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Tyrosine Phosphorylation of the vav Proto-oncogene Product Links FcεRI to the Rac1-JNK Pathway

1997; Elsevier BV; Volume: 272; Issue: 16 Linguagem: Inglês

10.1074/jbc.272.16.10751

1083-351X

Hidemi Teramoto, P Salem, Keith C. Robbins, Xosé R. Bustelo, J. Silvio Gutkind,

Receptor Mechanisms and Signaling

Stimulation of high affinity IgE Fc receptors (FcεRI) in basophils and mast cells activates the tyrosine kinases Lyn and Syk and causes the tyrosine phosphorylation of phospholipase C-γ, resulting in the Ca2+- and protein kinase C-dependent secretion of inflammatory mediators. Concomitantly, FcεRI stimulation initiates a number of signaling events resulting in the activation of mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK), which, in turn, regulate nuclear responses, including cytokine gene expression. To dissect the signaling pathway(s) linking FcεRI to MAPK and JNK, we reconstructed their respective biochemical routes by expression of a chimeric interleukin-2 receptor α subunit (Tac)-FcεRI γ chain (Tacγ) in COS-7 cells. Cross-linking of Tacγ did not affect MAPK in COS-7 cells, but when coexpressed with the tyrosine kinase Syk, Tacγ stimulation potently induced Syk and Shc tyrosine phosphorylation and MAPK activation. In contrast, Tacγ did not signal JNK activation, even when coexpressed with Syk. Ectopic expression of a hematopoietic-specific guanine nucleotide exchange factor (GEF), Vav, reconstituted the Tacγ-induced, Syk- and Rac1-dependent JNK activation; and tyrosine-phosphorylation of Vav by Syk stimulated its GEF activity for Rac1. Thus, these data strongly suggest that Vav plays a critical role linking FcεRI and Syk to the Rac1-JNK pathway. Furthermore, these findings define a novel signal transduction pathway involving a multimeric cell surface receptor acting on a cytosolic tyrosine kinase, which, in turn, phosphorylates a GEF, thereby regulating its activity toward a small GTP-binding protein and promoting the activation of a kinase cascade. Stimulation of high affinity IgE Fc receptors (FcεRI) in basophils and mast cells activates the tyrosine kinases Lyn and Syk and causes the tyrosine phosphorylation of phospholipase C-γ, resulting in the Ca2+- and protein kinase C-dependent secretion of inflammatory mediators. Concomitantly, FcεRI stimulation initiates a number of signaling events resulting in the activation of mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK), which, in turn, regulate nuclear responses, including cytokine gene expression. To dissect the signaling pathway(s) linking FcεRI to MAPK and JNK, we reconstructed their respective biochemical routes by expression of a chimeric interleukin-2 receptor α subunit (Tac)-FcεRI γ chain (Tacγ) in COS-7 cells. Cross-linking of Tacγ did not affect MAPK in COS-7 cells, but when coexpressed with the tyrosine kinase Syk, Tacγ stimulation potently induced Syk and Shc tyrosine phosphorylation and MAPK activation. In contrast, Tacγ did not signal JNK activation, even when coexpressed with Syk. Ectopic expression of a hematopoietic-specific guanine nucleotide exchange factor (GEF), Vav, reconstituted the Tacγ-induced, Syk- and Rac1-dependent JNK activation; and tyrosine-phosphorylation of Vav by Syk stimulated its GEF activity for Rac1. Thus, these data strongly suggest that Vav plays a critical role linking FcεRI and Syk to the Rac1-JNK pathway. Furthermore, these findings define a novel signal transduction pathway involving a multimeric cell surface receptor acting on a cytosolic tyrosine kinase, which, in turn, phosphorylates a GEF, thereby regulating its activity toward a small GTP-binding protein and promoting the activation of a kinase cascade. Activation of high affinity IgE Fc receptors (FcεRI) in basophils and mast cells induces the rapid release of histamine and other inflammatory mediators from secretory granules, and initiates a cascade of signal transduction events leading to enhanced production and secretion of various biologically active cytokines (1Scharenberg A.M. Kinet J.P. Chem. Immunol. 