(Received for publication, December 18, 1995; and in revised form, February 1, 1996)
From the
B cell antigen receptor (BCR)-mediated signal transduction controls B cell proliferation and differentiation. The BCR activates Ras, presumably by the formation of a Shc-Grb2 adaptor complex, which recruits the Grb2-associated guanine nucleotide exchange factor Sos to the plasma membrane. In order to reveal additional BCR-induced signaling events involving the Grb2 adaptor, we undertook the isolation of Grb2-binding proteins. Using the yeast two-hybrid system and bacterial fusion proteins, Vav and C3G were identified as Grb2 binders. Vav is a putative nucleotide exchange factor and a target for BCR-induced tyrosine phosphorylation. C3G exerts nucleotide exchange activity on the Ras-related Rap1 protein. While Sos binds to both Grb2 Src homology-3 (SH3) domains, Vav was found to associate selectively with the carboxyl-terminal SH3 domain, while C3G bound selectively to the amino-terminal SH3 domain of bacterially expressed Grb2. Despite the association of Vav with Grb2 in vitro, we could not demonstrate an interaction between endogenous Vav and Grb2 molecules in primary B cells. Instead, Vav was found to inducibly associate with the Grb2-related adaptor protein Crk upon BCR stimulation. C3G did not bind to either Grb2, Shc, or Crk in vivo. Instead, C3G was found in association with the Crk-L adaptor, both before and after BCR stimulation. We show that Crk-L also participates in BCR signaling, since it inducibly interacts with tyrosine-phosphorylated Cbl. We conclude that, in addition to Sos, Vav and C3G play a role in BCR-mediated signal transduction. These guanine nucleotide exchange factors selectively associate with Grb2, Crk, and Crk-L, respectively, which may serve to direct them to different target molecules. Since Cbl binds to Grb2, Crk, as well as Crk-L, we hypothesize that Cbl may affect the function of all three exchangers.
Src homology-3 (SH3) ()domains are found in a variety
of signaling molecules, involved in the response of cells to external
stimuli(1) . SH3 domains specifically interact with proline
rich sequences (2) and thereby control cellular localization (3) , substrate specificity(4) , or catalytic activity (5) of associating proteins. One example illustrating the
importance of SH3 domain-mediated intermolecular interactions is the
complex formation between the adaptor protein Grb2 and the guanine
nucleotide exchange factor (GEF) son of sevenless (Sos)(6) .
Grb2 consists of an SH2 domain, flanked by two SH3 domains. Sos is
constitutively associated with the SH3 domains of Grb2 and regulates
Ras activity by converting GDP-Ras to the GTP-bound
state(7, 8) . Translocation of Sos from the cytosol to
the plasma membrane is thought to play a key role in Ras
activation(9) . Recruitment of the Grb2-Sos complex to the
plasma membrane may be mediated by direct association of the Grb2 SH2
domain with a tyrosine-phosphorylated membrane molecule or by indirect
association via the tyrosine-phosphorylated adaptor protein Shc.
Receptors with intrinsic tyrosine kinase
activity(7, 8) , as well as receptors that activate
associated cytoplasmic protein-tyrosine kinases, such as cytokine
receptors(10) , the T cell antigen receptor (11) , and
the BCR (12, 13, 14) induce association of
Grb2-Sos with tyrosine-phosphorylated Shc. In lymphocytes, stimulation
of T cell antigen receptor and BCR induces Ras activation, which most
likely involves Sos.
Vav is exclusively found in hematopoietic
cells. It contains multiple structural motifs, including one SH2 and
two SH3 domains, a pleckstrin homology domain, as well as a region
found in the human Dbl oncogene product(15, 16) . Dbl
is a GDP-GTP exchange factor for the Ras-like polypeptide
Cdc42(18) , but Cdc42 has not been defined as a target for Vav.
