Syk-dependent phosphorylation of Shc. A potential link between FcepsilonRI and the Ras/mitogen-activated protein kinase signaling pathway through SOS and Grb2.

Antigen receptors on T- and B-cells activate Ras through a signaling pathway that results in the tyrosine phosphorylation of Shc and the formation of a complex of Shc with the Grb2 adaptor protein. The high affinity receptor for immunoglobulin E (FcεRI) in cultured mast (RBL-2H3) cells has been reported to function differently. Here we show to the contrary that engagement of FcεRI with antigen leads to increased tyrosine phosphorylation of Shc and the association of Shc with Grb2 and other proteins (p120 and p140). Like the FcεRI-mediated activation of the mitogen-activated protein kinase cascade, these responses are dependent on the tyrosine kinase Syk; they are enhanced by overexpression of Syk and are blocked by expression of dominant-negative Syk. Sos is constitutively associated with Grb2 in these cells but dissociates from Shc on stimulation with antigen. These reactions are rapid, reversible, and associated with the activation of Ras. Therefore, the Syk-dependent tyrosine phosphorylation of Shc and its association with Grb2 may provide a pathway through Sos for activation of Ras by FcεRI.

Tyrosine kinase-dependent receptors, such as the EGF 1 receptor, utilize an intermediate protein, Shc, the adaptor protein, Grb2, and the guanine nucleotide exchange factor, Sos, to convert Ras (p21 ras ) to its active GTP-bound state (1)(2)(3)(4) which, in turn, initiates signals for cell growth and differentiation via MAP kinase and other pathways (5). Two scenarios are possible. In one, Sos, which is constitutively bound to the SH3 domain of Grb2 (6 -9), is translocated to the membrane by binding of the SH2 domains of Grb2 to tyrosine-phosphorylated motifs of activated EGF receptors (6,10). This translocation does not enhance the exchange activity of Sos (7,11), but it is thought, instead, to allow Sos to interact with membrane-bound Ras because Ras can be activated by fusing Sos directly with the plasma membrane (12,13).
In the second scenario, the Grb2⅐Sos complex binds to receptor-bound phosphorylated Shc (13,14). Shc is tyrosine-phosphorylated after binding, via its SH2 domain, to the phosphorylated motifs of the activated EGF receptor (1). The formation of the Shc⅐Grb2⅐Sos complex correlates with the formation of the GTP-bound state of Ras (15), but other reactions take place. These include formation of ternary complexes of phosphorylated Shc-Grb2 with other proteins, namely p120 and p140 (16,17) and the phosphorylation of Sos by protein kinase C (18) and MAP kinase (19,20) with an associated retardation in the electrophoretic mobility of Sos (20,21). It is unclear whether the above two scenarios provide alternate or redundant mechanisms for activation of Ras.
The multimeric B-cell and T-cell antigen receptors appear to utilize similar mechanisms for the sustained activation of the p21 ras /MAP kinase signaling pathway. These receptors, unlike growth factor receptors, recruit the cytosolic tyrosine kinases, Src and Syk/ZAP70, which bind to tyrosine-phosphorylated motifs (ITAMs) that are present in the cytosolic tail of various subunits of these receptors (22). The subsequent activation of these kinases results in tyrosine phosphorylation of cellular proteins including Shc, formation of phosphorylated Shc⅐Grb2⅐Sos complexes (23,24), and the activation of the MAP kinase pathway via Ras (4).