1995; 61: 72-87Crossref PubMed Google Scholar). One of the earliest events induced upon FcεRI aggregation is the activation of the nonreceptor tyrosine kinases Lyn and Syk, and the tyrosine phosphorylation of cytoplasmic molecules, including phospholipase C-γ (2Eisenman E. Bolen J.B. Nature. 1992; 355: 78-80Crossref PubMed Scopus (417) Google Scholar). Phosphorylated phospholipase C-γ hydrolyses phosphatidylinositol 4,5-bisphosphate and liberates inositol 1,4,5-trisphosphate and diacylglycerol, which mobilizes Ca2+ from intracellular and extracellular sources and activates protein kinase C (3Ozawa K. Yamdada K. Kazanietz M.G. Blumberg P.M. Beaven M.A. J. Biol. Chem. 1993; 268: 1749-1756Abstract Full Text PDF PubMed Google Scholar), respectively. Whereas these second-messenger generating systems appear to be sufficient for the FcεRI-mediated secretory response (4Jabril-Cuenod B. Zhang C. Scharenberg A.M. Paolini R. Numerof R. Beaven M.A. Kinet J.P. J. Biol. Chem. 1996; 271: 16268-16272Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), how signals initiated by FcεRI aggregation at the plasma membrane are transmitted to the nucleus thereby controlling cytokine gene expression is much less understood. Recently, it has been shown that stimulation of FcεRI in mast cell lines, such as RBL-2H3 cells, leads to the activation of members of the mitogen-activated protein kinase (MAPK) 1The abbreviations used are: MAPK, mitogen-activated protein kinase; JNK, c-Jun NH2-terminal kinase; RBL, rat basophilic leukemia; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal calf serum; TNP, trinitrophenyl; DNP, dinitrophenyl; GEF, guanine nucleotide exchange factor. superfamily of serine-threonine kinases. The function of these enzymes is to convert extracellular stimuli to intracellular signals which, in turn, participate in gene expression regulation. In particular, engagement of FcεRI receptors in mast cell lines has been shown to result in the activation of MAPK and JNK (5Hirasawa N. Andrew S. Yamamura H. Beaven M.A. Kinet J.P. J. Biol. Chem. 1995; 270: 10960-10967Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 6Ishizuka T. Oshiba A. Sakata N. Terada N. Johnson G.L. Gelfand E.W. J. Biol. Chem. 1996; 22: 12762-12766Abstract Full Text Full Text PDF Scopus (65) Google Scholar). In this regard, recently available evidence suggests that engagement of FcεRI with antigen leads to the increased tyrosine phosphorylation of Shc and the association of Shc with Grb2, thus resulting in the recruitment of Sos and the stimulation of the Ras-MAPK pathway. Furthermore, Shc phosphorylation and MAPK activation was shown to be diminished upon overexpression of a dominant negative mutant of Syk, thus suggesting a central role for this kinase in the biochemical route communicating FcεRI to MAPK (5Hirasawa N. Andrew S. Yamamura H. Beaven M.A. Kinet J.P. J. Biol. Chem. 1995; 270: 10960-10967Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). In contrast, how FcεRI stimulation activates JNK is still unknown. In this study, we thought to dissect the signaling pathway(s) linking FcεRI to MAPK and JNK by reconstructing their respective biochemical routes upon ectopic expression of signaling molecules in COS-7 cells. Using this experimental approach, we provide evidence that whereas Syk and Shc connect FcεRI to the Ras-MAPK pathway, signaling from FcεRI to JNK involves the tyrosine phosphorylation by Syk of a hematopoietic specific guanine-nucleotide exchange factor, Vav, the exchange of GDP for GTP-bound to Rac1, and the consequent stimulation of a kinase cascade leading to JNK activation. RBL-2H3 cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS). Before cross-linking of IgE, cells were incubated overnight in DMEM containing 0.1% FBS. Sensitization with anti-trinitrophenyl (TNP) IgE ascites fluid (1:5,000) at 37 °C for 2 h and cross-linking with 0.1 μg/ml dinitrophenyl-coupled to human serum albumin were described previously (7Benhamou M. Gutkind J.S. Robbins K.C. Siraganian R.