Vav was reported to exert nucleotide exchange activity on Ras (19) . However, other authors found that Vav cooperates with
Ras to transform fibroblasts, but is not an exchange factor for
Ras(20, 21) . Therefore, the target of the Dbl domain
in Vav remains unidentified. Vav is phosphorylated on tyrosine upon T
cell antigen receptor, Fc receptor(22, 23) ,
BCR(24) , and cytokine receptor (25) stimulation. Mice
with a disrupted Vav gene die early during development, indicating that
Vav has an essential function prior to the onset of hematopoiesis (26) . Studies in chimeric mice have indicated that Vav also
plays a role in the differentiation and function of T and B
cells(27, 28, 29) .
C3G was identified as a protein binding to a bacterially expressed SH3 domain of the adaptor protein Crk (30) and it associates with endogenous Crk in PC12 cells(31) . In vitro, C3G can also interact with SH3 domains of Grb2(30) . The carboxyl terminus of C3G has homology with GEFs for Ras and C3G can complement Cdc25 function in yeast(30) . Recently, however, Rap1 rather than Ras, was identified as a target for C3G in mammalian cells(32) . The function of C3G in lymphocytes has not yet been addressed.
In this study, we have identified Vav and C3G as Grb2 binders, using the yeast two-hybrid system and bacterial fusion proteins, respectively. However, in vivo in B lymphocytes, only Sos participates in a Shc-Grb2 complex. Vav does not bind detectably to Shc or Grb2, but inducibly associates with Crk upon BCR stimulation. C3G was not detected in Shc, Grb2, or Crk complexes, but constitutively interacts with Crk-L. We demonstrate that Grb2, Crk, as well as Crk-L interact with the Cbl proto-oncogene product(33) , which is a major target for BCR-induced protein-tyrosine kinase activity(34) . This indicates that all three adaptors participate in BCR-induced signaling. Given their differential interaction with Sos, Vav, and C3G, we postulate that Grb2, Crk, and Crk-L selectively target the functions of these nucleotide exchangers in response to BCR stimulation.
Figure 1:
A, the
product of the Vav oncogene isolated from a human Epstein-Barr
virus-transformed B cell cDNA library interacts with the
carboxyl-terminal SH3 domain of Grb2. A human B cell-derived cDNA
library fused to the GAL4 TAD was expressed in yeast, together with the
full-length Grb2 cDNA, fused to the GAL4 DBD. The yeast strain
contained his3 and lacZ reporter genes and clones
were selected for growth on medium without histidine and
-galactosidase activity (blue color). Yeast colonies expressing
the Vav cDNA fragment, together with full-length (FL) Grb2, or
the carboxyl-terminal SH3 domain of Grb2 grow and stain blue. No growth
is detected of yeast cells expressing the amino-terminal SH3 domain of
Grb2 (SH3-C-Grb2) or the B29 molecule (FL-B29). B, Vav protein structure and description of the Vav fragment
isolated by the yeast two-hybrid screen with full-length Grb2.
Structural motifs shown in the Vav protein include a leucine-rich
region (LR), an acidic domain (AD), a Dbl homology
domain (DH), a pleckstrin homology domain (PH), a
zinc finger-like cysteine rich region (CR), and SH3 and SH2
domains(15, 16) . Nucleotide sequence analysis of the
1461-base pair cDNA fragment isolated by the yeast two-hybrid screen
indicated that this fragment encoded the 487 carboxyl-terminal amino
acids of the Vav protein, including a portion of the DH domain, and the
complete pleckstrin homology, CR, SH3, and SH2
domains.
Figure 2: Vav, Sos, and C3G from resting and activated B cells selectively interact with SH3 domains of bacterial GST-Grb2 fusion proteins. A, primary B cells were isolated by immunodepletion of T cells from tonsil cell suspensions. Cells were incubated in medium(-) or with anti-human Ig polyclonal antibody to trigger the BCR (+). Nonidet P-40 lysates were prepared, precleared with bacterial GST protein and incubated with GST fusion proteins of full-length Grb2, its SH2 domain, its carboxyl-terminal (SH3-C) or amino-terminal (SH3-N) SH3 domains. Part of the lysates was used for immunoprecipitation with anti-Vav mAb. Isolated proteins were separated by SDS-PAGE transferred to nitrocellulose sheets, incubated with anti-Vav mAb, and second step antibody-conjugated to horseradish peroxidase and developed by enhanced chemiluminescence. The arrow indicates the 95-kDa Vav protein. The asterisks indicate the positions of the fusion proteins. B, Ramos B cells were treated as described for A, lysates were incubated with GST-Grb2 fusion proteins, and samples were immunoblotted with anti-Sos polyclonal antibody. C, Ramos B cells were treated as described for A, lysates were incubated with GST-Grb2 fusion proteins or with anti-C3G polyclonal serum, and samples were immunoblotted with anti-C3G mAb. The asterisks indicate the positions of the fusion proteins.
