Identification of Two Tyrosine Phosphoproteins, pp70 and pp68, Which Interact with Phospholipase Cγ, Grb2, and Vav after B Cell Antigen Receptor Activation*

Tyrosine phosphorylation of cellular proteins mediates the assembly and localization of effector proteins through interactions facilitated by modular Src homology 2 (SH2) and phosphotyrosine binding domains. We describe here two tyrosine-phosphorylated proteins with M r values of 70,000 and 68,000 that interact with Grb2, phospholipase C (PLCγ1 and PLCγ2), and Vav after B cell receptor cross-linking. The interaction of pp70 and pp68 with PLC and Vav is mediated by the carboxyl-terminal SH2 domain of PLC and the SH2 domain of Vav. In contrast, the interaction of pp70 and pp68 with Grb2 requires cooperative binding of the SH2 and SH3 domains of Grb2. Western blot analysis demonstrated that neither pp70 nor pp68 represented the recently described linker protein SLP-76, which binds Grb2, PLC, and Vav in T cells after T cell receptor activation. Moreover, SLP-76 protein was not detected in a number of B cell lines or in normal mouse B cells. Hence, we propose that pp70 and pp68 likely represent B cell homologs of SLP-76 which facilitate and coordinate B cell activation.

Antigen binding to surface Ig of the B cell receptor (BCR) 1 complex results in activation of receptor-associated proteintyrosine kinases (PTKs). Three families of PTKs have been implicated in BCR-mediated signal transduction: the Src family PTKs (Lyn, Fyn, and Blk), Syk, and Bruton's tyrosine kinase (for review, see Refs. [1][2][3]. These PTKs are activated sequentially after BCR cross-linking and play requisite roles in B cell development and function (4 -16). These activated PTKs phosphorylate a variety of cellular substrates and regulate divergent signaling pathways including increases in free cytoplasmic calcium ([Ca 2ϩ ] i ), activation of Ras, and activation of Ras-related small G proteins. Activation of these signaling pathways is mediated by phospholipase C (PLC), the Shc and Grb2 adaptor proteins, and the vav proto-oncogene product, respectively.
Activation of the Ras pathway is mediated by protein kinase C-dependent and protein kinase C-independent mechanisms (25)(26)(27). The latter is thought to be mediated by two adaptor proteins, Shc and Grb2 (for review, see Ref. 28). Shc undergoes tyrosine phosphorylation after BCR cross-linking and is recruited to the BCR complex (29 -33). The phosphorylated tyrosine residues on Shc mediate its interaction with the SH2 domain of Grb2. Grb2 is constitutively associated with SoS, a guanine nucleotide exchange factor that facilitates exchange of GDP to GTP for Ras. Hence, tyrosine phosphorylation of Shc can result in the formation of a Shc⅐Grb2⅐SoS complex to regulate Ras-mediated cellular proliferation (30). In addition to SoS, recent studies have demonstrated that Vav serves as a guanine nucleotide exchange factor for the Rho family of small GTPases (34,35). Vav undergoes tyrosine phosphorylation after BCR cross-linking and is required for B cell development and BCR function (36 -41). Studies by Altman and colleagues (42) have demonstrated that Vav can bind tyrosine-phosphorylated Syk to mediate activation of the nuclear factor of activated T cells. Moreover, tyrosine phosphorylation of Vav results in c-Jun N-terminal kinase activation (34,35).
Although proximal PTKs are required for these three signaling pathways, the mechanisms by which the proximal PTKs regulate these distinct signaling pathways remain uncertain. To investigate these mechanisms, we analyzed the spectrum of tyrosine-phosphorylated proteins that interact with PLC␥, Grb2, and Vav. We describe here the identification of two tyrosine-phosphorylated proteins with apparent M r values of 70,000 and 68,000 which interact with all three signaling proteins. Together, these data suggest that these two proteins may facilitate the integration of multiple signal transduction pathways to coordinate B cell activation.

