Tyrosine phosphorylation of the vav proto-oncogene product links FcepsilonRI to the Rac1-JNK pathway.

Stimulation of high affinity IgE Fc receptors (FcepsilonRI) in basophils and mast cells activates the tyrosine kinases Lyn and Syk and causes the tyrosine phosphorylation of phospholipase C-gamma, resulting in the Ca2+- and protein kinase C-dependent secretion of inflammatory mediators. Concomitantly, FcepsilonRI 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 FcepsilonRI to MAPK and JNK, we reconstructed their respective biochemical routes by expression of a chimeric interleukin-2 receptor alpha subunit (Tac)-FcepsilonRI gamma chain (Tacgamma) in COS-7 cells. Cross-linking of Tacgamma did not affect MAPK in COS-7 cells, but when coexpressed with the tyrosine kinase Syk, Tacgamma stimulation potently induced Syk and Shc tyrosine phosphorylation and MAPK activation. In contrast, Tacgamma did not signal JNK activation, even when coexpressed with Syk. Ectopic expression of a hematopoietic-specific guanine nucleotide exchange factor (GEF), Vav, reconstituted the Tacgamma-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 FcepsilonRI 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 (Fc⑀RI) 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 (1). One of the earliest events induced upon Fc⑀RI aggregation is the activation of the nonreceptor tyrosine kinases Lyn and Syk, and the tyrosine phosphorylation of cytoplasmic molecules, including phospholipase C-␥ (2). Phosphorylated phospholipase C-␥ hydrolyses phosphatidylinositol 4,5-bisphosphate and liberates inositol 1,4,5-trisphosphate and diacylglycerol, which mobilizes Ca 2ϩ from intracellular and extracellular sources and activates protein kinase C (3), respectively. Whereas these second-messenger generating systems appear to be sufficient for the Fc⑀RI-mediated secretory response (4), how signals initiated by Fc⑀RI 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 Fc⑀RI in mast cell lines, such as RBL-2H3 cells, leads to the activation of members of the mitogen-activated protein kinase (MAPK) 1 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 Fc⑀RI receptors in mast cell lines has been shown to result in the activation of MAPK and JNK (5,6). In this regard, recently available evidence suggests that engagement of Fc⑀RI 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 Fc⑀RI to MAPK (5). In contrast, how Fc⑀RI stimulation activates JNK is still unknown.
In this study, we thought to dissect the signaling pathway(s) linking Fc⑀RI 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 Fc⑀RI to the Ras-MAPK pathway, signaling from Fc⑀RI 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
Cell Stimulation-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 g/ml dinitrophenyl-coupled to human serum albumin were described previously (7). COS-7 Cell Transfection and Stimulation-Expression plasmids (1 g/plate) were transfected into subconfluent COS-7 cells by the DEAEdextran technique (8), adjusting the total amount of DNA to 5 g/plate with vector DNA (pcDNA3, Invitrogen) when necessary. Forty-eight hours later, cells were cultured overnight in DMEM containing 0.1% * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
FBS. Cells were then left unstimulated or stimulated with EGF (100 ng/ml). Stimulation with antibodies to Tac was performed using 5 g/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 g/ml of avidin (Sigma). After incubation for the times indicated, cells were lysed.
Immunoprecipitation, Immunoblotting, and in Vitro Kinase Assays-Cell lysis, immunoprecipitation, immunoblotting, MAPK, and JNK assays were performed as described previously (8). 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.
Expression Plasmids-Syk was cloned from a cDNA library prepared from purified human monocyte poly(A) ϩ 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 (8).
Subcellular Localization-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 (9). Each fraction was immunoprecipitated with antibodies to Src (Santa Cruz) and immunoblotted with antiserum to Syk (Santa Cruz) and antibody to Tyr(P) (ICN).

RESULTS AND DISCUSSION
To begin dissecting the signaling pathway(s) linking Fc⑀RI to MAPK and JNK, we initially studied the temporal relationship between MAPK and JNK activation in RBL-2H3 cells. As expected, engagement of Fc⑀RI by addition of dinitrophenyl (DNP) coupled to human serum albumin to anti-TNP IgEprimed 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, Fc⑀RI 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 (10). Consistent with that, we observed that in RBL-2H3 cells Fc⑀RI 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, C and D).