Proline-rich motifs in the guanine nucleotide exchange factor Sos are known to interact with Grb2. In contrast to Vav, Sos derived from human tonsillar B cells, did not discriminate between the SH3-N and SH3-C domains of Grb2. It bound to both SH3 domains, and not to the SH2 domain (Fig. 2B), as expected(41) .
The C3G protein is known to bind to Grb2 in vitro(30) . Its expression and function in lymphocytes has thus far not been examined. Fig. 2C shows that the 145-kDa C3G molecule can effectively be isolated from Ramos B cells with specific antibody. Endogenous C3G from both resting and activated B cells bound with equal efficiency to GST-Grb2 fusion proteins (Fig. 2C). Upon stimulation of the BCR, no tyrosine phosphorylation of the C3G molecule could be detected (data not shown). Interestingly, C3G showed yet another Grb2 binding specificity than Vav and Sos. It preferentially interacted with the amino-terminal SH3 domain and to a lesser extent with the carboxyl-terminal SH3 domain. It did not bind at all to the Grb2 SH2 domain (Fig. 2C). We conclude that in vitro, Vav, Sos, and C3G proteins from human B cells all have the ability to bind to Grb2, which is not significantly affected by BCR stimulation. The three proteins selectively interact with the two SH3 domains of Grb2. While Sos binds with similar efficiency to both domains, Vav preferentially associates with SH3-C and C3G with SH3-N.
Figure 3: Sos, but not Vav or C3G, associates with Shc-Grb2 in vivo, and Vav associates with Crk. A, Ramos and Daudi Burkitt lymphoma B cells were incubated with medium(-) or stimulated with anti-BCR reagent (+), cells were lysed with Nonidet P-40 and subjected to immunoprecipitation with anti-Shc polyclonal serum. Immunoprecipitates were separated by SDS-PAGE, transferred to nitrocellulose sheets, and immunoblotted with anti-Sos antibody. B, tonsillar B cells were treated as described for A, and lysates were subjected to immunoprecipitation with anti-Shc, -Grb2, and -Crk antibodies. Samples were immunoblotted with anti-Vav mAb. NRS, normal rabbit serum control precipitate. C, Ramos B cells were treated as described for A, and lysates were subjected to immunoprecipitation with anti-Shc, -Grb, -Crk, and -C3G antibodies. Samples were immunoblotted with anti-C3G mAb. NRS, normal rabbit serum control precipitate. The asterisk indicates the position of the Ig heavy chains of the immunoprecipitating antibodies. The monoclonal mouse anti-Crk Ig reacts stronger with the second step reagent used for immunoblotting than the polyclonal rabbit Ig used for immunoprecipitation of Grb2, Shc, and C3G.
In contrast to Sos, Vav could not be detected by immunoblotting in anti-Shc or -Grb2 immunoprecipitates from Burkitt lymphoma cell lines (results not shown), or from primary tonsillar B cells either prior to, or after BCR stimulation (Fig. 3B). Similarly, C3G was not found in either anti-Grb2 or -Shc immunoprecipitates from resting or BCR-activated B cells (Fig. 3C). We conclude that, despite the shared in vitro reactivity of Sos, Vav, and C3G with Grb2-SH3 domains, only Sos has detectable affinity for Grb2 in human B lymphocytes and is found in a BCR-inducible Shc-Grb2 complex.
In PC12 pheochromocytoma cells, C3G was shown to constitutively interact with endogenous Crk(31) . However, we could not identify such an association in B cells, either before or after BCR stimulation (Fig. 3C). We conclude that Sos, Vav, and C3G differentially interact with SH3 domain containing adaptor proteins in intact human B lymphocytes. Whereas Sos participates in a BCR-induced Shc-Grb2 complex, Vav and C3G do not. Vav instead associates with Crk, but C3G does not.
