Association of Activated Phosphatidylinositol 3-Kinase with p120 cbl in Antigen Receptor-ligated B Cells*

A 120-kDa protein that is tyrosine-phosphorylated upon antigen receptor ligation in B lymphocytes has been identified as the product of the c- cbl protoonco- gene. Tyrosine phosphorylation of Cbl depends on the efficient association of membrane immunoglobulin heavy chains with the Ig (cid:97) / (cid:98) heterodimer but is unimpaired in splenic B cells from the Xid mouse. Cross- linking of membrane IgM and membrane IgG, but not of CD40, leads to the tyrosine phosphorylation of Cbl. In receptor-ligated B lymphocytes, p120 cbl associates with an 85-kDa protein that has been identified as the 85-kDa subunit of phosphatidylinositol 3-kinase. The antigen receptor on B lymphocytes is made up of membrane immunoglobulins associated with the Ig (cid:97) / (cid:98) heterodimer (1, 2). This heterodimer serves to link the receptor with associated Src family kinases such as Blk, Lyn, Fyn, and Fgr (3–6), and also with the Syk tyrosine kinase (7, 8). Tyrosine phosphorylation appears to be an obligatory event (9) in the initiation of signal transduction pathways that promote B lymphocyte proliferation and differentiation. A number of signaling events downstream of this receptor depend on the interaction of membrane immunoglobulin heavy chains with the associated Ig (cid:97) / (cid:98) heterodimer (10–12), while some are initiated independently of these associated glycoproteins vitro kinase assays were performed as described previously (17, 34). GST-Grb2 Association with Cbl— Glutathione S -transferase fusion proteins including Grb2 and a mutant Grb2 protein in which the N- terminal SH3 domain is non-functional (a tryptophan to lysine substi-tution in codon 36) were kindly provided by Dr. Bruce Mayer. Lysates from non-stimulated and stimulated A20.25 B cells were made using 1% Nonidet P-40 in 20 m M Tris, pH 7.5, 150 m M NaCl, and 2 m M phenylmethylsulfonyl fluoride. Binding to immobilized GST fusion proteins, elution, and analysis on polyacrylamide/SDS gels was performed as described earlier (34). Cbl associated with Grb2 was revealed by an immunoblot assay. and essentially , M

A 120-kDa protein that is tyrosine-phosphorylated upon antigen receptor ligation in B lymphocytes has been identified as the product of the c-cbl protooncogene. Tyrosine phosphorylation of Cbl depends on the efficient association of membrane immunoglobulin heavy chains with the Ig␣/␤ heterodimer but is unimpaired in splenic B cells from the Xid mouse. Crosslinking of membrane IgM and membrane IgG, but not of CD40, leads to the tyrosine phosphorylation of Cbl. In receptor-ligated B lymphocytes, p120 cbl associates with an 85-kDa protein that has been identified as the 85-kDa subunit of phosphatidylinositol 3-kinase.
The antigen receptor on B lymphocytes is made up of membrane immunoglobulins associated with the Ig␣/␤ heterodimer (1,2). This heterodimer serves to link the receptor with associated Src family kinases such as Blk, Lyn, Fyn, and Fgr (3)(4)(5)(6), and also with the Syk tyrosine kinase (7,8). Tyrosine phosphorylation appears to be an obligatory event (9) in the initiation of signal transduction pathways that promote B lymphocyte proliferation and differentiation. A number of signaling events downstream of this receptor depend on the interaction of membrane immunoglobulin heavy chains with the associated Ig␣/␤ heterodimer (10 -12), while some are initiated independently of these associated glycoproteins (13). Activated protein tyrosine kinases are presumed to participate in triggering the Ras pathway, in activating PI 1 3-kinase, and in initiating the hydrolysis of phosphatidylinositol 4,5-bisphosphate by specific isoforms of phosphatidylinositol-specific phospholipase C.
