SLP-76 Is a Substrate of the High Affinity IgE Receptor-stimulated Protein Tyrosine Kinases in Rat Basophilic Leukemia Cells*

(Received for publication, August 29, 1996, and in revised form, November 1, 1996)

L. Ranee Hendricks-Taylor Dagger , David G. Motto Dagger , Juan Zhang §, Reuben P. Siraganian § and Gary A. Koretzky Dagger §

From the Dagger  Departments of Internal Medicine and Physiology and Biophysics and Graduate Program in Immunology, University of Iowa College of Medicine, Iowa City, Iowa 52246 and the § Laboratory of Immunology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

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.


INTRODUCTION

The high affinity receptor for IgE (Fcepsilon RI),1 found on basophils and mast cells, mediates cell activation in type I allergic reactions (1). Fcepsilon RI is a member of a family of receptors expressed on immune effector cells that lack intrinsic enzymatic activity but possess cytoplasmic immune receptor tyrosine-based 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), Fcepsilon RI is composed of multiple subunits including a ligand binding component (alpha  chain) noncovalently associated with transmembrane proteins that bear the immune receptor tyrosine-based activation motifs (beta  chain and a homodimer of disulfide-linked gamma  chains) (4, 5). The beta  and gamma  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 Fcepsilon RI in the rat basophilic leukemia cell line RBL-2H3 (7). Furthermore, stimulation of a chimeric receptor containing the cytoplasmic domain of the TCR zeta  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 Fcepsilon 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 Fcepsilon RI, whereas the interaction between Syk and the beta and gamma  subunits requires tyrosine phosphorylation of the immune receptor tyrosine-based activation motifs after receptor ligation (15, 16). In addition, the phosphorylated beta  and gamma  subunits precipitate Shc, Grb2, and phospholipase C-gamma 1 (PLCgamma 1) from RBL-2H3 cell lysates (17).

Downstream signaling events after Fcepsilon RI engagement include PLCgamma 1 activation resulting in intracellular calcium release and protein kinase C activation (18, 19), activation of the Ras/mitogen-activated protein kinase pathway (20, 21), and activation of nuclear factor of activated T-cells (22), c-fos, and c-jun (23). Proteins phosphorylated upon Fcepsilon RI aggregation include the Fcepsilon RI itself, PLCgamma 1, Nck, vav, HePTP, paxillin, and several unidentified molecules of 72-78 kDa (9, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35). Recently we began the characterization of SLP-76, a 76-kDa substrate of TCR-stimulated PTKs (36, 37). We speculated that SLP-76 was one of the unidentified substrates of Fcepsilon RI-activated PTKs.

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 tyrosine-phosphorylated 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 motif-containing receptors including the Fcepsilon 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 Fcepsilon RI ligation. SLP-76 becomes rapidly tyrosine-phosphorylated after Fcepsilon RI engagement and remains phosphorylated for over 1 h. Furthermore, SLP-76 phosphorylation requires Syk activity but is not dependent on extracellular Ca+2. 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.


EXPERIMENTAL PROCEDURES

Cell Culture and Antibodies

RBL-2H3 cells were maintained in Eagle's minimal essential medium supplemented with 15% fetal calf serum. Jurkat (E6-1) T cells were grown in RPMI 1640 medium with 10% fetal calf serum. All media contained penicillin (1000 units/ml), streptomycin (1000 units/ml), and glutamine (20 mM). The following monoclonal antibodies (mAbs) were used: anti-Fcepsilon RI mAb, BC4 (30); anti-TCR mAb, C305 (40) (gift of A. Weiss, San Francisco, CA); anti-phosphotyrosine mAb, 4G10 (gift of B. Drucker, Portland, OR); and anti-flag mAb M2 (International Biotechnology Inc., Rochester, NY). Anti-SLP-76 sheep antiserum 0083 was generated against amino acids 136-235 of murine SLP-76 expressed as a GST fusion protein. For immunoprecipitation, the antiserum was bound to GammaBind Plus Sepharose (Pharmacia Biotech Inc.) and used at 0.5 µl/20 × 106 cell equivalents. The antiserum was diluted 1:500 for Western blotting.

GST Fusion Proteins

GST fusion proteins were produced in bacteria as described (41). Fusion proteins containing the SH2 domain of human SLP-76 (39), the R448K mutant variant of the SLP-76 SH2 (39), and a full-length human Grb2, SH2 domain of Grb2, and full-length Grb2 with a SH2 domain loss of function mutation (R86K) have been described previously (37).

