Interactions of Cbl with two adapter proteins, Grb2 and Crk, upon T cell activation.

Several recent studies have demonstrated that Grb2, composed entirely of SH2 and SH3 domains, serves as an adaptor protein in tyrosine kinase signaling pathways. Cb1, the protein product of c-cbl proto-oncogene, has been reported to be phosphorylated on tyrosine residues upon T cell receptor (TCR) engagement. Here we show that in unstimulated Jurkat cells Cbl is co-immunoprecipitated with monoclonal antibody against Grb2. However, in lymphocytes activated through the TCR, Cbl loses its ability to bind to Grb2 precipitated either with anti-Grb2 antibody or with an immobilized tyrosine phosphopeptide, Y1068-P, derived from the epidermal growth factor receptor. In vitro studies confirm that the ability of Cb1 to bind to both SH3 domains of Grb2 is strongly reduced in activated T lymphocytes. Investigation of the time course of Cbl dissociation from Grb2 reveals that it is transient and correlates with the kinetics of tyrosine phosphorylation of Cbl. Moreover, Cb1 is co-immunoprecipitated with Crk, another SH2/SH3 domain-containing protein, upon TCR stimulation. Tyrosine-phosphorylated Cbl binds exclusively to the SH2 domain of Crk. These results suggest that different adaptor proteins may have different roles in the regulation of c-cbl proto-oncogene product.

Several recent studies have demonstrated that Grb2, composed entirely of SH2 and SH3 domains, serves as an adaptor protein in tyrosine kinase signaling pathways. Cbl, the protein product of c-cbl proto-oncogene, has been reported to be phosphorylated on tyrosine residues upon T cell receptor (TCR) engagement. Here we show that in unstimulated Jurkat cells Cbl is co-immunoprecipitated with monoclonal antibody against Grb2. However, in lymphocytes activated through the TCR, Cbl loses its ability to bind to Grb2 precipitated either with anti-Grb2 antibody or with an immobilized tyrosine phosphopeptide, Y1068-P, derived from the epidermal growth factor receptor. In vitro studies confirm that the ability of Cbl to bind to both SH3 domains of Grb2 is strongly reduced in activated T lymphocytes. Investigation of the time course of Cbl dissociation from Grb2 reveals that it is transient and correlates with the kinetics of tyrosine phosphorylation of Cbl. Moreover, Cbl is co-immunoprecipitated with Crk, another SH2/SH3 domain-containing protein, upon TCR stimulation. Tyrosine-phosphorylated Cbl binds exclusively to the SH2 domain of Crk. These results suggest that different adaptor proteins may have different roles in the regulation of c-cbl proto-oncogene product.
Recent studies by a number of laboratories have characterized the mechanism by which growth factors activate the Ras signaling pathway. This mechanism involves formation of complexes of the Sos guanine nucleotide exchange protein, and Grb2, an SH2 1 and SH3 domain-containing adaptor protein with autophosphorylated growth factor receptors (1)(2)(3). The SH3 domains of Grb2 bind to the carboxyl-terminal proline-rich domain of Sos (2), whereas the SH2 domain binds to tyrosinephosphorylated sites (1)(2)(3). Complex formation of Sos with autophosphorylated receptor tyrosine kinases via Grb2 results in the translocation of Sos from the cytosol to the plasma membrane, where its substrate Ras is localized (1).
The Crk protein, a homologue of the product of the v-crk oncogene, is an SH2 and SH3 domain-containing adaptor protein related to Grb2 and Nck (4). Two forms of cellular Crk proteins have been found: Crk II has the domain structure SH2-SH3-SH3, while the shorter Crk I consists of one SH2 and one SH3 domains (5,6). Recently, a third member of the Crk family has been cloned from chronic myelogenous leukemia cells and referred to as Crk-L (7). It has been shown that Crk proteins associate with two guanine nucleotide exchange proteins for Ras, Sos, and C3G (8 -10). In addition, they interact via their SH2 domains with both phosphorylated paxillin and p130 CAS (11,12).
Stimulation of T lymphocytes via their TCR results in phosphorylation of multiple intracellular proteins on tyrosine residues (13,14). We and others have shown that in the lysates of activated T cells Grb2 associates with several phosphotyrosine proteins (15)(16)(17)(18), including proteins with molecular masses of 36, 52, 75, and 120 kDa. The 36-kDa membrane-bound phosphoprotein binds to the SH2 domain of Grb2. In UCHT1-stimulated T cells, p36 was shown to be associated with the complex of Sos and Grb2 and thus implicated in Ras activation (15,16). The 52-kDa tyrosine phosphoprotein is identical with Shc adaptor protein (19). The 75-kDa tyrosine kinase substrate, that has been recently cloned and referred to as SLP-76, interacts with the SH3 domains of Grb2 (17,20).