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5327-5330Crossref PubMed Scopus (200) Google Scholar). Expression plasmids (1 μg/plate) were transfected into subconfluent COS-7 cells by the DEAE-dextran technique (8Coso O.A. Chiariello M. Yu J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1570) Google Scholar), adjusting the total amount of DNA to 5 μg/plate with vector DNA (pcDNA3, Invitrogen) when necessary. Forty-eight hours later, cells were cultured overnight in DMEM containing 0.1% FBS. Cells were then left unstimulated or stimulated with EGF (100 ng/ml). Stimulation with antibodies to Tac was performed using 5 μg/ml of biotinylated monoclonal antibody to Tac, B1.49.9 (Amac). After washing with phosphate-buffered saline twice, cells were stimulated in serum-free medium containing 12 μg/ml of avidin (Sigma). After incubation for the times indicated, cells were lysed. Cell lysis, immunoprecipitation, immunoblotting, MAPK, and JNK assays were performed as described previously (8Coso O.A. Chiariello M. Yu J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1570) Google Scholar). Antiserum to MAPK and to Syk were purchased from Santa Cruz. Antibodies to Shc and to phosphotyrosine (anti-Tyr(P)) were purchased from Transduction Laboratories and ICN Biochemicals, respectively. Syk was cloned from a cDNA library prepared from purified human monocyte poly(A)+ mRNA templates by using a fragment of the porcine Syk cDNA (a gift from H. Yamamura) as a probe. An in frame BamHI site was generated immediately upstream of the initiation codon of Syk using polymerase chain reaction techniques and subcloned into pcDNA3. pcDNA3 Myr-Syk was generated by subcloning the Syk cDNA into pcDNA3-Myr (8Coso O.A. Chiariello M. Yu J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1570) Google Scholar). pcDNA3 Myr-Syk was transfected into COS-7 cells. After 48 h, cells were lysed in a hypotonic buffer, and proteins were isolated as cytosolic and membrane fractions, as described (9Fazioli F. Minichiello L. Matoskova B. Wong W.T. Di Fiore P.P. Mol. Cell. Biol. 1993; 13: 5814-5828Crossref PubMed Scopus (238) Google Scholar). Each fraction was immunoprecipitated with antibodies to Src (Santa Cruz) and immunoblotted with antiserum to Syk (Santa Cruz) and antibody to Tyr(P) (ICN). COS-7 cells were transfected using DEAE-dextran method, and cultured for 48 h, serum-starved in phosphate-free DMEM for 18 h, labeled with [32P]orthophosphate (100 μCi/ml) for 1 h for [32P]GDP accumulation and for 6 h for [32P]GDP and [32P]GTP determinations. Cells were disrupted in 50 mm Tris-HCl (pH 7.5), 20 mm MgCl2, 150 mm NaCl, 0.5% Nonidet P-40, 1 mm sodium orthovanadate, 1 mmphenylmethylsulfonyl fluoride, 25 μg/ml leupeptin, and 25 μg/ml aprotinin. Lysates were immunoprecipitated with a monoclonal antibody to AU5 (Babco) for 1 h and immunocomplexes recovered using gamma-binding G-Sepharose beads (Pharmacia Biotech Inc.). Immunoprecipitates were washed twice in lysis buffer, twice in 50 mm Tris-HCl (pH 7.5), 20 mm MgCl2, 500 mm NaCl, and resuspended in 1 mKH2PO4, 5 mm EDTA (pH 8.0). Bound nucleotides were released by heating and fractionated using polyethyleneimine thin layer chromatography plates (J. T. Baker). To begin dissecting the signaling pathway(s) linking FcεRI to MAPK and JNK, we initially