase other than Eyk, since lack of Eyk kinaseactivity has little effect on the endogenous STAT3 activ-ation in COS cells. Our preliminary data suggested thatSTAT3 may constitutively bind to the kinase-negativeCDc-EykM in COS cells, indicating a possible phosphoryl-ation-independent mechanism for their interaction (data

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Activation of the JAK-STAT pathway by Eyk

not shown). In addition, JAK 1 is probably associated withCDc-EykM (data not shown). There is a significant bandin the anti-PY Western blot running at the positioncorresponding to JAKI (Figure 6B). Furthermore, theactivation pattern of JAKI is similar to that of STAT3,which is activated by CDc-Eyk, CDv-Eyk and wt c-Eyk.Therefore, it is possible that JAKI is responsible forphosphorylating STAT3 in their complexes with thedimerized Eyk kinases. However, we have not been ableto demonstrate directly so far that the band on the anti-PY Western blot is indeed JAKI co-immunoprecipitatingwith CDc-EykM, due to the quality of the anti-JAKIantiserum. Direct evidence from an anti-JAK1 Westernblot using a better antibody will verify whether thehypothesis is correct.

Prolonged JAK-STAT pathway activation may bethe cause of Eyk transformationThe transient STATs activation stimulated by cytokinesenable cells to respond to the extracellular stimuli in atimely but restricted manner (Schindler et al., 1992; Shuaiet al., 1992; Hou et al., 1994; Wakao et al., 1994; Zhonget al., 1994). The transcriptional activation stimulated bySTATs leads to gene expression in a specific combination,eliciting specific cellular responses. In v-Eyk-transformedcells or in the cells overexpressing CDv-Eyk or CDc-Eyk,JAK1, STAT 1 and STAT3 are constitutively activated. Theprolonged activation of STATs may, however, promote adifferent pattern of gene expression from the transientstimulation, leading to the constitutive transcriptionalactivation of some genes critical for cell growth. Thiscould result in dramatically different cellular responsesfrom those initiated by the transient activation, and ultim-ately lead to transformation as a result of losing controlover cell growth. Although speculative, such a hypothesisis not unfounded. Evidence on control of PC12 celldifferentiation demonstrates that the short- and long-termstimulation of a certain pathway elicits different cellularresponses (reviewed by Marshall, 1995). Differentiationor proliferation is largely due to the quantitative differencesin the duration of the RasGTP stimulation and the MAPkinase activation by nerve growth factor (NGF) and EGF(Heasley and Johnson, 1992; Muroya et al., 1992; Traverseet al., 1992; Buday and Downward, 1993; Nguyen et al.,1993; Obermeier et al., 1994; Stephens et al., 1994; Tenget al., 1995). By analogy, it is reasonable to assume thatthe prolonged JAK-STAT pathway stimulation will alsoelicit different cellular responses, and it may cause trans-formation. Recently it has been shown that constitutiveactivation of the kinase activity of the hopscotch geneproduct, a Drosophila homolog of JAK kinase, resultedin hematopoietic neoplasia (Harrison et al., 1995; Louet al., 1995). Evidence from HTLV-l-transformed T cellsalso showed that the JAK-STAT pathway was constitut-ively activated (Migone et al., 1995). All this indicatesthat the JAK-STAT pathway plays an important role incell proliferation and transformation. In fact, many genescrucial for cell growth contain STAT-responsive elementsin their promoter regions, such as fos (Wagner et al.,1990; Sadowski and Gilman, 1993), junB (Bonni et al.,1993) and GBP (guanylate binding protein, not related toG proteins of the plasma membrane, Decker et al., 1991).Also, many cytokine-responsive genes have specific STAT

binding sites (reviewed by Darnell et al., 1994). Thepleiotropic effects of the long-term activation of the JAK-STAT pathway should be assessed in the context ofconstitutive activation of these genes. Future nuclear run-on experiments or Northern analysis will provide usefulinformation for determining the expression patterns ofsome important cellular genes such as fos, jun and mvcand IRF during evk transformation.