It has been shown that a 116-kDa tyrosine phosphoprotein implicated in the TCR signaling pathway binds to the aminoterminal SH3 domain of Grb2 (18). Recently, a 120-kDa tyrosine phosphoprotein which in vitro binds to SH3 domains of Fyn, Lck, and Grb2, has been identified as Cbl (21). The v-cbl oncogene is the transforming gene of the murine Cas NS-1 retrovirus which induces pre-B cell lymphomas and myeloid leukemias (22). The homologue of v-cbl in mammalian cells is the c-cbl proto-oncogene that encodes Cbl, a 120-kDa protein predominantly localized in the cytoplasm (23). Furthermore, in vivo association of Nck, another SH2 and SH3 domain-containing adaptor protein with Cbl, was also demonstrated (24).
In this paper, we characterize the in vivo interaction of Cbl with two adaptor proteins, Grb2 and Crk. We report that, upon T cell activation, Cbl rapidly and transiently dissociates from Grb2. Tyrosine-phosphorylated Cbl then binds to the SH2 domain of Crk. We provide evidence that Grb2 and Crk have a critical role in the regulation of Cbl proto-oncogene product.
Antibodies, Fusion Proteins, and Peptides-Anti-CD3 antibody UCHT1 was from D. A. Cantrell, ICRF. Monoclonal anti-phosphoty-* This work was supported in part by Research Grants OTKA F13166, OTKA T 013165, and ETT T-11682. 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.
¶ Supported by the Medical Research Council (UK). 1 The abbreviations used are: SH2, Src homology 2; SH3, Src homology 3; TCR, T cell antigen receptor; EGF, epidermal growth factor; GST, glutathione S-transferase; GRF, guanine nucleotide releasing factor; PAGE, polyacrylamide gel electrophoresis. rosine antibody 4G10 was obtained from Upstate Biotechnology, Inc. Polyclonal anti-Grb2 antibody immobilized on Sepharose beads and polyclonal anti-Cbl antibody were purchased from Santa Cruz Biotechnology. Monoclonal anti-Grb2 antibody was obtained from Transduction Laboratories. Anti-GRF antibody was raised against a peptide corresponding to a region of the carboxyl-terminal domain (74 -91) of GRF. Polyclonal anti-Crk antibody was generated against the first SH3 domain of Crk II.
c-DNA encoding full-length GST fusion proteins c-Crk II and c-Crk II ⌬SH2 (SH2-deleted mutant) were donated by Dr. H. Hanafusa (25). The SH2 fragment of c-Crk (amino-terminal 146 amino acids) was isolated by BamHI/BglII digestion of full-length c-Crk II and subsequent ligation into BamHI-digested pGEX2TK (Pharmacia Biotech Inc.). The GST fusion proteins encoding both SH3 domains of Grb2 have been described previously (17). The Y1068 phosphopeptide derived from the autophosphorylated EGF receptor was synthesized with the sequence PVPEY(phos)INQS. The unphosphorylated peptide, PVPEYINQS, was also made (1,15).