studied the temporal relationship between MAPK and JNK activation in RBL-2H3 cells. As expected, engagement of FcεRI by addition of dinitrophenyl (DNP) coupled to human serum albumin to anti-TNP IgE-primed RBL-2H3 cells potently activated MAPK and JNK; however, each followed a distinct temporal pattern (Fig.1, A and B). These data suggested that MAPK and JNK might be activated by different signaling pathways. For MAPK, FcεRI cross-linking is known to activate the nonreceptor tyrosine kinase Syk, and it has been suggested recently that Syk phosphorylates the adapter protein Shc, thereby stimulating the Ras-MAPK pathway through Grb2 and Sos (10Jabril-Cuenod B. Zhang C. Scharenberg A.M. Paolini R. Robert N. Beaven M.A. Kinet J.P. J. Biol. Chem. 1996; 271: 16268-16272Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Consistent with that, we observed that in RBL-2H3 cells FcεRI activation leads to the rapid tyrosine phosphorylation of Syk and the adapter protein Shc, following a time course similar to that of MAPK stimulation (Fig. 1, Cand D). FcεRI is a multimeric receptor containing a single α and β subunit and a homodimer of γ subunits (11Ravetch J.V. Kinet J.P. Annu. Rev. Immunol. 1991; 9: 457-492Crossref PubMed Scopus (1286) Google Scholar). Both β and γ chains exhibit a structural motif termed ITAM, for immunoreceptor tyrosine-based activation motif (12Weiss A. Littman D.R. Cell. 1994; 76: 263-274Abstract Full Text PDF PubMed Scopus (1957) Google Scholar), which participate in the recruitment of cytoplasmic tyrosine kinases and in the consequent tyrosine phosphorylation of their downstream targets (1Scharenberg A.M. Kinet J.P. Chem. Immunol. 1995; 61: 72-87Crossref PubMed Google Scholar). Studies with chimeric molecules containing the extracellular and transmembrane domains of the interleukin-2 receptor α subunit (Tac) fused to the cytosolic domain of β (Tacβ) and γ (Tacγ) chains of FcεRI have helped simplify the analysis of early signaling events provoked by FcεRI activation (13Letourneur F. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8905-8909Crossref PubMed Scopus (245) Google Scholar). When expressed in RBL-2H3 cells, cross-linking of the Tacγ chimera is sufficient to mimic the majority of the biochemical and biological responses triggered by FcεRI stimulation. In contrast, cross-linking of Tacβ does not appear to elicit signaling responses (13Letourneur F. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8905-8909Crossref PubMed Scopus (245) Google Scholar). Therefore, to investigate whether activation of Tacγ is sufficient to activate Syk, both were expressed in COS-7 cells, which lack endogenous FcεRI or Syk (see below). Transfected Tacγ was efficiently expressed, as judged by immunofluorescence labeling techniques (data not shown). Cross-linking of Tacγ chimeras with biotinylated anti-Tac antibodies followed by streptavidin induced the rapid tyrosine phosphorylation of a coexpressed epitope-tagged Syk (Fig. 2 A). When coexpressed with an epitope-tagged form of Shc, cross-linking of Tacγ induced only a limited increase in Shc tyrosine phosphorylation (Fig. 2 B). However, when Syk was coexpressed, Tacγ engagement provoked a rapid and substantial increase in Shc tyrosine phosphorylation (Fig.2 B). Paralleling Shc phosphorylation, cross-linking of Tacγ induced a very poor MAPK response, but when coexpressed with Syk, Tacγ potently elevated the phosphorylating activity of MAPK to an extent comparable with that elicited in response to EGF (Fig.2 C). Taken together, these results support a central role for the γ subunit of FcεRI and Syk in signaling from IgE receptors to the MAPK pathway. Surprisingly, however, cross-linking of Tacγ chimeras did not result in JNK activation, even when coexpressed with Syk. As a control, EGF effectively