Further studies are needed to investigate the relationshipbetween Eyk and other JAK family kinases and otherSTAT factors in order to establish a broader understandingas to how Eyk kinase is involved in the activation of theJAK-STAT pathway. In addition, the mechanism of v-Eyktransformation, whether the constitutive activation of theJAK-STAT pathway is indeed responsible and whetherthe Ras-MAP kinase pathway modestly activated by Eykplays any role in cooperating with the JAK-STAT pathwaysynergistically to promote cell growth and transformation,as suggested by recent evidence (David et al., 1995; Wenet al., 1995; Zhang et al., 1995), remains to be elucidated.

Materials and methodsTissue cultureCEFs were prepared and maintained as previously described (Hanafusa,1969), using either Scherer's or F-10 medium containing 5% calf serum.COS7 cells and 3Y1 cells were cultured in Dulbecco's modified Eagle'smedium with 10% fetal calf serum. All cells were cultured at 37°C with5% CO2. All sera were heat inactivated at 56°C for 20 min before use.

DNA transfectionThe transient transfections of the chimeric molecules CDc-eyk and CDv-eyk and other expression constructs into COS7 cells were achieved byusing the calcium phosphate precipitation method (Sambrook et al.,1989). In the case of selection of 3Y1 stable transfectants, after thenormal transfection procedures, cells were transferred 1:10 and culturedin regular medium containing 0.2 mg/ml G418 until colonies of suitablesize for cloning appeared.

Colony formation assay for transformed cellsCells were trypsinized and resuspended in 6.5 ml of medium. Fifteenml of bottom layer agar (0.7% agar/medium) were transferred to 100 mmdishes and allowed to solidify. Then 1 ml of resuspended cells weremixed with 10 ml of top agar (0.4% agar/medium), and pooled onto thebottom layer. The entire culture was allowed to solidify at roomtemperature for 15-30 min. The recipes for both top and bottom layersof agar/medium were described previously (Hanafusa, 1969). The plateswere incubated at 40°C for 1-2 weeks for transformed CEFs. For 3Y1cells, the culture was incubated at 37C.

Plasmid constructionFor CDc-eyk, c-evk cDNA was cut with Earl and NotI, with the formerend filled in using the Klenow enzyme. The resulting fragment was thenligated into pAO11 (CD8 cDNA in pBluescript), which was pre-digestedwith EcoRV and NotI. This resulted in a chimeric gene coding for amolecule consisting of the CD8 EC domain and the c-Eyk TM and ICdomains. The chimeric cDNA was then cut out by Sall and NotI, withthe latter site blunted. It was then ligated with pMEX digested with SalIand NotI (blunted). For CDv-eyk, a fragment corresponding to the v-evknucleotides 770-2140 was obtained by PCR with the amplified sequenceconfirmed by sequencing. The fragment was cut with BamnHI at its 5'end and ligated into pAlOl 1. cut by BacmiHI and NotI (blunted). Theresulting construct encodes a chimeric molecule with the CD8 EC regionand TM domain and the v-Eyk kinase domain and C-terminus. For wtc-evk expression vector, the full-length cDNA was cut with BamnHI andNotI (blunted) before it was ligated into pMEX pre-digested with BamnHIand EcoRI (blunted). All three constructs are pMEX based. For the sakeof convenience, the names of non-italic CDc-eyk. CDv-eyk and c-eykare used when referring to these constructs, and CDc-Eyk. CDv-Eyk and c-Eyk when referring to the corresponding protein products(Figure 2A).

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Immunoprecipitation, SDS-PAGE, Western blot and in vitrokinase assayThe protocol for immunoprecipitation has been reported previously (Jiaet al., 1992). The SDS-PAGE gel electrophoresis and electroblotting ofproteins to a supporting material were carried out as described (Sambrooket al., 1989). The filter was either nitrocellulose (Schleicher & Schuell) orPVDF Immobilon membrane (Millipore). The membrane was developedusing Enhanced Chemiluminescence (ECL) reagents (DuPont). Thein vitro kinase assay was performed as previously described (Jiaetal., 1992).