Following B cell receptor ligation, cytoplasmic tyrosines in the ITAMs (immunoreceptor tyrosine-associated motifs) of Ig␣ and Ig␤ are tyrosine-phosphorylated, presumably by activated Src family kinases. The subsequent recruitment of Syk to phosphorylated ITAMs (14,15), and possibly an interaction between Src family kinases and Syk (16 -18), leads to the activation of the latter. Another protein tyrosine kinase that is activated following receptor ligation and which plays a role in antigen receptor mediated signaling is Btk (19 -21). Splenic B cells in Xid mice, which carry a point mutation in the PH domain of Btk, appear to be identical to gene-targeted mice in which no Btk protein is synthesized (22). Btk appears to be critical for the entry into S phase of B lymphocytes that have been stimulated by antigen receptor cross-linking (22,23).
An alternative pathway by which antigen-selected B lymphocytes may be induced to proliferate is mediated by T lymphocyte-derived cytokines and the triggering of CD40 on B cells by its ligand on activated T cells (24 -26). Cross-linking of CD40 can lead to the activation of tyrosine and serine/threonine kinases, the activation of PI 3-kinase, the activation of phospholipase C␥ isoforms, and the nuclear translocation of the NFB transcription factor. While the molecular consequences of activating B lymphocytes via the antigen receptor and by CD40-mediated signaling are similar, the mechanisms by which these individual pathways are activated by these different ligands may well be distinct.
Cbl is the cellular homolog of the v-Cbl oncoprotein (27,28) and is a predominantly cytosolic protein, which contains 17 proline-rich motifs potentially capable of binding a range of SH3 domains. Tyrosine phosphorylation of Cbl has been described in response to receptor occupancy of a number of receptors including the antigen receptor on T cells, the erythropoietin receptor, the granulocyte/macrophage colony-stimulating factor receptor, the Fc␥ receptor, the colony-stimulating factor-1 receptor, and the epidermal growth factor receptor (29 -32). Tyrosine phosphorylation of Cbl has also been observed in v-src-and v-abl-transformed cells (32,33), and this modification may be a critical event in mitogenic signaling.
We describe here the identification of Cbl as a prominent substrate for tyrosine phosphorylation in antigen-receptorligated B lymphocytes. This phosphorylation event requires the efficient interaction of membrane immunoglobulin heavy chains with the Ig␣/␤ heterodimer, and is unimpaired in splenic B cells from the Xid mouse. Following receptor ligation, Cbl is seen to associate with an 85-kDa phosphoprotein, identified as the 85-kDa subunit of PI 3-kinase. Our studies suggest that a major role of Cbl in B cells may be the recruitment and activation of PI 3-kinase following antigen receptor ligation.

EXPERIMENTAL PROCEDURES
Cells-Cell lines used included WEHI 231 (IgM, immature B), BAL 17 (IgM, mature B), and A20.25 (IgG 2a , mature B), as well as transfectants of A20.25 described below. The source for WEHI 231 cells was described earlier (34). BAL 17 cells were kindly provided by Dr. W. E. Paul. A20.25 transfectants expressing wild type human IgM and the YS/VV transmembrane human IgM mutant were kindly provided by Drs. A. Abbas and R. Mitchell (10). Splenic B cells from CBA/CaHNxid/J mice (Jackson Laboratory, Bar Harbor, ME) were purified as described in Ref. 17.
Lymphocyte Activation-Anti-IgM and anti-IgG stimulation of B cells was performed as described earlier (17). Anti-CD40 stimulation was performed incubating 2 ϫ 10 7 BAL 17 B cells in 200 l of serumfree RPMI medium at 37°C with anti-mouse CD40 at a concentration known to induce proliferation (2 g/ml; Serotec).
Immunoprecipitations, anti-phosphotyrosine immunoblots, and in vitro kinase assays were performed as described previously (17,34). GST-Grb2 Association with Cbl-Glutathione S-transferase fusion proteins including Grb2 and a mutant Grb2 protein in which the Nterminal SH3 domain is non-functional (a tryptophan to lysine substitution in codon 36) were kindly provided by Dr. Bruce Mayer. Lysates from non-stimulated and stimulated A20.25 B cells were made using 1% Nonidet P-40 in 20 mM Tris, pH 7.5, 150 mM NaCl, and 2 mM phenylmethylsulfonyl fluoride. Binding to immobilized GST fusion proteins, elution, and analysis on polyacrylamide/SDS gels was performed as described earlier (34). Cbl associated with Grb2 was revealed by an immunoblot assay.