EXPERIMENTAL PROCEDURES
Cells and Antibodies-Ramos, Daudi, and Raji Burkitt lymphoma cells (ATCC) were maintained in RPMI 1640 supplemented with 10% fetal calf serum. A20, WEHI-231, and 70Z/3 mouse B cell lines (ATCC) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Goat anti-human IgM F(ab)Ј 2 , anti-mouse IgM F(ab)Ј 2 , and anti-mouse IgG F(ab)Ј 2 fragments were purchased from Jackson Immunoresearch Laboratories (West Grove, PA). Additional antibodies used in this study included PY20, an anti-phosphotyrosine (Tyr(P)) monoclonal antibody (mAb, Signal Transduction Laboratories, Lexington, KY); an anti-Grb2 mAb (Signal Transduction Laboratories); an anti-Vav mAb (Signal Transduction Laboratories); an anti-PLC␥1 mAb (Upstate Biotechnology, Inc., Lake Placid, NY); an anti-PLC␥1 antiserum (Santa Cruz Biotechnology, Santa Cruz, CA); * 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.
Constructs and GST Fusion Proteins-GST fusion protein constructs were generated in the pGEX-KT vector by in-frame ligation of polymerase chain reaction fragments (46). Grb2 mutants were kindly provided by Dr. Bruce Mayer and subcloned into the pGEX-KT vector (47). Specifically, Trp-36, Arg-86, and Trp-193 were mutated to lysine to disrupt the function of the NH 2 -terminal SH3, the SH2, and the COOHterminal SH3 domains, respectively. A myc epitope-tagged version of Grb2 was generated by adding the myc epitope sequence (SMEQKLI-SEEDLN) to the NH 2 terminus of Grb2. All constructs were verified by standard dideoxy sequencing. Induction and purification of GST fusion proteins were performed according to the manufacturer's recommendations (Pharmacia Biotech Inc.).
BCR Stimulation, Immunoprecipitation, and Protein Analysis-Cells were washed and resuspended in phosphate-buffered saline at a concentration of 10 8 cells/ml. Prior to stimulation, cells were rested at 37°C for 15 min. Cells were then stimulated with an anti-surface Ig F(ab)Ј 2 fragment (24 g/ml) for 1.5 min and lysed immediately in 10 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40 (lysis buffer) containing protease and phosphatase inhibitors for 15 min (44). Cell lysates were cleared by centrifugation (15,000 ϫ g for 10 min) at 4°C, and the supernatants were analyzed.
For binding studies to fusion proteins, BCR-stimulated cell lysates (5 ϫ 10 6 cells) were incubated with 2 g of fusion protein for 1 h at 4°C and captured with 20 l of glutathione-Sepharose beads (Pharmacia). For in vitro competition experiments, BCR-activated cell lysates (2 ϫ 10 6 cells) were mixed with either unphosphorylated or tyrosine-phosphorylated SLP-76 produced from 2 ϫ 10 6 Sf9 cells and incubated with 0.5 g of fusion protein as described above (44). Precipitates were washed three times with lysis buffer, resolved by SDS-polyacrylamide gel electrophoresis and analyzed by Western blot analysis. Protocols for immunoprecipitation and Western blotting have been described previously (44).
Stable Transfection of Cells-Stable transfection of cells was performed as described previously (44). In brief, 10 7 Daudi cells were electroporated with 25 g of linearized cDNA using a BTX 600 Electroporator (Biotechnologies and Experimental Research Inc., San Diego). Conditions used for electroporation were 1,250 microfarads, 250 V, and a resistance setting of R6. Cells at limiting dilution were then selected in 0.5 g/ml puromycin to generate stable clones.
Ion Exchange Chromatography-Cell lysates were diluted 6-fold with 10 mM Tris-HCl, pH 8.0, resulting in a buffer concentration of 10 mM Tris, pH 8.0, 25 mM NaCl. The diluted sample was loaded onto a Mono Q anion exchange column (Pharmacia) and eluted with a linear NaCl gradient (0.025-1 M) in 10 mM Tris, pH 8.0, 0.1% Nonidet P-40 at a flow rate of 1 ml/min at 4°C. 20 fractions (1 ml each) were collected. Samples (50 l) from each fraction were then assayed for PLC␥1binding phosphoproteins using a GST-PLC␥1 fusion protein as described above.

