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

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 32Porthophosphate (100 μCi/ml) for 1 h for 32PGDP accumulation and for 6 h for 32PGDP and 32PGTP 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. and cross-linking of Tacγ failed to induce Vav tyrosine phosphorylation when coexpressed in COS-7 cells (Fig. B). However, when Syk and Vav were each coexpressed in these cells, Tacγ aggregation in enhanced Vav tyrosine phosphorylation and a activation of JNK. These data together with results in RBL-2H3 cells the of Vav in signaling from to JNK. whether recruitment of Syk to the plasma membrane upon aggregation of FcεRI or cross-linking of Tacγ chimeric molecules is the activity of Syk downstream signaling pathways. To that we the of a form of Syk to the of Tacγ engagement for signaling to the MAPK and JNK pathway. A chimeric protein containing the NH2-terminal signal of Src fused to Syk to the plasma membrane when expressed in COS-7 cells, the cytosolic of wild-type Syk T. T. J. K. H. J. K. T. S. Yamamura H. J. Biol. Chem. 1991; Full Text PDF PubMed Google and data not shown). Furthermore, this form of Syk was (Fig.3 and its expression was sufficient to the activity of a epitope-tagged MAPK (Fig. D). However, Myr-Syk alone did not JNK activity when with Vav, it effectively induced the tyrosine phosphorylation of Vav and potently activated the JNK pathway, to an extent comparable with that provoked by expression of the (Fig.3 D). These data that Syk is activated upon recruitment to the plasma other kinases are to signal to MAPK or to activate JNK in a have recently that JNK activation by can be by expression of a dominant negative mutant of (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 that as a GEF for In of results and we whether expression of Vav proteins guanine nucleotide exchange on In this regard, the high activity of Rho, Rac1, and Cdc42 has the in cells of their GTP-bound forms C. 1996; 271: PubMed Scopus Google Scholar). Thus, for these we of a recently described technique that the of GDP to these small a to medium as an to their nucleotide exchange in we expressed in COS-7 cells Rac1, and Cdc42 H. Coso O.A. H. T. Miki T. Gutkind J.S. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, P. Gutkind J.S. Bustelo X.R. Nature. PubMed Scopus Google Scholar), together with expression vector a form of the domain of 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 (Fig. A). small GTP-binding proteins were efficiently expressed, as judged by with the Furthermore, when transfected cells were and then cultured for a of time in the of each small labeled as by thin layer chromatography analysis of these experimental labeled nucleotides were observed in cells and enhanced the of GDP to Ras, on the other small GTP-binding proteins left As a control, we used the prolonged incubation with 32Porthophosphate containing induced a increase in GTP-bound Ras (Fig. In contrast, under incubation time expression of did not affect Ras, but increased the of labeled GDP to Rac1 A). Collectively, these results that can guanine nucleotide exchange in on Rac1. identical experimental wild-type Vav Myr-Syk induced nucleotide exchange on Rac1 (Fig. which was with the of each alone to induce JNK activity (see However, when Myr-Syk was coexpressed with Vav, we observed a increase in the of labeled GDP into Rac1. These two JNK activation provoked by coexpression of Myr-Syk together with Vav or upon cross-linking of Tacγ when coexpressed with Syk and Vav and to effectively Vav in strongly suggest that tyrosine phosphorylation of Vav its GEF toward Rac1, leading to JNK activation. Consistent with this JNK stimulation induced by Tacγ cross-linking in and COS-7 cells was by the dominant negative mutant of Rac1, (Fig. C). Moreover, we have recently observed that tyrosine phosphorylation of purified Vav protein its GEF activity on expressed Rac1 when in in assays P. Gutkind J.S. Bustelo X.R. Nature. PubMed Scopus Google Scholar), the that Vav as a tyrosine GEF for Rac1. A number of GEFs for small GTP-binding proteins of the Rho family have been by of their in F. Nature. 1993; PubMed Scopus Google Scholar). the function of these as as the controlling their activity in their is still unknown. In this regard, findings provide evidence that whereas is wild-type Vav only guanine nucleotide exchange in Rac1 upon activation of an upstream tyrosine kinase, Syk, and that Vav in this are by tyrosine Thus, these findings define a novel signal transduction pathway involving a cell surface receptor a nonreceptor tyrosine kinase, which, in turn, phosphorylates a GEF in tyrosine thereby regulating its activity toward a small GTP-binding protein and promoting the activation of a kinase cascade. A of such a biochemical Syk, Vav, Rac1, and its downstream JNK, as as the pathway connecting Syk to MAPK is in findings might have the of other multimeric antigen As in mast cells evidence that the γ subunit of FcεRI signals Syk activation. The FcεRI γ chain is to the chain of the antigen cell and whereas subunits Syk, the cell receptor subunits with M. A. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar, M. A. 1994; PubMed Scopus Google Scholar). Furthermore, cell receptor and cell receptor activation both to Vav tyrosine phosphorylation X.R. Barbacid M. Nature. 1992; PubMed Scopus (245) Google Scholar, B. Nature. 1992; PubMed Scopus Google Scholar) and JNK activation B. E. M. T. M. Y. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar). upon it is that Vav plays a role in mast cells, cells, and cells, linking multimeric antigen receptors and their downstream nonreceptor tyrosine kinases to the Rac1-JNK signaling pathway. D. Klausner for Tacγ cDNA and Yamamura for the fragment of the porcine Syk