Cbl Is Tyrosine-phosphorylated following Membrane IgM or
Membrane IgG Cross-linking of B Cells-Membrane immunoglobulins were cross-linked on an IgM-expressing B cell line (WEHI 231) and an IgG-expressing B cell line (the 4JJ subclone of A20.25, which also expresses a transfected human IgM mutant; Ref. 10) using anti-mouse IgM and IgG, respectively. Lysates from stimulated cells contained a 120-kDa tyrosinephosphorylated protein (Fig. 1, upper panel), which was depleted by preclearing with anti-Cbl, but not by control antibodies. Depletion of Cbl was confirmed by an immunoblot assay (lower panel). Tyrosine phosphorylation of Cbl is seen as early as 10 s after cross-linking and peaks at about 5 min. Similar results were observed with the surface IgM-positive BAL 17 line and untransfected A20.25 cells.
In Fig. 2A, Cbl tyrosine phosphorylation is apparent from the 30-s time point. In a slightly longer exposure, phosphorylation was apparent from the 10-s time point. Tyrosine-phosphorylated proteins with a slightly slower mobility than the major Cbl band probably represent Cbl species phosphorylated on multiple sites. These bands were depleted by anti-Cbl as seen in Figs. 1 and 2A. Similar bands were observed on anti-Cbl immunoblots of lysates from activated B cells (data not shown). The tyrosine-phosphorylated 70-kDa species seen both in Figs. 1 and 2A, exactly comigrates with, and probably represents, tyrosine-phosphorylated Syk. Phosphorylation of Cbl is observed soon after the cross-linking of membrane immunoglobulins but is not observed after cross-linking CD40 (Fig. 2B), although both stimuli can independently contribute to the initiation of similar signaling pathways.
Cross-linking of a Transmembrane IgM Mutant Fails to Initiate the Tyrosine Phosphorylation of Cbl-Mutations in the the transmembrane domain of membrane IgM have been demonstrated to impair signal transduction. A Tyr/Ser 3 Val/Val mutation (10) in human membrane IgM expressed in a murine B cell line (A20.25) has been examined in a number of studies. While cross-linking of a wild type human IgM transfectant leads to the effective initiation of intracellular signaling, this mutant human membrane IgM associates poorly with the murine Ig␣/␤ heterodimer and is compromised in terms of its ability to initiate a calcium flux (10) or to lead to the tyrosine phosphorylation of a number of unidentified cellular proteins (11,12). We immunoprecipitated Cbl following cross-linking of the above A20.25 transfectants either with anti-mouse IgG or anti-human IgM. As seen in Fig. 3 (20), while the activation of Syk (20) and Btk peaks a few minutes later (19,20). The dependence of Cbl tyrosine phosphorylation on the Ig␣/␤ heterodimer and the time course of Cbl phosphorylation suggest that antigen receptor-associated Src family kinases and Syk probably play a role in this tyrosine phosphorylation event. Xid mice harbor a point mutation in the PH domain of Btk (36,37). In response to antigen receptor ligation, splenic B cells from Xid mice appear to initiate signal transduction in a manner similar to B cells from wild type mice, but the Xid B cells, in contrast to wild type B cells, fail to enter S phase following stimulation (23). While Xid B cells are defective in terms of T-independent responses, they respond normally to protein antigens. Splenic B cells from gene-targeted mice, which fail to synthesize any Btk, cannot be functionally distinguished from Xid B cells (22), suggesting that the Xid point mutation completely disrupts Btk function.
Receptor ligation of Xid B cells, however, leads to the efficient tyrosine phosphorylation of Cbl (Fig. 4), suggesting that the phosphorylation of Cbl occurs independently of Btk. Shc-Grb2-Cbl Complexes in Lysates from Activated B Lymphocytes-Cbl contains multiple proline-rich stretches, which presumably constitute sites for SH3 interactions, and also contains a number of tyrosine residues, which, if phosphorylated, could serve as sites for SH2 binding. While the N-terminal SH3 domain of Grb2 has been demonstrated to associate with Cbl in lysates from T lymphocytes (29), the significance of this association remains unclear. One possibility could involve the recruitment of Cbl to the antigen receptor via Shc and Grb2. While it has been suggested that Shc-Grb2-SOS complexes contribute to the recruitment of SOS to the membrane in activated T cells (38), this remains an unresolved issue. Recruitment of Grb2-SOS to the membrane may depend on its association in activated T cells with a 36-kDa membrane-associated phosphoprotein (39). Complex formation between T cell recep-  Fig. 1. B, Cbl is tyrosine-phosphorylated after antigen receptor ligation but not following CD40 cross-linking. BAL 17 B cells were either not stimulated (N) or were stimulated with anti-IgM for 5 min (S1), anti-CD40 for 30 s (S2), or anti-CD40 for 5 min (S3). In leftmost four lanes, total lysates were separated on SDS-PAGE. In rightmost four lanes, anti-Cbl immunoprecipitates were separated. Proteins were visualized by an antiphosphotyrosine immunoblot. tor-associated ITAMs and Shc may be relatively inefficient (40).