Analogous to the T-and B-cell antigen receptors, antigeninduced aggregation of the mast cell Fc⑀RI, the multimeric high affinity receptor for IgE, leads to tyrosine kinase-dependent activation of the MAP kinase pathway (25), but the pathways leading to the activation of Ras and the activation of Ras itself have not been established. The ␤ and ␥ chains of Fc⑀RI contain ITAMs (22) that allow recruitment of the tyrosine kinases, Lyn and Syk (26 -32), and the tyrosine phosphorylation of various proteins (33)(34)(35), including Fc⑀RI ␤ and ␥ chains (28,30,31,36) and Vav (25) which has been implicated in the activation of Ras in B-and T-cells (37,38). Expression of porcine Syk, or a dominant-negative truncated Syk (Syk-T) that lacks the kinase domain (25,32), in a vaccinia expression system, has indicated that Syk is essential for activation of the MAP kinase-phospholipase A 2 pathway in cultured RBL-2H3 mast cells (25). This activation is apparent from an increase in MAP kinase activity (25,39,40) and the tyrosine phosphorylation and shift in electrophoretic migration of p42 mapk (41).
As reported here, we have established that Ras and the Shc-Grb2-Sos pathway are activated in RBL-2H3 cells via Fc⑀RI and that the constitutive phosphorylation of Shc, as noted by others (17), could be minimized by reducing the serum content of the medium. Furthermore, overexpression of active or dominant negative forms of Syk demonstrate that tyrosine * 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 1 The abbreviations used are: EGF, epidermal growth factor; ITAM, immunoreceptor tyrosine-based activation motif (22); Fc⑀RI, high affinity receptor for IgE; DNP-HSA, dinitrophenylated human serum albumin; DNP-lysine, ⑀-dinitrophenyl-lysine; Syk-T, truncated Syk that lacks the kinase domain (25,32); MAP, mitogen-activated protein; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol. phosphorylation of Shc and its association with Grb2 and the proteins p120 (possibly c-Cbl) and p140 are dependent on Syk. The studies provide the first definitive indication of communication between Fc⑀RI and the Ras/MAP kinase cascade through Shc, Grb2, and Sos via Syk.
Cell Culture and Infection with Vaccinia Expression System-RBL-2H3 cells were maintained in monolayer culture in minimal essential medium supplemented with Earle's salts, 15% heat-inactivated fetal bovine serum, and 1% L-glutamine at 37°C in 5% CO 2 . Recombinant vaccinia viruses were prepared, and infection of RBL-2H3 cells was performed as described previously (25,32). Cultures were incubated with wild type, Syk or Syk-T recombinant viruses (7-10 plaque-forming units/cell) in minimal essential medium supplemented with Earle's salts with 2.5% fetal bovine serum and 1 g/ml anti-DNP IgE (to achieve 100% occupancy of Fc⑀RI) for 12 h. The cells were then detached with 1 mM EDTA in phosphate-buffered saline (pH 7.4), washed twice in DMEM that contained 0.1% BSA and 25 mM HEPES (DMEM/BSA medium), and then suspended in the same medium. Immunoprecipitations, Electrophoresis, and Immunoblotting-Protein A-Sepharose beads, coated with the indicated antibodies, were washed three times with the above lysis buffer and incubated with the cell lysate for 2-14 h. The beads were then washed five times for SDS-PAGE. Where stated, anti-phosphotyrosine immunoprecipitates were first eluted from the beads with 10 mM phenyl phosphate in lysis buffer at 4°C for 15 min; the eluted proteins were reprecipitated with rabbit anti-Shc antibody. Proteins were separated by SDS-PAGE in 8 -16% gradient mini-gels or 12.5 and 10% large format gels as described elsewhere (32). Proteins were transferred by Western technique onto Immobilon polyvinylidene difluoride membranes (Millipore, Bedford, MA) and probed with the indicated antibodies. For anti-phosphotyrosine blots, blots were stripped and reprobed with anti-Shc antibody to confirm identity and equal loading of Shc. Immunoblotting was performed according to the protocols supplied with the Enhanced Chemiluminescent detection kit. Radioactive proteins were visualized by autoradiography.

RESULTS AND DISCUSSION
Activation of Ras, Tyrosine Phosphorylation of Shc, and Association of Grb2 with Phosphorylated Shc and Sos-As shown in Fig. 1, stimulation of RBL-2H3 cells for 1 min with the antigen, DNP-BSA, resulted in an increase in GTP-bound Ras (panel A). This increase was apparent within 15 s, reached a maximum by 1 min, and was sustained for at least 10 min (panel B).