Distinct Sets of Tyrosine-phosphorylated Proteins Interact with Grb2 and PLC␥-Given the critical roles that PLC␥1 and
Grb2 play in regulating [Ca 2ϩ ] i mobilization and Ras activation, respectively, we analyzed tyrosine-phosphorylated proteins that interact with these molecules after BCR stimulation. GST fusion proteins containing either full-length Grb2 or the two SH2 domains of PLC␥1 were incubated with either resting or BCR-activated Ramos B cell lysates. Bound proteins were analyzed by immunoblotting with an anti-Tyr(P) mAb (Fig. 1A, left panel). Four major tyrosine-phosphorylated proteins with M r values of 150,000, 120,000, 70,000, and 68,000 bound a full-length Grb2 fusion protein from BCR-activated cell lysates ( To test whether pp70 and pp68, which interact with the Grb2 fusion protein, were identical to proteins 3 and 4, which interact with the PLC␥1 fusion protein, we depleted activated B cell lysates with the Grb2 fusion protein and then analyzed the ability of PLC␥1 to bind proteins 3 and 4. Sequential precipitation by the Grb2 fusion protein depleted proteins 3 and 4, which interacted with PLC␥1 (Fig. 1B, lanes 1-4). In contrast, proteins 1 and 2 still bound PLC␥1 after depletion by Grb2 (Fig.  1B, lane 4). Conversely, the PLC␥1 fusion protein depleted the Grb2-binding proteins pp70 and pp68, but not pp150 or pp120 (Fig. 1B, lanes 5-8). Together, these data demonstrate that pp70 and pp68, which bind Grb2, are identical to phosphoproteins 3 and 4, respectively, which interact with PLC␥1.
Although these in vitro binding experiments demonstrated common and unique phosphoproteins that bound PLC␥1 and Grb2, we determined whether these interactions also occurred in vivo. PLC␥1 was immunoprecipitated from either resting or BCR-stimulated Ramos cells and analyzed by Western blot analysis with an anti-Tyr(P) mAb. Consistent with the in vitro binding experiments, proteins 1-4 were detected in PLC␥1 immunoprecipitates from BCR-activated cell lysates, although the stoichiometry of binding of proteins 1 and 2 was lower in the PLC␥1 immunoprecipitates than the in vitro binding experiments ( Fig. 2A, lanes 2 and 5). In addition to tyrosinephosphorylated PLC␥1, tyrosine phosphoproteins with M r values of 160,000, 150,000, and 120,000 also coimmunoprecipitated with PLC␥1 after BCR cross-linking. pp120 was identified as the c-cbl proto-oncogene product by Western blot analysis (data not shown). No tyrosine phosphoproteins were detected in PLC␥1 immunoprecipitates from unstimulated cells or in control immunoprecipitates with normal rabbit serum (NRS, Fig. 2A, lanes 1, 3, and 4).
Because many B cells utilize the ␥2 isoform of PLC (PLC␥2) (18,19,48), we also analyzed the ability of proteins 1-4 to associate with PLC␥2 in vivo. Proteins 1, 3, and 4 were detected in immunoprecipitates of PLC␥2 from BCR-activated Daudi B cells. Protein 2 was below the level of detection and/or resolution in these immunoprecipitates (Fig. 2B). Together, these results demonstrate that phosphoproteins 1, 3, and 4 interact with both PLC␥1 and PLC␥2 after BCR activation.
To test whether proteins 3 and 4 also interacted with Grb2 in vivo, we generated Daudi B cell lines that stably express a myc epitope-tagged form of Grb2. This strategy was utilized to exclude the possibility that the immunoprecipitating antibody may inhibit the binding of Grb2-interacting proteins. Two representative clones, D28 and D35, which overexpress Grb2, are shown in Fig. 2C, lanes 2 and 3. Similar to the in vitro binding experiments, proteins 3 and 4 coimmunoprecipitated with Grb2 after BCR cross-linking (Fig. 2D, lane 4). Taken together, these in vitro binding and in vivo immunoprecipitation studies demonstrate that phosphoproteins 3 and 4 represent Grb2-and PLC␥-binding proteins, whereas phosphoproteins 1 and 2 interact with PLC␥.