Gel mobility shift analysis for the activation of STATtranscription factorsCells, confluent and starved in 0.1 or 0% serum, were washed twicewith phosphate-buffered saline (PBS) and scraped into Eppendorf tubes.The tubes were spun at 2500 g for 30 s and excess washing bufferremoved. The cell pellet was resuspended in three volumes of hypotonicbuffer [20 mM HEPES, pH 7.9, 10 mM KCI, I mM EDTA. 1 mMEGTA, 0.2% NP-40, 10% glycerol, 0.1 mM orthovanadate, 1 mMdithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF) and1% aprotinin] and incubated at 4°C for 10 min before being spun at15 000 g for 0.5-1 min. The supernatant was removed, which was thecytoplasmic extract, and the pellet was resuspended in three volumes ofhigh salt extraction buffer (420mM NaCl, 10% glycerol, 20 mM HEPES,pH 7.9, 10mM KCl, 1 mM EDTA, 1 mM EGTA, 0.1 mM orthovanadate,1 mM DTT, 1 mM PMSF and 1% aprotinin). After thorough mixing ona rotating wheel at 4°C for 30 min, the suspension was centrifuged at15 000 g for 5 min. The supernatant was the nuclear extract for the gelmobility shift assay.One p1 (100 ng/pl) of oligonucleotide, M67 (GATCCATTTCCCGTA-

AATCAT), which contains a STAT-specific binding sequence, was labeledwith 100 tCi each of [x-32P]dATP, dGTP, dCTP and dTTP at roomtemperature for 15 min using the Klenow fragment. Then 2.5 pM colddNTP was added, and the reaction was continued for 5 min more before11.1 of 0.5 M EDTA was added to stop the reaction. The labeled probewas passed through a G-50 column to remove the unincorporated dNTPbefore being adjusted to a final volume of 100 p.l.The shift reaction mixture consisted of 1 p1 of dI-dC (2 mg/ml),

2.5 p1 of 5X shift buffer (100 mM HEPES, pH 7.9, 20% Ficoll, 5 mMMgClb, 200 mM KCI, 0.5 mM EGTA, 2.5 mM EDTA), 1 p.1 of labeledM67 probe, 1-2 ,ul of nuclear extract and 6-7 p1 of ddH,O. The rea!ctionmixture was incubated at room temperature for 20 min before 1 iul ofthe reaction mixture was loaded onto a 4.5% shift gel [1.2 nil of 10"TBE, 6.0 ml of 40% acrylamide:bis (29:1), 1.0 ml of 10% amnont.ium11persulfate, 35 p.1 of TEMED, and 42 ml of ddH2O]. Before thc '..inpleswere loaded, pre-electrophoresis was carried out in 0.25X T13Blf at 4'Cuntil the conductivity at 400 V fell below 10 mA. The gel was usuallyrun until the xylene cyanol FF dye ran past the mid-line of the full gellength (1.5 h) or to close to the bottom of the gel (normally 3 h). Thegel was then dried and subjected to autoradiography.

AcknowledgementsThe authors wish to thank Ran Jia for his help and discussions andZilong Wen for his generous gift of antibodies against STATs. We greatlyappreciate Kurt Horvath, Uwe Vinkemeier and Jacqueline Bromberg forgenerously providing us with the FLAG-tagged STAT 1 expression vector,antisera against the JAK and STAT proteins and oligonucleotidescontaining the consensus binding sites for STATs. Our appreciation alsogoes to Alvaro Monteiro, Xiaoqiao Jiang, Xin Ye, Shinya Tanaka andother members of the Hanafusa Laboratory for their discussions anddirect contribution towards this project. This work was supported byresearch grants from NIH CA44356 (to H.H.), A134420 and A132489(to J.D.) and NIH training grant CA09673 (to A.A.). R.Y. is an AaronDiamond Foundation Fellow.

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Received on October 6, 1995; revised on March 28, 1996

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