Protein Precipitations and Immunoprecipitations

RBL-2H3 cells were stimulated with anti-Fcepsilon RI mAb (BC4; 0.03 µg/ml) in Eagle's minimal essential medium with 0.1% bovine serum albumin for 10 min unless otherwise noted. Jurkat cells were stimulated with anti-TCR mAb (C305; 1:1000) for 1 min. For Ca2+ depletion, cells were washed with 5 mM EGTA in PBS and stimulated in PIPES AC (0.025 M PIPES, 0.119 M NaCl, 0.005 M KCl, 0.04 M NaOH, and 0.1% bovine serum albumin, pH 7.4) with 0.001 M CaCl2 plus 50 µM EGTA. Cell lysates prepared in Nonidet P-40 lysis buffer (1% Nonidet P-40, 150 mM NaCl, and 10 mM Tris, pH 7.4) including protease (50 µg/ml aprotinin, 10 µg/ml leupeptin, 50 µg/ml pepstatin A, and 1 mM phenylmethylsulfonyl fluoride) and phosphatase (400 µM sodium vanadate, 10 mM sodium fluoride, and 10 mM sodium pyrophosphate) inhibitors were subjected to precipitation with GST fusion proteins or antibodies for 2 h at 4 °C. Protein complexes were washed in high-salt lysis buffer (500 mM NaCl), resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with mAb or antisera followed by a horseradish peroxidase-conjugated secondary antibody (Bio-Rad). Immunoreactive proteins were detected by ECL (Amersham Life Science, Inc.).

In Vitro Kinase Assay

Immunoprecipitation complexes were washed in 500 mM LiCl, 5 mM Tris (pH 7.4), and water. The samples were resuspended in kinase reaction buffer (10 mM MnCl2 and 20 mM Tris, pH 7.4) containing [32P]ATPgamma (10 µCi/sample) for 10 min at room temperature, washed, subjected to SDS-8% PAGE, and visualized by autoradiography.


RESULTS AND DISCUSSION

Association of Phosphotyrosine-containing Proteins with GST-Grb2 in Stimulated RBL-2H3 Cell Lysates

Ligation of the Fcepsilon 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-Fcepsilon 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 Fcepsilon RI-stimulated cells.


Fig. 1. GST/Grb2 associates with multiple tyrosine-phosphorylated proteins in Fcepsilon RI-stimulated RBL-2H3 cells. Nonidet P-40 lysates of resting or Fcepsilon RI-stimulated RBL-2H3 cells were precipitated with fusion proteins consisting of GST/Grb2 (full-length Grb2), GST/Grb2SH2 (SH2 domain only of Grb2), or GST/Grb2R86K (full-length Grb2 containing a loss of function mutation in the SH2 domain). For each condition 10 µg of fusion protein and 10 × 106 cell equivalents were used. The resulting protein complexes were washed, resolved by SDS-10% PAGE, transferred to nitrocellulose, and immunoblotted with 4G10 anti-phosphotyrosine mAb. The migrations of pp76, pp31, pp36, and pp116 are indicated.
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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 tyrosine-phosphorylated Shc in resting and Fcepsilon 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 PLCgamma 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 Fcepsilon RI

The Grb2/phosphoprotein associations 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 Fcepsilon 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.


Fig. 2. Time course of tyrosine phosphorylation of SLP-76 and its associated proteins in stimulated RBL-2H3 cells. A, RBL-2H3 cells (10 × 106 cell equivalents/condition) were left unstimulated or were stimulated with anti-Fcepsilon RI mAb for the indicated lengths of time. Nonidet P-40 lysates were prepared and subjected to immunoprecipitation with anti-SLP-76 antisera; the resulting protein complexes were washed, resolved by SDS-8% PAGE, transferred to nitrocellulose, and immunoblotted with 4G10 anti-phosphotyrosine mAb. SLP-76 and the tyrosine-phosphorylated proteins with which it associates are indicated. B, subsequently, the immunoblot was stripped and reprobed with anti-SLP-76 antisera. Lysates from resting and anti-TCR stimulated Jurkat T cells are shown for comparison.
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SLP-76 co-immunoprecipitates with two additional tyrosine-phosphorylated proteins migrating at ~62 and ~130 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 anti-phosphotyrosine 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.