Structural Domains within Grb2 and PLC␥1 Required for Their Interaction with Phosphoproteins 1-4 -Grb2 is composed of a single SH2 domain flanked by two SH3 domains. To identify the structural domains within Grb2 which interact with proteins 3 and 4, we generated GST-Grb2 fusion proteins in which each domain was disrupted by a single point mutation. Interestingly, Grb2 mutants with a single domain disrupted retained their ability to interact in vitro with both proteins 3 and 4, although protein 3 appeared to have a slight preference for the COOH-terminal SH3 domain and protein 4 for the NH 2 -terminal SH3 and SH2 domains (Fig. 3A, top  panel, lanes 2-4). Mutation of any combination of SH2 or SH3 domains eliminated the binding of proteins 3 and 4 to Grb 2 FIG. 2. A, in vivo interaction of proteins 1-4 with PLC␥1. Resting  (lanes 1 and 3) or BCR-stimulated (lanes 2 and 4) Ramos cell lysates were immunoprecipitated (2 ϫ 10 7 cells/lane) with an anti-PLC␥1 antiserum (lanes 1 and 2), normal rabbit serum (NRS, lanes 3 and 4), or precipitated ( 2) or the D35 clone (lanes 3 and 4) were immunoprecipitated with an anti-myc epitope mAb (9E10) and analyzed by Western blotting with an anti-Tyr(P) mAb (PY20). Similar data were obtained with two additional clones (D28 and D17). (Fig. 3A, top panel, lanes 5-7). Hence, cooperation of SH2 and SH3 domains is required for the efficient interaction of Grb2 with proteins 3 and 4.
To identify the structural domains within PLC␥1 which mediate its interaction with proteins 1-4, we analyzed the ability of individual SH2 domains to bind these proteins. The COOHterminal, but not the NH 2 -terminal SH2 domain of PLC␥1 was sufficient to bind proteins 1-4 (Fig. 3B, top panel, lanes 2 and  3). In this experiment, proteins 2 and 3 comigrated, although in many other experiments these two proteins could be resolved (see Fig. 5). In contrast, the SH3 domain of PLC␥1 failed to interact with any of these four phosphoproteins (Fig. 3B, lane  4). Rather, the SH3 domain of PLC␥1 interacted with pp120 cbl and pp150 (Fig. 3B, lane 4, and data not shown).
Analysis of Phosphoproteins 1-4 -The binding characteristics of proteins 3 and 4 are similar to SLP-76, a M r 76,000 tyrosine phophosprotein recently described in T cells which binds PLC␥1 and Grb2 in vitro (49). Low levels of SLP-76 mRNA and protein have been described in B cells (49,50). To determine whether proteins 3 or 4 represented SLP-76, we analyzed B cell lysates by immunoprecipitation and Western blotting with anti-SLP-76 antibodies. SLP-76 protein was not detected in any of the six B cell lines examined (Fig. 4A, lanes  1-6). In contrast, SLP-76 was readily detected in the Jurkat T cell line (Fig. 4A, lane 7). In addition, although proteins 3 and 4 bound both Grb2 and PLC␥1 fusion proteins (Fig. 1A), they failed to be recognized by an anti-SLP-76 mAb (Fig. 4B, lanes  1-4 and 6 -9). In contrast, SLP-76 from activated Jurkat T cells readily bound Grb2 and PLC␥1 (Fig. 4B, lanes 5 and 10). Similar data were obtained using normal mouse B cells (data not shown).
To resolve better the separation of proteins 1-4 to facilitate their identification, we separated BCR-activated Ramos cell lysates by anion exchange chromatography. Eluted proteins were analyzed by binding to the PLC␥1 fusion protein and immunoblotting with an anti-Tyr(P) mAb (Fig. 5, left panel). Protein 2 was eluted in fractions 7 and 8 (ϳ0.3 M NaCl), proteins 3 and 4 in fractions 9 and 10 (ϳ0.4 M NaCl), and protein 1 in fractions 10 and 11 (ϳ0.45 M NaCl). These fractions were then immunoblotted with antibodies against molecules implicated in BCR activation with similar M r mobilities. Protein 2 was recognized by an anti-Syk antiserum (Fig. 5, right  panel, lane 1) and is consistent with previous reports that Syk interacts with PLC␥1 after BCR cross-linking (51,52). Western blot analysis with antibodies directed against SHP-1, SHP-2, Bruton's tyrosine kinase, and Sam68 failed to recognize protein 1, 3, or 4 (data not shown). Hence, proteins 3 and 4 do not represent any of these signaling molecules.