Fc⑀RI is a multimeric receptor containing a single ␣ and ␤ subunit and a homodimer of ␥ subunits (11). Both ␤ and ␥ chains exhibit a structural motif termed ITAM, for immunoreceptor tyrosine-based activation motif (12), which participate in the recruitment of cytoplasmic tyrosine kinases and in the consequent tyrosine phosphorylation of their downstream targets (1). Studies with chimeric molecules containing the extracellular and transmembrane domains of the interleukin-2 receptor ␣ subunit (Tac) fused to the cytosolic domain of ␤ (Tac␤) and ␥ (Tac␥) chains of Fc⑀RI have helped simplify the analysis of early signaling events provoked by Fc⑀RI activation (13). When expressed in RBL-2H3 cells, cross-linking of the Tac␥ chimera is sufficient to mimic the majority of the biochemical and biological responses triggered by Fc⑀RI stimulation. In contrast, cross-linking of Tac␤ does not appear to elicit signaling responses (13). Therefore, to investigate whether activation of Tac␥ is sufficient to activate Syk, both were expressed in COS-7 cells, which lack endogenous Fc⑀RI or Syk (see below). Transfected Tac␥ was efficiently expressed, as judged by immunofluorescence labeling techniques (data not shown). Crosslinking of Tac␥ chimeras with biotinylated anti-Tac antibodies followed by streptavidin induced the rapid tyrosine phosphorylation of a coexpressed epitope-tagged Syk ( Fig. 2A). When coexpressed with an epitope-tagged form of Shc, cross-linking of Tac␥ induced only a limited increase in Shc tyrosine phosphorylation (Fig. 2B). However, when Syk was coexpressed, Tac␥ engagement provoked a rapid and substantial increase in Shc tyrosine phosphorylation (Fig. 2B). Paralleling Shc phosphorylation, cross-linking of Tac␥ induced a very poor MAPK response, but when coexpressed with Syk, Tac␥ potently elevated the phosphorylating activity of MAPK to an extent comparable with that elicited in response to EGF (Fig. 2C). Taken together, these results support a central role for the ␥ subunit of Fc⑀RI and Syk in signaling from IgE receptors to the MAPK pathway. Surprisingly, however, cross-linking of Tac␥ 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. 2C). Collectively, these data established that coexpression of Tac␥ and Syk in COS-7 cells is sufficient to reconstitute the MAPK response to Fc⑀RI stimulation, while suggesting that additional molecules not endogenously expressed in COS-7 cells were necessary to link Fc⑀RI 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 (8). 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 (14). 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 (5) product (Vav) (Fig. 3A), which is preferentially expressed in cells of the hematopoietic lineage. Moreover, Vav exhibits structural motifs frequently found in GEFs for small GTPbinding proteins of the Ras and Rho families (14), 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 (15). These results prompted us to explore the possibility that wild-type Vav serves as a link between Fc⑀RI and the Rac-1-JNK pathway.
Expression of Vav alone (15) or together with the Tac␥ chimera failed to induce JNK activation (Fig. 3B), and crosslinking of Tac␥ failed to induce Vav tyrosine phosphorylation when coexpressed in COS-7 cells (Fig. 3B). However, when Tac␥, Syk and Vav were each simultaneously coexpressed in these cells, Tac␥ 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 Fc⑀RI/Syk to JNK.
We next asked whether recruitment of Syk to the plasma membrane upon aggregation of Fc⑀RI or cross-linking of Tac␥ chimeric molecules is the determining step initiating activity of Syk downstream signaling pathways. To that end, we exam-ined the ability of a membrane-targeted form of Syk to bypass the requirement of Tac␥ engagement for signaling to the MAPK and JNK pathway. A chimeric protein containing the NH 2 -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. 16 and data not shown). Furthermore, this membrane-targeted form of Syk was heavily tyrosine-phosphorylated (Fig. 3C), and its expression was sufficient to elevate the activity of a cotransfected epitopetagged MAPK (Fig. 3D). 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. 3D). These data indicate that once Syk is activated upon recruitment to the plasma mem-

FIG. 2. Cross-linking of Tac␥ induces MAPK activation and phosphorylation of Syk and Shc in COS-7 cells. A, COS-7 cells
were transfected with pcDNA3 Tac␥ and pcDNA3 HA-Syk, as indicated. The cells were lysed, HA-Syk was immunoprecipitated (IP) with antibodies to HA and analyzed by immunoblotting with antibodies to phosphotyrosine (Anti-pTyr) and antiserum to Syk. B, COS-7 cells were transfected with pcDNA3 Tac␥, pcDNA3 HA Shc, and pcDNA3 Syk or pcDNA3 alone (vector), as indicated. Cell lysates were immunoprecipitated with antibodies to HA and analyzed by immunoblotting with antibodies to phosphotyrosine or to HA. Total cell lysates were immunoblotted with antiserum to Syk. C, COS-7 cells were transfected with pcDNA3-HA-MAPK or pcDNA3-HA-JNK for, respectively, MAPK and JNK assays, together with pcDNA3 alone, pcDNA3 Tac␥, or pcDNA3 Tac␥ ϩ pcDNA3 Syk, as indicated. COS-7 cells were left unstimulated (0 min) or treated with IgE ϩ DNP or anti-Tac. MAPK and JNK activity was assayed in cellular lysates. These experiments were repeated five times with similar results.