Figure 4: Crk-L is expressed in B cells and interacts with C3G. Ramos B cells were incubated in medium(-) or stimulated with anti-BCR reagent (+), Nonidet P-40 lysates were prepared, and Crk-L was isolated by immunoprecipitation. Samples were separated by SDS-PAGE, transferred to nitrocellulose, and either incubated with anti-Crk-L antibody or with anti-C3G mAb. NRS, normal rabbit serum control precipitate. The asterisk indicates the position of the Ig heavy chains of the immunoprecipitating antibodies.
To determine whether Crk-L is regulated by the BCR, we first investigated whether it is phosphorylated in response to receptor stimulation. However, no inducible tyrosine phosphorylation of either Crk or Crk-L was observed (results not shown). We were able to link Crk and Crk-L to BCR-induced signaling by the following observation. The Cbl proto-oncogene product inducibly associates with both adaptors upon B cell activation (Fig. 5). We conclude that Crk-L, and possibly C3G, which constitutively interacts with this adaptor, participate in a BCR-induced signaling pathway.
Figure 5: Crk and Crk-L associate with Cbl upon BCR stimulation. Ramos B cells were incubated with medium(-) or stimulated with anti-BCR reagent (+), and Nonidet P-40 lysates were prepared and subjected to immunoprecipitation with anti-Crk-L and anti-Crk-I and -II antibodies. Samples were transferred to nitrocellulose sheets and immunoblotted with anti-Cbl reagent. NRS, normal rabbit serum control precipitate.
In this paper, we show that Sos, Vav, and C3G all bind to Grb2 in vitro, with different specificities for the amino-terminal or carboxyl-terminal SH3 domains. However, the endogenous GEFs in B lymphocytes bind to different adaptor proteins. Only Sos binds to Grb2, whereas Vav binds to Crk and C3G to Crk-L. The BCR communicates with all three adaptor proteins. BCR stimulation induces recruitment of Grb2 to Shc and the association of Crk with Vav. Crk-L was shown to be connected to BCR signaling by its inducible interaction with the Cbl molecule.
It is of interest that Sos, Vav, and C3G associate with different adaptor proteins in B lymphocytes. The Grb2, Crk, and Crk-L adaptors may serve to differentially regulate intracellular targeting, substrate specificity, and/or catalytic activity of Sos, Vav, and C3G. Sos is known to activate Ras and therewith the MAP kinase pathway leading to cell proliferation. Sos is constitutively associated with Grb2 SH3 domains, leaving the SH2 domain available for interaction with other proteins. The BCR-inducible Shc-Grb2 association is mediated by the SH2 domain of Grb2 and presumably serves to recruit the Grb2-Sos complex to the plasma membrane where Ras is located. It is much less clear which are the in vivo targets of the GEF activity of Vav and C3G and which cellular responses are regulated by these two proteins.
The homology of Vav with Dbl and Cdc24 suggests specificity for the Cdc25, Rho, and/or Rac small GTP-binding proteins, which play a role in cytoskeletal organization(16) . However, no activity of Vav on these molecules has been demonstrated. Its specificity for Ras is debated, but Vav has been implicated in cellular activation and mitogenesis. Its overexpression can enhance MAP kinase activity in fibroblasts, in a Ras-independent manner(21) . In T lymphocytes, Vav overexpression can activate the NFAT nuclear factor, involved in interleukin-2 expression (43) . In this case, Vav function is dependent on Ras. It is not known whether the effects of Vav on mitogenic pathways can be ascribed to its GEF domain or to functions exerted by other domains.
Originally, C3G was thought to regulate Ras, based on the sequence homology of its carboxyl-terminal domain with Cdc25 and Sos and on the suppression of a Cdc25 loss of function mutation in yeast by this domain(30) . Recently, however, C3G was shown to have GEF activity for Rap1, rather than Ras(32) . Rap1 has been implicated in negative regulation of Ras(44) . It will be of interest to determine whether C3G may have targets other than Rap1 and whether it is involved in the regulation of mitogenesis and/or other cellular responses.