Interaction of Phosphoproteins 3 and 4 with Vav-Although SLP-76 was not expressed in normal murine B cells or the B cell lines analyzed, the similar abilities of proteins 3 and 4 to interact with Grb2 and PLC␥1 suggested that these two proteins may represent B cell homologs of SLP-76. Because SLP-76 has been recently demonstrated to interact with the vav proto-oncogene product (53-55), we analyzed the ability of proteins 3 and 4 also to interact with Vav. Vav immunoprecipitates from BCR-activated cells demonstrated the association of Vav with two tyrosine-phosphorylated proteins with mobilities identical to the Grb2-and PLC-binding phosphoproteins 3 and 4 (Fig. 6A, lane 4). These two phosphoproteins were not detected in Vav immunoprecipitates from resting B cells or in control immunoprecipitates (Fig. 6A, lanes 1-3). Mapping studies using GST fusion proteins containing the Vav SH2 or SH3 domains demonstrated that the Vav SH2 domain was sufficient for its interaction with these two phosphoproteins (Fig. 6B,  lane 3). Although a tyrosine-phosphorylated protein with mobility identical to that of protein 1 was also precipitated by the SH2 domain of Vav (Fig. 6B, lane 3), this interaction was not observed in vivo (Fig. 6A, lane 4).
To confirm that the two Vav-binding phosphoproteins were the same as the Grb2-and PLC␥1-binding proteins 3 and 4, we again performed sequential depletion studies using Grb2, PLC␥, and Vav fusion proteins. Sequential precipitation with the Grb2 fusion protein depleted proteins 3 and 4, which interact with the Vav SH2 domain (Fig. 6C, lanes 1-4). In addition, sequential precipitation with the PLC␥1 SH2 domains depleted the Vav-interacting proteins 3 and 4 (Fig. 6C, lanes 6 -9). Together, these experiments demonstrate that phosphoproteins 3 and 4 interact with at least three distinct signaling adaptor/ effector molecules, Grb2, PLC␥, and Vav.

SLP-76 Competes with Proteins 3 and 4 in Their
Interaction with Vav-To provide further evidence that proteins 3 and 4 may be functionally homologous to SLP-76, we analyzed whether SLP-76 could compete with proteins 3 and 4 in their interaction with the SH2 domain of Vav. BCR-activated cell lysates were incubated with a Vav SH2 domain fusion protein in the absence (Fig. 7, top panel, lane 1) or in the presence of unphosphorylated (lane 2) or phosphorylated (lane 3) forms of SLP-76. We have demonstrated previously that SLP-76 serves as a downstream substrate of the ZAP-70 and Syk PTKs and can be phosphorylated by coinfection of SLP-76 with activated ZAP-70 or Syk PTKs in insect Sf9 cells (44). Only tyrosinephosphorylated SLP-76 significantly decreased the ability of proteins 3 and 4 to bind the Vav SH2 domain (Fig. 7, top panel,   FIG. 6. In vitro and in vivo interactions of proteins 3 and 4 with Vav. A, in vivo interaction of tyrosine-phosphorylated proteins with Vav. Resting (lanes 1 and 3) or BCR-stimulated (lanes 2 and 4) Ramos cell lysates (2 ϫ 10 7 cells/lane) were immunoprecipitated with an anti-Vav antiserum (lanes 3 and 4) or control NRS (lanes 1 and 2).  4 and 9). GST mock-depleted lysates were also precipitated with the GST-Vav SH2 domain fusion protein (lanes 5 and 10) for comparison. Bound proteins were analyzed by Western blotting with an anti-Tyr(P) mAb (PY20).  7, bottom panel, lanes 2 and 3) and is consistent with the SH2-phosphotyrosine based interaction of Vav with proteins 3 and 4 (Fig. 6B). A similar requirement for tyrosine-phosphorylated SLP-76 was observed in competition experiments with the PLC␥1 SH2 domain (data not shown). Together, these data further support the hypothesis that proteins 3 and 4 likely represent B cell counterparts of SLP-76. DISCUSSION Tyrosine phosphorylation of cellular proteins after BCR activation plays critical roles in PLC␥-, Vav-, and Shc/Grb2mediated [Ca 2ϩ ] i mobilization, c-Jun N-terminal kinase activation, and Ras activation, respectively. Phosphorylation can directly mediate activation of enzymes, such as PLC␥ and Vav (20,35). In addition, phosphorylation can mediate the assembly and/or localization of effector proteins through interactions facilitated by SH2 and PTB domains (for review, see Refs. 56 and 57). An example of the latter is represented by the tyrosine phosphorylation of Shc to recruit Grb2⅐SoS to the membrane and mediate Ras activation (28). Although activation of the proximal Src family, Syk, and Bruton's tyrosine kinases are required for the efficient function of all three signaling pathways, the integration of these distinct signaling pathways is also critical for efficient BCR-mediated function.