FIG. 3. Phosphorylation of Vav in RBL-2H3 cells and COS-7 cells.
A, RBL-2H3 cells were left unstimulated (0 min) or treated with IgE ϩ DNP. Cells were lysed, and Vav was immunoprecipitated with antiserum to Vav and analyzed by immunoblotting with antibodies to phosphotyrosine (Anti-pTyr) or with antiserum to Vav. B, phosphorylation of Vav and activation of JNK in COS-7 cells. COS-7 cells were transfected with pcDNA3-HA-JNK and pcDNA3, pcDNA3 Tac␥, pcDNA3 Tac␥ ϩ pcDNA3 Syk, or pcDNA3 Tac␥ ϩ pcDNA3 Syk ϩ Vav, as indicated. Cells were left unstimulated (0 min) or treated with antibodies to Tac for 15 min, as depicted. COS-7 cells were lysed, and Vav was immunoprecipitated with antiserum to Vav and analyzed by immunoblotting with antibodies to phosphotyrosine (Anti-pTyr) or antiserum to Vav. Total cell lysates were analyzed by immunoblotting with antiserum to Syk. JNK assays were performed. C, localization and phosphorylation of Myr-Syk in COS-7 cells. Total cellular extracts were immunoprecipitated with antiserum to Src (anti-Myr) and immunoblotted with antiserum to Syk and antibodies to phosphotyrosine. D, activation of MAPK and JNK by overexpression of Myr-Syk. COS-7 cells were transfected with pcDNA3 expression vector carrying cDNAs for the wild-type or myristoylated forms of Syk together with pcDNA3-HA-MAPK or pcDNA3-HA-JNK, as indicated, and cells were processed as above. The experiments were repeated three times with identical results. brane, no other Fc⑀RI-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 (15), 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 Rac1 in 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 (17). Thus, for these experiments we took advantage of a recently described technique that uses the levels of 32 P-labeled GDP bound to these small GTPases after a brief exposure to [ 32 P]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 (18,19), together with empty expression vector (control), a membrane-targeted form of the catalytic domain of Sos (Myr-Sos) (8), or Onco-Vav (Fig. 4A). 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 [ 32 P]orthophosphate, each small GTPase incorporated labeled GDP, as determined by thin layer chromatography analysis of anti-AU5 immunoprecipitates. Under these experimental con-ditions, 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 GTPbinding proteins (Fig. 4A, left panel). As a control, we used the standard, more prolonged incubation with [ 32 P]orthophosphate containing medium. Under those conditions, Myr-Sos induced a dramatic increase in GTP-bound Ras (Fig. 4A, 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. 4A). 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.  4B), 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 Tac␥ when coexpressed with Syk and Vav and 2) Syk's ability to effectively tyrosine-phosphorylate Vav in vivo, strongly suggest that Sykinduced tyrosine phosphorylation of Vav increases its GEF toward Rac1, leading to JNK activation. Consistent with this conclusion, JNK stimulation induced by Tac␥ cross-linking in Tac␥-, Syk-, and Vav-transfected COS-7 cells was blocked by the dominant negative mutant of Rac1, N17 rac1 (Fig. 4C). 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 (19), 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 (20). 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 transduc-  Tac (15 min). COS-7 cells were lysed, and JNK assays were performed as described (11). Data represent the average Ϯ S.E. of three independent experiments, expressed as fold increase in JNK activity with respect to vector transfected cells (vector).
FIG. 5. The pathway linking Fc⑀RI to MAPK and JNK. Schematic representation of molecules participating in the signaling pathway leading to the activation of MAPK and JNK. We postulate that Syk acts on Shc, Grb2, and Sos to induce guanine nucleotide exchange on Ras leading to MAPK activation, and on Vav to induce guanine nucleotide exchange on Rac1, thus activating the JNK pathway.
tion 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, Fc⑀RI, 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 ␥ subunit of Fc⑀RI signals Syk activation. The Fc⑀RI ␥ chain is functionally analogous to the chain of the antigen T cell receptor, and whereas Fc⑀RI␥ subunits recruit Syk, the T cell receptor subunits interact with Zap70 (21,22). Furthermore, T cell receptor and B cell receptor activation both lead to Vav tyrosine phosphorylation (23,24) and JNK activation (25). 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.