Immunoprecipitation and Western Blotting-Immunoprecipitation and Western blotting were performed as described previously (15). Briefly, cell lysates were precleared with protein A-agarose, then proteins were immunoprecipitated with either 10 g of polyclonal anti-Grb2 antibody, or 5-5 g of anti-Crk and anti-Cbl antibodies. All immunoprecipitating antibodies were cross-linked to protein A-agarose. Washing was performed with ice-cold 50 mM HEPES buffer, pH 7.4, containing 250 mM NaCl, 0.2% Triton X-100, 0.1 mM Na 3 VO 4 . Bound proteins were separated by SDS-polyacrylamide gel electrophoresis (in 7.5 and 12% gels), transferred to nitrocellulose membrane, and immunoblotted with the indicated antibodies. Blots were developed by the enhanced chemiluminescence (ECL) system (Amersham). For precipitation with GST fusion proteins, 4 g of different Grb2 and Crk fusion proteins were used as noncovalently bound adducts to glutathioneagarose beads. Separation of bound proteins and immunoblotting were performed as described above. RESULTS We have recently used an immobilized tyrosine phosphopeptide derived from the Y1068 autophosphorylation site of the EGF receptor, to precipitate the complex of Grb2 and Sos from the lysates of T lymphocytes (15). It has been demonstrated that the phosphopeptide, Y1068-P binds to the SH2 domain of Grb2 with high affinity, whereas the SH3 domains of Grb2 are available for protein-protein interaction (1,15). To test if the phosphopeptide could precipitate proteins other than Sos in complex with Grb2, both the phosphorylated and unphosphorylated forms of Y1068 peptide immobilized on beads were incubated with lysates from untreated and TCR cross-linked Jurkat cells. Probing of Y1068-P precipitates with anti-phosphotyrosine antibody, 4G10, demonstrates that a 120-kDa phosphoprotein is present in the lysate of unstimulated cells (Fig. 1). Interestingly, upon T cell activation, the amount of this phosphoprotein is strongly decreased in the Y1068-P precipitate, while the appearance of a 75-kDa tyrosine phosphoprotein was detected in the same precipitate. Immunoblotting with an antibody against the 120 kDa c-cbl proto-oncogene product shows the presence of Cbl in the Y1068-P precipitates from unstimulated cells and a decreased amount of Cbl in the precipitates from UCHT1-stimulated cells (Fig. 1). These data suggest that the 120-kDa tyrosine phosphoprotein observed in our experiment is identical with Cbl or at least Cbl is a component of the 120-kDa tyrosine phosphoprotein band. The Y1068-P phosphopeptide precipitates equal amounts of Grb2 from either unstimulated or activated T lymphocytes (15); thus, the decreased amount of Cbl in the precipitates indicates that the association of Grb2 with Cbl is strongly inhibited in the lysates of activated cells. Identical results were obtained using human peripheral blood T lymphoblasts (data not shown). It is noteworthy that while Cbl disappeared from the Grb2 complex, in the 4G10 blot a relatively large amount of a 120-kDa tyrosine phosphoprotein still bound to Grb2 even in stimulated cells. It is likely that another tyrosine phosphoprotein exists in stimulated T cells with a molecular mass of 115-120 kDa. Analysis of the partial amino acid sequence of an unidentified 116-kDa phosphoprotein, that can also bind to Grb2, suggests that it is different from Cbl (20).
To prove the interaction of Grb2 with Cbl in another experimental system, immunoprecipitations with anti-Grb2 antibody covalently bound to Sepharose beads were performed. Jurkat cells were stimulated with UCHT1 antibody for 2 min or left untreated, proteins were immunoprecipitated with anti-Grb2, and then probed with anti-Cbl antibody. Fig. 2 demonstrates that in the lysate of resting cells Cbl co-immunoprecipitates with Grb2. In contrast, in the lysate of activated cells, Cbl is not associated with Grb2. Immunoblotting of whole cell lysates from quiescent or activated T cells with anti-Cbl antibody shows equal quantities of immunoreactive bands (Fig. 2). This excludes the possibility that the disappearance of Cbl from the Grb2 immunoprecipitates is due to the proteolytic degradation of the protein. For control immunoprecipitates, an antipeptide antibody raised against the guanine nucleotide releasing factor (GRF) that is exclusively present in neuron cells was used (Fig. 2) (26).
T cell stimulation results in a rapid and significant phosphorylation of Cbl detected with anti-phosphotyrosine antibody (21). Donovan et al. (21) have demonstrated Cbl phosphorylation only in whole cell lysates of Jurkat cells. Therefore, we immunoprecipitated Cbl and then performed immunoblotting with 4G10. Fig. 3 demonstrates that Cbl has a certain level of basal phosphorylation in quiescent cells. However, in response to TCR activation, Cbl is rapidly phosphorylated on tyrosine residues. Maximal phosphorylation is seen after 5 min of stimulation, with a reduction to background levels by 45 min.
We also investigated the kinetics of Cbl dissociation from Grb2 upon T cell activation. Jurkat cells were treated with UCHT1 antibody for 0, 5, 15, and 45 min; then Cbl was immunoprecipitated with anti-Grb2 antibody. Fig. 4 shows that after 5 min of stimulation Cbl is not co-immunoprecipitated with Grb2. However, after 15 min of UCHT1 treatment, the amount of Cbl is slightly increased in the Grb2 immunoprecipitate and returns to the level seen in unstimulated cells after 45 min. These data demonstrate a marked correlation between the kinetics of Cbl phosphorylation (Fig. 3) and the kinetics of dissociation of Cbl from Grb2 (Fig. 4A). To ensure that the loss of Cbl from Grb2 immunoprecipitates is not an artifact due to different amounts of Grb2 present in the immunocomplexes, Grb2 immunoprecipitates with monoclonal anti-Grb2 antibody were probed. The result clearly demonstrates that Grb2 precipitates contain equal immunoreactive bands (Fig. 4B). Grb2 was not found to undergo any phosphorylation on either tyrosine or serine/threonine residues in T lymphocytes activated via their TCR (15). Therefore, it is possible that the inability of phosphorylated Cbl to bind to Grb2 in T cells activated for a short time is due to conformational changes in the structure of Cbl upon increased tyrosine phosphorylation, although at present definitive proof of this hypothesis is not available.