Vav associated with Grb2 in yeast and with bacterial Grb2-GST fusion proteins, but we could not detect a Vav-Grb2 interaction in vivo. Vav was found earlier to bind to Grb2 SH3 domains in yeast and in transfected mammalian cells(45) . More importantly, coimmunoprecipitation experiments showed that Vav inducibly associates with Grb2 and Shc upon pervanadate treatment of T lymphocytes(46) . This suggests that in vivo, tyrosine phosphorylation of Vav is involved its binding to Grb2 an raises the question whether the interaction involves a Grb2 SH2 domain rather than an SH3 domain. We do not exclude the possibility that Vav can associate with Shc-Grb2 in B cells, but failed to detect it under conditions that allowed binding of Vav to Crk. Therefore, the Vav-Crk interaction might be prevalent in BCR-mediated signaling.
Vav was shown to bind in vivo to Crk-I and/or Crk-II proteins. The v-crk avian retroviral gene has transforming properties and induces tyrosine phosphorylation of intracellular substrates. Since Crk-I and -II do not contain enzymatic activity, these properties must be ascribed to associating proteins(17) . Crk can interact via one of its SH3 domains with the Abl tyrosine kinase, which might be involved in Crk-induced cellular transformation. In addition, Abl can phosphorylate Crk(4) , indicating that Crk function might be regulated by phosphorylation. In B cells, we have not observed BCR-induced tyrosine phosphorylation of Crk, but the Crk-Vav association was induced upon BCR stimulation. It is possible that BCR-induced tyrosine phosphorylation of Vav allows the Crk-Vav interaction to occur. We do not know whether this interaction involves Crk SH3 or SH2 domains.
The Crk SH3 domain can bind Sos as well as C3G and Crk overexpression enhanced nerve growth factor-induced Ras activity, indicating that Crk-associated proteins can regulate Ras(31) . We found that in B cells, C3G is not associated with Crk, but with the product of a related gene, Crk-L. Crk-L was identified at the genomic level, by cross-reactivity with a Crk cDNA (42) and contains one SH2 and two SH3 domains. Like Crk, Crk-L can associate with the Abl protein-tyrosine kinase and can be phosphorylated by it(47) . Crk-L is not phosphorylated in response to BCR stimulation (results not shown) and its association with C3G appears to be constitutive. Therefore, it was not immediately clear whether the Crk-L-C3G complex is linked to BCR signaling. However, we found that BCR stimulation induces association of Crk-L with the Cbl protein, indicating that Crk-L is regulated by the BCR. We don't know whether the pool of Crk-L that is associated with Cbl can simultaneously interact with C3G, but it is an interesting possibility. Thus far, we could not detect phosphorylation or plasma membrane translocation of C3G in response to BCR stimulation. Obviously, the most important aim is to define the in vivo activity of C3G and assess whether this can be modulated by BCR stimulation.
The Cbl protein has emerged as a major
target for antigen receptor-induced tyrosine phosphorylation both in T
cells (48) and in B cells(34) . Recently, epidermal
growth factor was also shown to induce tyrosine phosphorylation of Cbl
and its association with the epidermal growth factor
receptor(49) . Interestingly, Cbl associates with all three
adaptors described here: it constitutively interacts with Grb2 ()and inducibly associates with Crk-I and -II and Crk-L upon
BCR triggering. These three adaptors could bring Cbl in proximity of
Sos, Vav, and C3G, respectively. The function of Cbl is as yet unclear,
but it was identified as the transforming gene of a murine retrovirus,
which induces pre-B cell lymphomas and myeloid leukemias(50) .
In Caenorhabditis elegans, the Cbl homologue sli-1 was recently identified as a negative regulator of the Let-23 receptor tyrosine kinase pathway(51) , which involves Grb2
and Ras homologues. We hypothesize, therefore, that by participating in
the three BCR-regulated adaptor complexes, Cbl might affect the
function of associated Sos, Vav, and C3G and therewith regulate
mitogenesis and possibly other responses to BCR stimulation.