We wished to ascertain whether the formation of a complex between Shc, Grb2, and Cbl could be demonstrated in activated B lymphocytes. We were able to confirm, as described previously (41), the formation of Shc-Grb2 complexes after B cell activation (Fig. 5A) and also the association of fusion proteins containing an intact N-terminal SH3 domain of Grb2 with Cbl from B cell lysates (Fig. 5B). While these data suggest that Shc-Grb2-Cbl complexes may well be formed in activated B cells, in our hands we were able to observe an in vivo association between Grb2 and Cbl only in three murine pre-B cell lines. 2 Although we could detect phosphorylated Shc species in anti-Cbl immunoprecipitates from lysates of activated B cells (data not shown), we were unable, in any of the three activated B cell lines tested, to demonstrate an interaction in vivo between Grb2 and Cbl. However, Shc-Grb2-Cbl complexes are readily observed in activated human B cell lines. 3 The band that has been labeled as Shc in the lower panel of Fig. 5A is inferred to be the phosphorylated form of p52 Shc. Anti-Shc immunoblots detect the p52 and p46 species of Shc in B cells but fail to reveal p66. Although the p46 form of Shc is also phosphorylated in activated B cells, the band representing it is probably obscured in Fig. 5A by rabbit IgG detected by the second antibody.
In Receptor-ligated B Cells, Cbl Associates with the 85-kDa Subunit of Phosphatidylinositol 3-Kinase-Since Cbl is tyrosine-phosphorylated in response to receptor ligation, we wished to ascertain if Cbl is specifically associated with any proteins after antigen receptor cross-linking. Given the knowledge that Cbl associates constitutively with Src family kinases via the SH3 domains of the latter (29,31) and with Syk by a poorly understood mechanism (31), one of the approaches that was used to identify proteins associated with Cbl was an in vitro kinase assay on Cbl immunoprecipitates of detergent lysates from non-activated and activated B cells. As seen in Fig. 6, phosphorylation of Cbl as revealed by this assay is increased after receptor ligation. In addition, a prominent 85-kDa phosphoprotein was seen to be associated specifically with Cbl immunoprecipitated from lysates of activated B cells, but not from non-activated B cell lysates. Since this protein was of the predicted size for the 85-kDa subunit of phosphatidylinositol 3-kinase, we examined lysates of B cells before and after activation for Cbl-associated PI-3 kinase as well as for Btk. Lysates were immunoprecipitated from non-activated and activated B cells with anti-Cbl antibodies and were analyzed for associated PI-3 kinase using antibodies specific for the p85 subunit in an immunoblot assay. The same filter was also probed with anti-Btk but no significant association of Btk with Cbl was observed (data not shown). As seen in Fig. 7A, p85 was seen to be associated with Cbl only in activated B cells. Since B lymphocyte antigen receptor cross-linking leads to an increase in PI 3-kinase activity (42,43), we were keen to establish whether there was an increase in PI 3-kinase activity associated with Cbl following anti-IgM exposure. As seen in Fig. 7B, a dramatic increase in Cbl-associated phosphatidylinositol 3-kinase kinase activity was observed in activated B cell lysates.
