SLP-76 is a substrate of the high affinity IgE receptor-stimulated protein tyrosine kinases in rat basophilic leukemia cells.

Stimulation of the IgE high affinity receptor on rat basophilic leukemia RBL-2H3 cells results in activation of protein tyrosine kinases and rapid tyrosine phosphorylation of several substrates, many of which remain unidentified. In this report, we demonstrate that the Grb2 adapter protein, when expressed as a glutathione S-transferase fusion protein, associates with four tyrosine-phosphorylated molecules (116, 76, 36, and 31 kDa) from lysates of stimulated RBL-2H3 cells. We show further that the 76-kDa protein is SLP-76, a hematopoietic cell-specific protein first identified as a Grb2-binding protein in T cells. Upon stimulation of the high affinity receptor for IgE, SLP-76 undergoes rapid tyrosine phosphorylation and associates with two additional tyrosine phosphoproteins of 62 and 130 kDa via the SH2 domain of SLP-76. Additional studies demonstrate that the SLP-76 SH2 domain also binds a protein kinase from stimulated RBL-2H3 cell lysates. Furthermore, the phosphorylation of SLP-76 requires Syk activity but is not dependent on Ca+2 mobilization. These data, together with our previous work documenting its role in T-cell activation, suggest that SLP-76 and the proteins with which it associates may play a fundamental role in coupling signaling events in multiple cell types in the immune system.

The high affinity receptor for IgE (Fc⑀RI), 1 found on basophils and mast cells, mediates cell activation in type I allergic reactions (1). Fc⑀RI is a member of a family of receptors expressed on immune effector cells that lack intrinsic enzymatic activity but possess cytoplasmic immune receptor tyrosinebased activation motifs that bind cytoplasmic protein tyrosine kinases (PTKs) (2,3). Similar to other immune cell receptors (such as the T-cell antigen receptor (TCR) or B-cell antigen receptor), Fc⑀RI is composed of multiple subunits including a ligand binding component (␣ chain) noncovalently associated with transmembrane proteins that bear the immune receptor tyrosine-based activation motifs (␤ chain and a homodimer of disulfide-linked ␥ chains) (4,5). The ␤ and ␥ cytoplasmic domains are responsible for signal transduction (6,7). Chimeric receptors containing the cytoplasmic domains and an unrelated extracellular domain mimic most of the cellular signaling events triggered by the intact Fc⑀RI in the rat basophilic leukemia cell line RBL-2H3 (7). Furthermore, stimulation of a chimeric receptor containing the cytoplasmic domain of the TCR chain also results in rat basophilic leukemia cell activation, (8) suggesting a conservation of signaling properties between T cells and mast cells.
The interaction of IgE-bound antigens with the Fc⑀RI initiates intracellular signaling events leading to the generation of inflammatory mediators and cytokines. Proximal biochemical events include the rapid phosphorylation and activation of several PTKs including Lyn (a src family kinase) (9), p72-Syk (10,11), and focal adhesion kinase (12)(13)(14). Lyn associates constitutively with the Fc⑀RI, whereas the interaction between Syk and the ␤ and ␥ subunits requires tyrosine phosphorylation of the immune receptor tyrosine-based activation motifs after receptor ligation (15,16). In addition, the phosphorylated ␤ and ␥ subunits precipitate Shc, Grb2, and phospholipase C-␥1 (PLC␥1) from RBL-2H3 cell lysates (17).
SLP-76, a novel hematopoietic cell-specific protein, was identified as a protein that associates with the Grb2 adapter protein and becomes phosphorylated after TCR ligation (36,37). SLP-76 has several potential tyrosine phosphorylation sites within its amino terminus (38), a central region rich in proline residues that mediates the Grb2 association (39), and a carboxyl-terminal SH2 domain that binds to at least two tyrosinephosphorylated proteins and a serine/threonine kinase after TCR ligation (39). SLP-76 seems to play a key role in T-cell activation because its overexpression results in dramatically enhanced TCR-mediated induction of nuclear factor of activated T-cells and interleukin 2 promoter activity (39). It seemed likely that SLP-76 also plays a role in signaling through other immune receptor tyrosine-based activation mo-tif-containing receptors including the Fc⑀RI. Here we report that SLP-76, as well as phosphotyrosine-containing proteins of 31, 36, and 116 kDa, associate with Grb2 in the mast cell analog RBL-2H3 after Fc⑀RI ligation. SLP-76 becomes rapidly tyrosine-phosphorylated after Fc⑀RI engagement and remains phosphorylated for over 1 h. Furthermore, SLP-76 phosphorylation requires Syk activity but is not dependent on extracellular Ca ϩ2 . Stimulation of RBL-2H3 cells also results in the phosphorylation of several other proteins and their association with the SLP-76 SH2 domain. In addition, the SLP-76 SH2 domain binds a kinase in stimulated RBL-2H3 cells. Together these data suggest that SLP-76 functions in several cell types to couple signaling pathways leading to immune cell effector function.