Fig. 3. The SH2 domain of SLP-76 associates with two phosphoproteins after Fcepsilon RI aggregation. Nonidet P-40 lysates of resting or Fcepsilon RI-stimulated RBL-2H3 cells were precipitated with the indicated GST/SLP-76 SH2 domain or GST/SLP-76 SH2 R448K loss of function fusion proteins (10 µg of fusion protein, 20 × 106 cell equivalents/condition). The resulting protein complexes were washed, resolved by SDS-8% PAGE, transferred to nitrocellulose, and immunoblotted with 4G10 anti-phosphotyrosine mAb. Lysates from Jurkat T cells stimulated with anti-TCR are shown for comparison. The migrations of pp62 and pp130 are indicated.
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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 Fcepsilon 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 receptor stimulation. Attempts are currently underway to identify the relevant kinase in both T cells and rat basophilic leukemia cells.


Fig. 4. In vitro kinase assay of GST/SLP-76 SH2 domain fusion protein precipitations. Nonidet P-40 lysates of resting or Fcepsilon RI-stimulated RBL-2H3 cells were precipitated with the indicated GST/SLP-76 SH2 domain fusion protein (10 µg of fusion protein, 20 × 106 cell equivalents/condition), and the resulting protein complexes were washed and subjected to an in vitro kinase assay with [32P]ATPgamma . The resulting radiolabeled reaction products were resolved by SDS-8% PAGE and detected by autoradiography. The 100-kDa radiolabeled phosphoprotein is indicated by an arrow.
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Phosphorylation of SLP-76 Requires Syk Activity but Is Not Dependent on Extracellular Ca2+

We next performed experiments to determine the requirements for SLP-76 tyrosine phosphorylation in RBL-2H3 cells after Fcepsilon 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 Fcepsilon 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.


Fig. 5. Phosphorylation of SLP-76 requires Syk kinase. Nonidet P-40 lysates of unstimulated or Fcepsilon RI-stimulated wild type RBL-2H3 cells, Syk-deficient RBL-2H3 cells (TB1A2), and the Syk-negative cell reconstituted with Syk cDNA (3A1) were immunoprecipitated with anti-SLP-76 antisera, washed, and resolved by SDS-8% PAGE. A, precipitates were immunoblotted with 4G10 anti-phosphotyrosine mAb. B, subsequently, the immunoblot was stripped and reprobed with anti-SLP-76 antisera.
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Mast cell activation after Fcepsilon RI engagement depends upon several downstream signaling pathways including the activation of PLCgamma 1 and the release of intracellular calcium. A sustained increase in intracellular Ca2+ is required for the tyrosine phosphorylation of some substrates (such as focal adhesion kinase) but not others (such as the Fcepsilon RI) (14). We tested the dependence of SLP-76 phosphorylation on sustained increases in Ca2+ concentrations by depleting Ca2+ with EGTA treatment followed by receptor stimulation in Ca2+-free medium. Cell lysates were subjected to SLP-76 immunoprecipitation and immunoblot analysis with anti-phosphotyrosine antibody (Fig. 6). As shown, Fcepsilon RI stimulation leads to tyrosine phosphorylation of SLP-76 in the absence of extracellular Ca2+, demonstrating that SLP-76 phosphorylation is proximal to kinase activity dependent upon sustained increases in cellular calcium from extracellular sources.


Fig. 6. Phosphorylation of SLP-76 does not depend on calcium from external sources. RBL-2H3 cells were pretreated with EGTA and rested or Fcepsilon RI-stimulated in Ca2+-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.
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The Grb2 adapter protein links PTKs with the Ras/mitogen-activated 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 tyrosine-phosphorylated 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 Fcepsilon 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 Fcepsilon 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 Fcepsilon RI with distal events in mast cell activation.

RBL-2H3 cell activation after IgE binding requires the coordination of several signaling pathways including those mediated by PLCgamma 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 Ca2+ mobilization, suggesting that SLP-76 phosphorylation does not require the activity of Ca2+-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.


FOOTNOTES

*   This work was supported in part by Grant RO1GM 53256 from the National Institutes of Health (to G. A. K.). G. A. K. is an established investigator of the American Heart Association and is supported in part by the Roy J. Carver Charitable Trust. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   To whom correspondence should be addressed: 540 EMRB, University of Iowa College of Medicine, Iowa City, IA 52242. Tel.: 319-335-6844; Fax: 319-335-6887; E-mail: gary-koretzky{at}uiowa.edu.
1    The abbreviations used are: Fcepsilon R1, high affinity receptor for IgE; PTK, protein tyrosine kinase; TCR, T-cell antigen receptor; PLCgamma 1, phospholipase Cgamma 1; mAb, monoclonal antibody; GST, glutathione S-transferase; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; ATPgamma , adenosine 5'-O-(thiotriphosphate).
2    M. Musci, D. Motto, S. Ross, N. Fang, and G. Koretzky, submitted for publication.

Acknowledgments

We thank B. Drucker and A. Weiss for their gifts of reagents.


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