Three different cell lines representing distinct stages of differentiation were used in all the studies described above (other than those examining membrane IgM mutants). Most experiments were performed with all three cell lines, although in some only two of the three lines were used. No functional differences between these cell lines was observed in any of the above studies. DISCUSSION Since the tyrosine phosphorylation of Cbl is an early event downstream of the engagement of a number of mitogenic receptors, we sought to identify proteins that specifically associate with Cbl after the initiation of signal transduction. A number of proteins associate constitutively with Cbl usually via SH3 domain-mediated interactions. These proteins include Src family kinases, Grb2, and Nck (29,31,44). We have demonstrated here that an 85-kDa protein specifically associates with Cbl following B cell receptor ligation and that this 85-kDa protein is the p85 subunit of PI 3-kinase. In addition we have also demonstrated a convincing increase in PI 3-kinase activity associated with Cbl in activated B cells, suggesting that both subunits of this enzyme associate with Cbl and implying that Cbl might play a role in the activation of PI 3-kinase. It is quite likely that the major functional role of Cbl in activated B cells is to serve as a docking/activation site for PI 3-kinase. PI 3-kinase is known to be activated when one or both of the SH2 domains in the p85 subunit of this enzyme bind to tyrosine-phosphorylated proteins that contain Y-X-X-M motifs (45). The activation of PI-3 kinase in response to antigen receptor ligation in B cells has been described previously (42,43) and association of PI-3 kinase with CD19 (which contains a Y-X-X-M motif in its cytoplasmic tail) has also been demonstrated (46). Given the ease with which a Cbl-PI 3-kinase complex can be detected in vivo in activated B cells, and the dramatic increase in Cbl-associated PI-3 kinase catalytic activity following antigen receptor ligation, we postulate that the major functional consequence of Cbl tyrosine phosphorylation is the catalytic activation of PI 3-kinase. The 110-kDa catalytic subunit of this enzyme possesses both a lipid kinase activity and a serine/threonine protein kinase activity. The lipid kinase activity may, via PI intermediates phosphorylated in the 3position of inositol, contribute to the activation of the isoform of protein kinase C (47). Activation of Cbl presumably initiates a cascade of events including the activation of S6 kinase and the induction of proliferation (48,49). While this report was being prepared, the association of PI 3-kinase with Cbl was reported in receptor-ligated T cells (50), and we are aware of similar to be reported results from a second group (51). It is likely that the activation of PI-3 kinase by Cbl will prove to be a recurring theme in mitogenic signal transduction.
The tyrosine phosphorylation of Cbl in response to antigen receptor cross-linking requires the associated Ig␣/␤ complex and does not require functional Btk. While CD40 cross-linking can also lead to the activation of PI-3 kinase in B cells, this apparently occurs via a Cbl-independent mechanism. It is unclear as to whether there is a functional significance to the association of Cbl with Grb2 and the formation of complexes between Shc, Grb2, and Cbl following B cell activation. While such a complex could in theory promote the association of Cbl with the antigen receptor and therefore with cellular membranes, given the apparently low stoichiometry with which such complexes are generated, it is unclear whether such an event is necessary for the activation of PI 3-kinase activity. It is worth noting that previous attempts to demonstrate PI 3-kinase association in vivo with the Ig␣/␤ heterodimer have proved unsuccessful (44), suggesting that recruitment of PI 3-kinase via Shc, Grb2 and Cbl to the antigen receptor does not occur readily. However it has also been suggested, albeit based on indirect evidence, that membrane association of PI 3-kinase might be necessary for catalytic activation (52). The association of Cbl with Golgi membranes has been demonstrated in activated macrophages (32). This phenomenon, as well as the activation of PI 3-kinase, might be linked to the formation of Grb2-Cbl or Shc-Grb2-Cbl complexes in cells that have received a mitogenic stimulus. FIG. 7 A, the p85 subunit of PI 3-kinase associates with Cbl in receptor-ligated B cells. BAL 17 B cells were either not stimulated (N) or cross-linked with anti-mouse IgM (S) and immunoprecipitated with anti-Cbl. The presence of associated p85 was detected using an immunoblot assay. B, Anti-Cbl-associated PI 3-kinase activity in receptorligated B cells. BAL 17 B cells were either not stimulated (N) or cross-linked with anti-IgM (S) and immunoprecipitated with preimmune serum, anti-Cbl, or anti-phosphotyrosine antibodies. The presence of associated PI 3-kinase activity was detected by incubation with micellar PI and [␥-32 P]ATP and analysis on thin layer chromatography.