It has been reported that only the amino-terminal SH3 domain of Grb2 can bind Cbl (21). We investigated the in vitro binding of Cbl to GST fusion proteins of Grb2. Lysates of quiescent and activated Jurkat cells were mixed with both SH3 domains of GST-Grb2 immobilized on beads, and then protein precipitates were immunoblotted with anti-Cbl antibody. As shown in Fig. 5, both SH3 domains are able to bind to Cbl in lysates of unstimulated cells, although based on our several experiments, Cbl has an in vitro preference for the aminoterminal SH3 domain of Grb2. In addition, Fig. 5 demonstrates that in UCHT1-treated cells the amount of Cbl associated with both SH3 domains of Grb2 is decreased. Donovan et al. (21) also reported that the amino-terminal SH3 domain of Grb2 binds less Cbl from the lysate of vanadate-treated Jurkat cells than from the lysate of unstimulated cells (21). These data suggest that in activated Jurkat cells the affinity of tyrosine-phosphorylated Cbl for the SH3 domains of Grb2 is strongly reduced (Fig. 5).
It has been reported recently that Crk adaptor proteins associate via their SH2 domain with a tyrosine-phosphorylated 116-kDa protein and thus implicated in TCR signaling (27). One of the possible candidate proteins of this size is Cbl. Therefore, we probed immunoprecipitates of Crk with anti-phosphotyrosine antibody, 4G10, and anti-Cbl. As shown in Fig. 6, although some amount of Cbl is co-immunoprecipitated with Crk from the lysates of unstimulated cells, activation of Jurkat cells induces increased complex formation of Cbl with Crk. Immunoblot analysis with anti-phosphotyrosine antibody con-firms the increased association of Crk with the 120-kDa phosphoprotein (Fig. 6). Using different GST fusion proteins of Crk II, we have investigated which domain of Crk II interacts with Cbl. Lysates of unstimulated and activated Jurkat cells were mixed with fusion proteins immobilized on glutathione-agarose beads, and then protein precipitates were probed with anti-Cbl antibody. Consistent with the in vivo data, the full-length Crk II protein was able to precipitate a small amount of Cbl from unstimulated cells. Following TCR activation, we could detect an increased association of Crk II GST fusion protein with Cbl (Fig. 7). The SH2 domain of Crk interacted in a manner similar to the full-length protein with Cbl. Although we added equal amounts (4 g) of GST fusion proteins to cell lysates, the experiment shows that in activated cells the SH2 domain alone can bind more Cbl than the full-length protein. The affinity of the single SH2 domain of Crk for Cbl could be higher than that of the full-length Crk. Finally, the SH2-deleted Crk II fusion protein (⌬SH2) was unable to bind to Cbl either from the lysate of activated or unstimulated T cells (Fig. 7). Using full-length Lysates were then subjected to affinity purification with the indicated GST fusion proteins (4 g/point) immobilized on agarose beads. Bound proteins were then subjected to SDS-7.5% PAGE, transferred to nitrocellulose, and immunoblotted with anti-Cbl antibody.
GST protein of Crk-L, we have detected inducible association of Crk-L with Cbl upon T cell activation (data not shown), suggesting that all members of the Crk family are able to form complexes with Cbl via their SH2 domains. DISCUSSION Cbl, the protein product of the c-cbl proto-oncogene, has recently been implicated in several tyrosine kinase signaling pathways (21,28,29). It has also been reported that Cbl can bind to two adaptor proteins, Grb2 and Nck, via their SH3 domains in T lymphocytes and HL60 cells, respectively (21,24). Our data are consistent with these reports in that Cbl associates with Grb2 in unstimulated T cells in vitro and in vivo, in an SH3 domain-dependent manner. However, upon CD3 activation of Jurkat cells, Cbl rapidly and transiently dissociates from Grb2 (Figs. 1, 2, and 4A). Agonist-induced dissociation of a proline-rich domain-containing protein from Grb2 has not previously been reported for any protein in any cell type. Donovan et al. (21) have shown that Cbl undergoes a rapid tyrosine phosphorylation in response to TCR stimulation. We have demonstrated that the kinetics of Cbl phosphorylation correlate remarkably with the kinetics of Grb2/Cbl dissociation.