elevated JNK activity under identical experimental conditions (Fig. 2 C). Collectively, these data established that coexpression of Tacγ and Syk in COS-7 cells is sufficient to reconstitute the MAPK response to FcεRI stimulation, while suggesting that additional molecules not endogenously expressed in COS-7 cells were necessary to link FcεRI to JNK. Whereas Ras controls the activation of MAPK, we and others have recently observed that two members of the Rho family of small GTP-binding proteins, Rac1 and Cdc42, regulate JNK activity (8Coso O.A. Chiariello M. Yu J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1570) Google Scholar). Although most molecules connecting Syk to Ras, including Shc, Grb2, and Sos, are ubiquitously expressed, guanine nucleotide exchange factors (GEFs) for Rho, Rac1, and Cdc42 exhibit a very restricted cell type and tissue distribution (14Hart M.J. Eva A. Zangrilli D. Aaronson S.A. Evans T. Cerione R.A. Zheng Y. J. Biol. Chem. 1994; 269: 62-65Abstract Full Text PDF PubMed Google Scholar). Thus, we hypothesized that COS-7 cells might lack an exchange factor acting downstream from Syk in the Rac/Cdc42-JNK pathway. In this regard, as recently shown by others (5Hirasawa N. Andrew S. Yamamura H. Beaven M.A. Kinet J.P. J. Biol. Chem. 1995; 270: 10960-10967Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar), FcεRI activation in RBL-2H3 cells induces the rapid and prolonged tyrosine phosphorylation of the Vav proto-oncogene product (Vav) (Fig.3 A), which is preferentially expressed in cells of the hematopoietic lineage. Moreover, Vav exhibits structural motifs frequently found in GEFs for small GTP-binding proteins of the Ras and Rho families (14Hart M.J. Eva A. Zangrilli D. Aaronson S.A. Evans T. Cerione R.A. Zheng Y. J. Biol. Chem. 1994; 269: 62-65Abstract Full Text PDF PubMed Google Scholar), and we have shown recently that truncated, oncogenically active forms of Vav (Onco-Vav), can potently activate JNK, but not MAPK, acting on a Rac-1-dependent signaling pathway (15Crespo P. Bustelo X.R. Aaronson D.S. Coso O.A. Lopez-Barahona M. Barbacid M. Gutkind J.S. Oncogene. 1996; 13: 455-466PubMed Google Scholar). These results prompted us to explore the possibility that wild-type Vav serves as a link between FcεRI and the Rac-1-JNK pathway. Expression of Vav alone (15Crespo P. Bustelo X.R. Aaronson D.S. Coso O.A. Lopez-Barahona M. Barbacid M. Gutkind J.S. Oncogene. 1996; 13: 455-466PubMed Google Scholar) or together with the Tacγ chimera failed to induce JNK activation (Fig. 3 B), and cross-linking of Tacγ failed to induce Vav tyrosine phosphorylation when coexpressed in COS-7 cells (Fig. 3 B). However, when Tacγ, Syk and Vav were each simultaneously coexpressed in these cells, Tacγ aggregation resulted in enhanced Vav tyrosine phosphorylation and a remarkable activation of JNK. These data together with results obtained in RBL-2H3 cells demonstrate the importance of Vav in signaling from FcεRI/Syk to JNK. We next asked whether recruitment of Syk to the plasma membrane upon aggregation of FcεRI or cross-linking of Tacγ chimeric molecules is the determining step initiating activity of Syk downstream signaling pathways. To that end, we examined the ability of a membrane-targeted form of Syk to bypass the requirement of Tacγ engagement for signaling to the MAPK and JNK pathway. A chimeric protein containing the NH2-terminal myristoylation signal of Src fused to Syk (Myr-Syk), localized to the plasma membrane when expressed in COS-7 cells, rather than exhibiting the typical cytosolic location of wild-type Syk (Ref. 16Taniguchi T. Kobayashi T. Kondo J. Takahashi K. Nakamura H. Suzuki J. Nagai K. Yamada T. Nakamura S. Yamamura H. J. Biol. Chem. 1991; 266: 15790-15796Abstract Full Text PDF PubMed Google Scholar and data not