Studies with [ 32 P]orthophosphate-labeled cells and immunoprecipitation with anti-phosphotyrosine or anti-Shc antibodies ( Fig. 2A) revealed that stimulation of cells for 1 min also resulted in tyrosine phosphorylation of numerous proteins (compare lane 2 with lane 1, Fig. 2A) and a substantial increase in the state of phosphorylation of Shc (shown in Fig. 2A, lanes  5 and 6). In this and subsequent experiments including Western blots (data not shown), Shc migrated as two bands which by size corresponded to p46 shc and p52 shc (p66 shc was not detected) (1,44). Of these two forms, p52 shc was most heavily phosphorylated ( Fig. 2A, lane 6). Increased phosphorylation of two additional proteins (estimated sizes, 120 and 140 kDa), which co-immunoprecipitated with Shc, was also apparent (see Fig.   2A, lanes 4 and 5). Subsequent addition of excess monovalent hapten, DNP-lysine, caused rapid reversal in the state of phosphorylation of Shc (Fig. 2B) and p120 and p140 (not shown).
Consistent with the 32 P-labeling experiments, the stimulation with antigen resulted in increased tyrosine phosphorylation of Shc as indicated by anti-phosphotyrosine blots of the Shc immunoprecipitates (Fig. 2C). In this and four other experiments, p52 shc was prodominantly tyrosine-phosphorylated. In general the tyrosine phosphorylation of p46 shc was weaker but variable. Reprobing of blots with anti-Shc antibody confirmed the identity of Shc and that the total amount of Shc protein was unchanged. Immunoblotting with anti-Grb2 antibody also revealed an increased association with Grb2 with Shc (Fig. 2D).
Grb2 also co-immunoprecipitated with Sos, but this association was apparent in both unstimulated and stimulated cells (Fig. 2E). Although the association of Sos with Grb2 was enhanced in some experiments, in others the enhancement was minimal (as in Fig. 2E). Sos was also constitutively associated with Shc, as indicated by co-immunoprecipitation of Sos with Shc, but appeared to dissociate from Shc in stimulated cells (compare stimulated cells, lane 2, with unstimulated cells, lanes 1 and 3, in Fig. 2F). A small shift in migration of Sos was also apparent in these gels. A possible explanation would be the phosphorylation of Sos by either protein kinase C or MAP kinase (see Introduction).
Attempts were made to identify the p120 and p140 proteins. A likely candidate for p120 was the proto-oncogene product, c-Cbl, which was shown to be a substrate for receptor-activated tyrosine kinases in B-and T-cells and to associate with the SH3 domain of Grb2 and other proteins (45)(46)(47). In the Shc immunoprecipitates, phosphorylated p120 was found to co-migrate with c-Cbl. Immunoprecipitation of c-Cbl indicated that it was tyrosine-phosphorylated upon cell stimulation and that it coprecipitated tyrosine-phosphorylated p140 (data not shown). Thus, c-Cbl might associate with both Shc and p140; the identity of p140 and the relevance of this association are under investigation.
Dependence of Events on Syk as Demonstrated by Expression of Syk-T and Porcine Syk-In cells infected with wild type vaccinia virus and then labeled with [ 32 P]orthophosphate, 32 Pphosphorylation of p56 shc was still apparent in antigen-stimulated cells (Fig. 3A, compare lane 2 with 1). This phosphorylation was enhanced in cells that were infected with Sykrecombinant virus (Fig. 3A, lanes 3 and 4) but blocked in cells infected with Syk-T-recombinant virus (Fig. 3A, lanes 5 and 6). In a matching experiment both p46 shc and p52 shc were tyrosinephosphorylated in cells infected with either wild type or Sykrecombinant virus (Fig. 3B, lanes 1-4), but this phosphorylation was abrogated in the Syk-T-infected cells as was the tyrosine phosphorylation of p120 and p140 (Fig. 3B, lanes 5 and  6). In these two experiments there was a disparity in the extent of 32 P labeling and tyrosine phosphorylation of p46 shc which could be indicative of the phosphorylation of p52 shc by protein kinase C which was reported to enhance the interaction of p52 shc with cytosolic tyrosine phosphatase (48). The increased association of Shc with Grb2 was still observed in wild type infected cells, was enhanced in Syk-infected cells, and was blocked in Syk-T-infected cells (Fig. 3C).