RESULTS AND DISCUSSION
Association of Phosphotyrosine-containing Proteins with GST-Grb2 in Stimulated RBL-2H3 Cell Lysates-Ligation of the Fc⑀RI on RBL-2H3 cells leads to rapid tyrosine phosphorylation of numerous proteins and subsequent activation of the Ras/mitogen-activated protein kinase pathway. Because the Grb2 adapter protein (42) links proximal PTK activation with other signaling pathways in several systems, we examined the tyrosine phosphoproteins that inducibly associate with Grb2 in RBL-2H3 cells. Cells were left unstimulated or stimulated with anti-Fc⑀RI mAb for 10 min. Lysates were incubated with GST/ Grb2 fusion protein, and protein complexes were subjected to anti-phosphotyrosine Western blotting. As shown in Fig. 1, a full-length GST/Grb2 fusion protein associates with tyrosine phosphoproteins of 116, 76, 36, and 31 kDa from Fc⑀RI-stimulated cells.
Our results are similar to those of Turner et al. (43), who reported the association of 110 -120-, 76-, and 31-33-kDa phosphoproteins with a Grb2 fusion protein. These investigators also demonstrated an association between Grb2 and tyrosinephosphorylated Shc in resting and Fc⑀RI-stimulated RBL-2H3 cells. Although we detected tyrosine-phosphorylated Shc in both unstimulated and stimulated RBL-2H3 cells, we did not find evidence for an association between Shc and our GST-Grb2 fusion proteins in numerous in vitro assays.
Three of the Grb2-associated proteins we detected from RBL-2H3 lysates (pp116, pp76, and pp36) seem similar to phosphotyrosine-containing proteins from stimulated Jurkat T-cell lysates (37,44). In T cells, pp116 has been identified as c-cbl (45), an adapter protein with an unknown function; pp76 is SLP-76, a hematopoietic cell-specific protein (36) important in T-cell activation (39); and pp36 is a substrate of the TCR-stimulated PTKs whose binding to PLC␥1 (46) may be critical for its activity (47). pp36 may be lnk, a phosphotyrosine-containing protein recently cloned from rat lymph node (48).
A GST fusion protein containing the SH2 domain of Grb2 was used to further characterize the Grb2-binding proteins from RBL-2H3 lysates. The SH2 fusion protein bound only pp31 and pp36 (Fig. 1), suggesting that pp76 and pp116 bind to the Grb2 SH3 domains. It is likely that the interaction between pp31 and pp36 with Grb2 is phosphotyrosine-dependent because mutation of the predicted phosphate binding arginine residue of the Grb2 SH2 domain to lysine (GST/Grb2R86K) completely abrogates binding of pp36 and pp31 (Fig. 1).
SLP-76 Is Expressed in RBL-2H3 Cells and Is Phosphorylated after Ligation of the Fc⑀RI-The Grb2/phosphoprotein as- sociations suggested that SLP-76 was expressed and phosphorylated in RBL-2H3 cells. To address this possibility, lysates from unstimulated and stimulated cells were subjected to immunoprecipitation and immunoblot analysis with anti-SLP-76 antiserum. As shown in Fig. 2, SLP-76 is expressed in RBL-2H3 cells and undergoes an increase in tyrosine phosphorylation within 30 s of Fc⑀RI ligation. Phosphorylation peaks by 1 min and remains high after 30 min of stimulation. We did not observe a decrease in SLP-76 phosphorylation up to 60 min after stimulation (data not shown). The electrophoretic mobility of SLP-76 from RBL-2H3 cells is faster than that of SLP-76 isolated from Jurkat T cells. This may be due to differences in the posttranslational modification of SLP-76 or to species variation. We are currently investigating the sites of SLP-76 phosphorylation in RBL-2H3 and Jurkat cells to address this possibility.