We provide evidence here of two cellular tyrosine phosphoproteins, pp70 (protein 3) and pp68 (protein 4), which interact with PLC␥, Vav, and Grb2. A M r 70,000 tyrosine phosphoprotein, VAP-1, has been described previously to associate with Vav after BCR cross-linking (38). Given the similarities in binding of pp70 and pp68 to these three effector proteins, pp70 and pp68 may represent differentially phosphorylated forms of the same protein. However, the inability to abrogate the M r difference between pp70 and pp68 by treatment with the serine/threonine protein phosphatases 1 and their identical isoelectric points make this possibility less likely (data not shown). Alternatively, pp70 and pp68 may represent differentially spliced forms of the same gene. Finally, pp70 and pp68 may represent a family of homologous proteins. Molecular characterization of these two proteins is under way to elucidate further their mechanistic and functional roles in BCR activation.
We also provide evidence here of the in vivo interaction of a M r 76,000 tyrosine phosphoprotein and Syk with PLC␥1. The latter is consistent with previous observations implicating the biochemical and functional requirements of Syk in [Ca 2ϩ ] i mobilization (7,51,52). The interaction of the M r 76,000 phosphoprotein with PLC␥1 was not observed with Vav or Grb2 and likely selectively mediates the [Ca 2ϩ ] i pathway. Genetic studies have demonstrated the functional importance of PLC␥1, Vav, and Grb2 in B cell function and development. B cells deficient in PLC␥ demonstrate a lack of inositol 1,4,5-trisphosphate hydrolysis and an absence of [Ca 2ϩ ] i mobilization after BCR cross-linking (43). Deletion of vav in mice results in an arrest of both B and T cell development (39 -41). Finally, overexpression of Grb2 results in an augmentation of antigen receptor-mediated activation of cytokine synthesis. 2 Hence, these three signaling and adaptor proteins play requisite roles in normal B cell function.
The structural domains within PLC␥1, Vav, and Grb2 which mediate their binding to pp70 and pp68 have also been demonstrated to be functionally important in lymphocyte activation. Mutation of the SH2 domains of PLC␥2 or the SH2 domain of Vav abrogates BCR-or T cell receptor-mediated signaling functions (43,53). Hence, the ability of these structural domains within PLC␥ and Vav to interact with pp70 and pp68 are likely to be important in mediating BCR function. Although BCR activation in naive B cells or B cell lines results in activation of all three major signaling pathways, anergized B cells demonstrate a lack of coordination of these three signaling pathways. Studies by Goodnow and colleagues (58) have demonstrated that although anergized B cells activate the [Ca 2ϩ ] i and Ras pathways, no activation of c-Jun N-terminal kinase or nuclear localization of nuclear factor B was observed in these cells. Hence, the integration of these major signaling pathways, which potentially could be regulated by pp70 and pp68, is required for normal B cell function.
The interaction of pp70 and pp68 with Grb2, PLC␥, and Vav in B cells is similar to the interaction of a linker protein, SLP-76, with Grb2, PLC␥, and Vav in T cells. SLP-76 interacts in vitro with Grb2 fusion protein as well as with Vav and PLC␥1 after T cell receptor cross-linking (49,(53)(54)(55)59). Moreover, SLP-76 is phosphorylated by ZAP-70 and is required for T cell receptor-mediated activation of the nuclear factor of activated T cells and the IL-2 gene (44,59,60). Although SLP-76 mRNA is expressed in several B cell lines and SLP-76 protein has been reported in WEHI-231 cells (49,50), we failed to detect any SLP-76 protein in WEHI-231 B cells, in five additional B cell lines, or in normal mouse B cells using an anti-SLP-76 mAb or an anti-SLP-76 antiserum ( Fig. 4 and data not shown). In addition, pp70 and pp68 migrate faster on SDSpolyacrylamide gels than SLP-76. However, pp70 and pp68 interact with Grb2, PLC␥1, and Vav in a fashion similar to that of SLP-76. In addition, the ability of tyrosine-phosphorylated SLP-76 to compete with pp70 and pp68 for their interactions with Vav and PLC␥1 ( Fig. 7 and data not shown) suggests that pp70 and pp68 may contain structural motifs similar to those present within SLP-76 required for its interaction with Vav and PLC␥1. We therefore propose that these two proteins may represent B cell proteins that are related to SLP-76.