In contrast to the above results, the small amounts of Grb2 that were constitutively associated with Shc (i.e. unstimulated cells in (Fig. 3C, lanes 1, 3, and 5) were unaffected by any of the expression systems. A small increase in the association of Grb2 with Sos was noted in Syk-transfected cells after antigen stimulation (Fig. 3D, lane 4). In general, however, the association of Sos with Grb2 was minimally influenced by antigen stimula- Implications-The above results provide the first demonstration that engagement of Fc⑀RI by antigen in RBL-2H3 mast cells leads to activation of Ras and the apparent association of all components of Shc-Grb2-Sos pathway which precedes activation of Ras. Activation of this pathway is dependent on Syk (this paper) as is the activation of phospholipase A 2 through MAP kinase (25). Therefore, the Shc-Grb2-Sos pathway may provide one mechanism for the activation of Ras by Fc⑀RI and, in turn, the activation of the MAP kinase-phospholipase A 2 pathway by the interaction of Ras with Raf1 (49). We cannot exclude the possibility, however, that Vav may provide an alternate pathway (25).
As reported previously (17), our initial studies showed that Shc was tyrosine-phosphorylated constitutively when RBL-2H3 cells were maintained in 15% serum (data not shown). This constitutive phosphorylation was minimized by exposing cells to low serum concentrations (2.5%), and, as demonstrated here, the serum deprivation unmasked the previously undetected antigen-induced tyrosine phosphorylation of Shc. The linkage between Fc⑀RI and Shc phosphorylation was further confirmed by the rapid dephosphorylation of Shc when antigen was displaced by excess DNP-lysine (Fig. 2B). In addition, we included additional elution and immunoprecipitation steps which further enhanced the specificity of the isolation procedure for phosphorylated Shc (see "Materials and Methods").
The intent of this investigation was to determine whether or not Shc-Grb2-Sos pathway was activated by Fc⑀RI because of the previously noted analogies between Fc⑀RI and the T-and B-cell antigen receptors. There were, however, two incidental findings that may have general relevance. These were the apparent association of Shc with the proteins, p120 and p140, and the apparent modification of Sos (i.e. retardation in electrophoretic migration) and its dissociation from Shc. It would appear that p120 and p140 are engaged by receptors that possess intrinsic tyrosine kinase activity, such as the EGF receptor (16), or recruit tyrosine kinases, such as Fc⑀RI (this paper). The dissociation of Sos from Shc might be due to phosphorylation of Sos by protein kinase C or MAP kinase. As noted earlier, phosphorylation by either enzyme is associated with a retardation in electrophoretic migration of Sos. Phosphorylation by protein kinase C is thought to represent a step in the cycle of activation and inactivation of Sos (18) or, in the case of phosphorylation by MAP kinase, to promote dissociation of the Sos⅐Grb2 complex from tyrosine-phosphorylated peptides (20). These considerations suggest a variation of the second scenario that was discussed earlier in that tyrosine phosphorylation of Shc promotes formation of a ternary complex with pre-existing Grb2⅐Sos complexes and subsequent phosphorylation of Sos by protein kinase C promotes dissociation of this complex. Further investigation of these phenomena is clearly warranted, but they illustrate features shared by receptors that utilize intrinsic or extrinsic tyrosine kinase activities for initiating stimulatory signals.