shown). Furthermore, this membrane-targeted form of Syk was heavily tyrosine-phosphorylated (Fig.3 C), and its expression was sufficient to elevate the activity of a cotransfected epitope-tagged MAPK (Fig. 3 D). However, Myr-Syk alone did not enhance JNK activity but, when cotransfected with Vav, it effectively induced the tyrosine phosphorylation of Vav (not shown) and potently activated the JNK pathway, to an extent comparable with that provoked by expression of the fully active, transforming vav oncogene (Fig.3 D). These data indicate that once Syk is activated upon recruitment to the plasma membrane, no other FcεRI-associated kinases are required to signal to MAPK or to activate JNK in a Vav-dependent manner. We have reported recently that JNK activation by Onco-Vav can be blocked by expression of a dominant negative mutant of Rac-1, N17 Rac-1 (15Crespo P. Bustelo X.R. Aaronson D.S. Coso O.A. Lopez-Barahona M. Barbacid M. Gutkind J.S. Oncogene. 1996; 13: 455-466PubMed Google Scholar), thereby inferring that Onco-Vav acts as a GEF for Rac-1. In view of those results and our present data, we next asked whether expression of Vav proteins could promote guanine nucleotide exchange on Rac1in vivo. In this regard, the high intrinsic GTPase activity of Rho, Rac1, and Cdc42 has prevented the detection in living cells of their corresponding GTP-bound forms (17Laudanna C. Campbell J.J. Butcher E.C. Science. 1996; 271: 981-983Crossref PubMed Scopus (434) Google Scholar). Thus, for these experiments we took advantage of a recently described technique that uses the levels of 32P-labeled GDP bound to these small GTPases after a brief exposure to [32P]orthophosphate-containing medium as an approach to evaluate their nucleotide exchange in vivo. Initially, we expressed in COS-7 cells AU5-epitope-tagged Ha-Ras, RhoA, Rac1, and Cdc42 (18Teramoto H. Coso O.A. Miyata H. Igishi T. Miki T. Gutkind J.S. J. Biol. Chem. 1996; 271: 27225-27228Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 19Crespo P. Schuebel K.E. Ostrom A.A. Gutkind J.S. Bustelo X.R. Nature. 1997; 385: 169-172Crossref PubMed Scopus (682) Google Scholar), together with empty expression vector (control), a membrane-targeted form of the catalytic domain of Sos (Myr-Sos) (8Coso O.A. Chiariello M. Yu J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1570) Google Scholar), or Onco-Vav (Fig. 4 A). All tagged small GTP-binding proteins were efficiently expressed, as judged by Western blotting with the anti-epitope antibody. Furthermore, when transfected cells were starved and then cultured for a short period of time in the presence of [32P]orthophosphate, each small GTPase incorporated labeled GDP, as determined by thin layer chromatography analysis of anti-AU5 immunoprecipitates. Under these experimental conditions, no labeled nucleotides were observed in mock-transfected cells (not shown), and Myr-Sos consistently enhanced 2–3-fold the level of radioactive GDP bound to Ras, without displaying any demonstrable effect on the other small GTP-binding proteins (Fig.4 A, left panel). As a control, we used the standard, more prolonged incubation with [32P]orthophosphate containing medium. Under those conditions, Myr-Sos induced a dramatic increase in GTP-bound Ras (Fig. 4 A, right panel). In contrast, under either incubation time expression of Onco-Vav did not affect Ras, but increased the level of labeled GDP bound to Rac1 more than 8-fold (Fig.4 A). Collectively, these results indicate that Onco-Vav can promote guanine nucleotide exchange in vivo on Rac1. Under identical experimental condition, neither wild-type Vav nor Myr-Syk induced nucleotide exchange on Rac1 (Fig. 4 B), which was consistent with the failure of each one alone to induce JNK activity (see above). However, when Myr-Syk was coexpressed with Vav, we