The mechanism by which Cbl dissociates from Grb2 upon T cell activation is unknown but may relate to tyrosine phosphorylation of Cbl. Phosphorylation of Sos exchange protein on serine/threonine residues has been reported (30,31) to result in the disassembly of Sos from Grb2. This finding suggests that conformational changes in the structure of a proline-rich domain-containing protein due to phosphorylation may cause its dissociation from Grb2. In the case of Sos phosphorylation either in fibroblasts or in T cells, this mechanism seems to be involved in the negative feedback regulation of Ras signaling pathway (30 -32). By contrast, we show here that a TCR agonist induces a rapid and transient phosphorylation of Cbl and its dissociation from Grb2. Therefore, functionally the dissociation of Cbl from Grb2 is completely different from the disassembly of Sos from Grb2.
While this manuscript was in preparation, association of Cbl with Grb2 and phosphatidylinositol 3Ј-kinase in Jurkat cells has been reported (33). In contrast with our results, Cbl and Grb2 have been shown to form a constitutive complex regardless of the activation state of Jurkat cells. One possible explanation is that we stimulated the cells via their TCR, while Meisner et al. (33) used co-stimulation of the TCR and CD4 receptors. Cross-linking of both the TCR and CD4 receptors may result in a more intensive phosphorylation of Cbl, or alternatively, phosphorylation may occur on different sites. Based on in vitro data with SH2 and SH3 domains of Grb2 GST fusion proteins, tyrosine-phosphorylated Cbl can bind to both SH2 and SH3 domains of Grb2 in fully activated cells (33). In CD3-activated Jurkat cells, we have not detected binding of Cbl to the SH2 domain of Grb2 (data not shown).
In addition, we show that tyrosine-phosphorylated Cbl binds to another SH2/SH3 domain-containing adaptor protein, Crk, via its SH2 domain. Sawasdikosol et al. (27) have reported that in activated T lymphocytes a phosphotyrosine protein with a molecular mass of 116 kDa binds to the SH2 domain of Crk. Therefore, it is highly likely that this 116-kDa phosphotyrosine protein is identical with Cbl. Using SH2 mutant Crk for protein precipitation, we failed to detect the association of Cbl with the Crk SH3 domains. This suggests that the mechanism by which Cbl binds to Crk is completely different from the association of Cbl with Grb2 and Nck. At present, the role of Crk in regulation of Cbl is unclear. Two other phosphotyrosine proteins have been shown to be associated with Crk, in a similar SH2-dependent manner, paxillin and the Crk-associated substrate p130 CAS (11,12). In addition, Crk proteins can be associated, via their SH3 domains, with two Ras-specific guanine nucleotide exchange proteins, Sos and C3G (8 -10). Therefore, phosphorylated Cbl in complex with Crk/Sos or Crk/C3G might be involved in Ras signaling pathways in T cells.
It has been suggested that Cbl is a nuclear protein which may function as a transcripton factor (23): it contains a possible nuclear localization sequence, a putative leucine zipper at the carboxyl terminus, and a zinc finger-related protein motif (29). Therefore, Cbl or tyrosine-phosphorylated Cbl could be transported into the nucleus. This is supported by the fact that the truncated form of Cbl, the protein product of v-cbl oncogene, can enter the nucleus and bind DNA (23). Moreover, it has been recently suggested that the carboxyl terminus of Cbl is involved in the retention of Cbl in the cytoplasm and the inhibition of DNA binding (23). The truncation in the sequence of v-Cbl occurs at the carboxyl-terminal domain which contains several proline-rich motifs. These motifs are likely to be responsible for the interaction with the SH3 domains of Grb2. Taken together, this suggests that Grb2 might have a role in the regulation of Cbl in quiescent cells by acting as a retention factor. However, every effort so far to detect Cbl in the nucleus has been unsuccessful. Tanaka et al. (28) have very recently reported that in macrophages stimulated via their Fc␥ receptor or in HER14 cells activated with EGF, Cbl was found to translocate from the cytoplasm to the trans-Golgi region of the cells. Further studies will therefore elucidate the possible role of Cbl in complexes with different adaptor proteins in tyrosine kinase signaling pathways. GST, GST-Crk II, GST-Crk SH2, and GST-Crk II SH2 mutant fusion proteins bound to glutathione-agarose beads were added to cell lysates. Proteins bound to the beads were separated by SDS-7.5% PAGE, transferred to nitrocellulose membrane, and immunoblotted with anti-Cbl antibody.