SLP-76 co-immunoprecipitates with two additional tyrosinephosphorylated proteins migrating at ϳ62 and ϳ130 kDa after RBL-2H3 cell stimulation (Fig. 2). Similar SLP-76 protein associations are seen in activated Jurkat cells (Fig. 2) (38, 39); however, pp130 migrates as a doublet in RBL-2H3 cells but as a single band in Jurkat cells. The reason for this difference is not yet clear.
The Interaction between SLP-76 and pp130 and pp62 Is Mediated by the SH2 Domain of SLP-76 -We speculated that the association between SLP-76 and pp62 and pp130 may be mediated by an interaction of the SH2 domain of SLP-76 with tyrosine-phosphorylated residues of the other molecules. To address this possibility, GST fusion proteins containing either the wild type SLP-76 SH2 domain or the SLP-76 SH2 domain with a point mutation of a key arginine required for phosphotyrosine binding (R448K) were incubated with lysates from stimulated RBL-2H3 cells. Associated proteins were resolved by SDS-PAGE and subjected to immunoblot analysis with antiphosphotyrosine mAb (Fig. 3). Wild type GST/SLP-76/SH2 precipitated both pp130 and pp62 from activated lysates, whereas GST/SLP-76/SH2R448K failed to associate with either molecule. Thus, it seems that both pp130 and pp62 interact with SLP-76 through its SH2 domain. We are currently pursuing the identity of pp62 and pp130.
The possibility of other proteins binding to the SH2 domain of SLP-76 was addressed by performing an in vitro kinase assay on protein complexes associating with the GST SLP-76 fusion proteins. As shown in Fig. 4, the appearance of a 100-kDa phosphoprotein suggests that the GST/SLP-76/SH2 fusion protein associates with a protein kinase in Fc⑀RI-stimulated, but not unstimulated, RBL-2H3 cell lysates. No kinase activity precipitates with the GST/SLP-76/SH2R448K fusion protein.
The 100-kDa phosphoprotein seems to be similar to a SLP-76 SH2-associated molecule we found to be phosphorylated on phosphoserine and phosphothreonine, but not phosphotyrosine residues in stimulated Jurkat T cells (39). This analysis is supported by the lack of a 100-kDa molecule in anti-phosphotyrosine Western blot analysis of SLP-76 immunoprecipitations of Jurkat and RBL-2H3 cell lysates (Figs. 2 and 3). These results suggest that a serine/threonine kinase associates either directly or indirectly with the SLP-76 SH2 domain after recep- tor stimulation. Attempts are currently underway to identify the relevant kinase in both T cells and rat basophilic leukemia cells.
Phosphorylation of SLP-76 Requires Syk Activity but Is Not Dependent on Extracellular Ca 2ϩ -We next performed experiments to determine the requirements for SLP-76 tyrosine phosphorylation in RBL-2H3 cells after Fc⑀RI engagement. Tyrosine phosphorylation of many substrates in RBL-2H3 cells requires expression and activation of the proximal signaling kinase Syk (16,47). To test whether SLP-76 phosphorylation depends on Syk, we examined SLP-76 after receptor ligation in a Syk-deficient RBL-2H3 variant (TB1A2) (49). Wild type RBL-2H3 cells, the Syk-deficient mutant, and a Syk-reconstituted cell line (3A1) derived from TB1A2 cells were left unstimulated or were stimulated for 10 min with BC4. Cell lysates were subjected to immunoprecipitation with anti-SLP-76 antiserum and immunoblotted with anti-phosphotyrosine and anti-SLP-76 antibodies. Stimulation of the Fc⑀RI on wild type and Syk-reconstituted cells resulted in SLP-76 phosphorylation, whereas no phosphorylation was detected in the Syk-deficient mutant (Fig. 5A). However, each cell expressed similar amounts of SLP-76 protein (Fig. 5B). These data suggest that SLP-76 phosphorylation is dependent on prior activation of the Syk PTK.