observed a dramatic increase in the incorporation of labeled GDP into Rac1. These two observations, 1) potent JNK activation provoked by coexpression of Myr-Syk together with Vav or upon cross-linking of Tacγ when coexpressed with Syk and Vav and 2) Syk's ability to effectively tyrosine-phosphorylate Vav in vivo, strongly suggest that Syk-induced tyrosine phosphorylation of Vav increases its GEF toward Rac1, leading to JNK activation. Consistent with this conclusion, JNK stimulation induced by Tacγ cross-linking in Tacγ-, Syk-, and Vav-transfected COS-7 cells was blocked by the dominant negative mutant of Rac1, N17 rac1 (Fig. 4 C). Moreover, we have recently observed that tyrosine phosphorylation of purified Vav protein dramatically enhances its GEF activity on bacterially expressed Rac1 when analyzed in in vitro assays (19Crespo P. Schuebel K.E. Ostrom A.A. Gutkind J.S. Bustelo X.R. Nature. 1997; 385: 169-172Crossref PubMed Scopus (682) Google Scholar), further supporting the emerging notion that Vav behaves as a tyrosine phosphorylation-dependent GEF for Rac1. A number of GEFs for small GTP-binding proteins of the Rho family have been identified by virtue of their transforming potential in murine fibroblasts (20Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1762) Google Scholar). Nevertheless, the normal function of these GEFs, as well as the molecular mechanisms controlling their enzymatic activity in their natural setting, is still unknown. In this regard, our findings provide solid evidence that whereas Onco-Vav is constitutively active, wild-type Vav only promotes guanine nucleotide exchange in Rac1 upon activation of an upstream tyrosine kinase, Syk, and that Vav function(s) in this setting are controlled by tyrosine phosphorylation. Thus, these findings define a novel signal transduction pathway involving a cell surface receptor activating a nonreceptor tyrosine kinase, which, in turn, phosphorylates a GEF in tyrosine residues, thereby regulating its activity toward a small GTP-binding protein and promoting the activation of a kinase cascade. A schematic representation of such a likely biochemical route, including, sequentially, FcεRI, Syk, Vav, Rac1, and its downstream target, JNK, as well as the pathway connecting Syk to MAPK is depicted in Fig.5. Our present findings might also have important implications regarding the functioning of other multimeric antigen receptors. As discussed above, in mast cells accumulating evidence demonstrates that the γ subunit of FcεRI signals Syk activation. The FcεRI γ chain is functionally analogous to the ζ chain of the antigen T cell receptor, and whereas FcεRIγ subunits recruit Syk, the T cell receptor ζ subunits interact with Zap70 (21Chan A.C. Iwashima M. Turck C.W. Weiss A. Cell. 1992; 71: 649-662Abstract Full Text PDF PubMed Scopus (889) Google Scholar, 22Iwashima M. Irving B.A. van Oers N.S. Chan A.C. Weiss A. Science. 1994; 263: 1136-1139Crossref PubMed Scopus (2) Google Scholar). Furthermore, T cell receptor and B cell receptor activation both lead to Vav tyrosine phosphorylation (23Bustelo X.R. Barbacid M. Nature. 1992; 356: 68-71Crossref PubMed Scopus (245) Google Scholar, 24Margolis B. Nature. 1992; 356: 71-74Crossref PubMed Scopus (307) Google Scholar) and JNK activation (25Su B. Jacinto E. Hibi M. Kallunki T. Karin M. Ben-Neriah Y. Cell. 1994; 77: 727-736Abstract Full Text PDF PubMed Scopus (849) Google Scholar). Based upon our results, it is predictable that Vav plays a common role in basophils, mast cells, T cells, and B cells, linking multimeric antigen receptors and their associated downstream nonreceptor tyrosine kinases to the Rac1-JNK signaling pathway. We thank Dr. Richard D. Klausner for providing Tacγ cDNA and Dr. Hirohei Yamamura for providing the fragment of the porcine Syk cDNA.