Mast cell activation after Fc⑀RI engagement depends upon several downstream signaling pathways including the activation of PLC␥1 and the release of intracellular calcium. A sustained increase in intracellular Ca 2ϩ is required for the tyrosine phosphorylation of some substrates (such as focal adhesion kinase) but not others (such as the Fc⑀RI) (14). We tested the dependence of SLP-76 phosphorylation on sustained increases in Ca 2ϩ concentrations by depleting Ca 2ϩ with EGTA treatment followed by receptor stimulation in Ca 2ϩ -free medium. Cell lysates were subjected to SLP-76 immunoprecipitation and immunoblot analysis with anti-phosphotyrosine antibody (Fig.   6). As shown, Fc⑀RI stimulation leads to tyrosine phosphorylation of SLP-76 in the absence of extracellular Ca 2ϩ , demonstrating that SLP-76 phosphorylation is proximal to kinase activity dependent upon sustained increases in cellular calcium from extracellular sources.
The Grb2 adapter protein links PTKs with the Ras/mitogenactivated protein kinase cascade and the generation of phosphatidylinositol-derived second messengers in a variety of systems. Thus, characterizing molecules that associate with Grb2 should provide insight into the regulation of signal transduction. In this report, we describe the association of four tyrosinephosphorylated molecules with Grb2 in activated RBL-2H3 cells: pp116, pp76, pp36, and pp31. Phosphoproteins migrating at 116 kDa (c-cbl), 76 kDa (SLP-76), and 36 kDa have been shown to participate in signal transduction events in T cells. The identity of pp116 and pp36 has not yet been confirmed in RBL-2H3 cells. Ongoing experiments are addressing the possibility that the 31-kDa phosphoprotein is a component of the Fc⑀RI (17).
In this report, we identify the 76-kDa band in RBL-2H3 cells as SLP-76, an adapter protein described originally as a substrate of the TCR-stimulated PTKs. We show further that SLP-76 is a substrate of the PTKs activated after Fc⑀RI engagement on RBL-2H3 cells. Our data demonstrate that SLP-76, via its SH2 domain, associates with at least two unidentified tyrosine phosphoproteins, pp130 and pp62, in stimulated RBL-2H3 cells. These associations are similar to those seen after antigen receptor stimulation in T cells. However, pp130 appears as a doublet in RBL-2H3 cells but as a single molecule in T cells, suggesting RBL-specific modifications of pp130 or the existence of a second molecule. In addition, the SH2 domain of SLP-76 associates directly or indirectly with a kinase in stimulated RBL-2H3 cells, resulting in the appearance of a 100-kDa phosphoprotein. Together, these findings suggest that SLP-76 may play an important role in linking the Fc⑀RI with distal events in mast cell activation.
RBL-2H3 cell activation after IgE binding requires the coordination of several signaling pathways including those medi- ated by PLC␥1 and Ras. It is not yet clear in which biochemical pathway SLP-76 functions. Our data suggest that SLP-76 phosphorylation is dependent on Syk protein tyrosine kinase activation; thus, SLP-76 likely acts downstream of this proximal signaling event. In addition, SLP-76 becomes phosphorylated in RBL-2H3 cells in the absence of sustained Ca 2ϩ mobilization, suggesting that SLP-76 phosphorylation does not require the activity of Ca 2ϩ -dependent kinases. Recent studies from our laboratory suggest that in T cells, SLP-76 potentiates signaling in the extracellular signal-regulated kinase pathway, 2 and it is likely that it has a similar function in RBL-2H3 cells.
These data along with our studies in T cells suggest that SLP-76 plays a key role in receptor-mediated signal transduction leading to immune cell activation. We are performing structure/function analyses of SLP-76 to determine the functional significance of its associations with other potential signaling molecules in both T cells and rat basophilic leukemia cells. These experiments should help to further elucidate the role of SLP-76 in the process of immune cell activation.
FIG. 6. Phosphorylation of SLP-76 does not depend on calcium from external sources. RBL-2H3 cells were pretreated with EGTA and rested or Fc⑀RI-stimulated in Ca 2ϩ -free medium. Control stimulation of untreated cells was as described above. Nonidet P-40 lysates were subjected to immunoprecipitation with anti-SLP-76 antisera, resolved by SDS-8% PAGE, transferred to nitrocellulose, and immunoblotted with 4G10 anti-phosphotyrosine mAb.