SOS, VAV, AND C3G PARTICIPATE IN B-CELL RECEPTOR-INDUCED SIGNALING PATHWAYS AND DIFFERENTIALLY ASSOCIATE WITH SHC-GRB2, CRK, AND CRK-L ADAPTERS

B cell antigen receptor (BCR)-mediated signal transduction controls B cell proliferation and differentiation. The BCR activates Ras, presuma bly 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 i nvolving the Grb2 adaptor, we undertook the isolation of Grb2-binding proteins. Using the yeast two-hybrid system and bacterial fusion prote ins, Vav and C3G were identified as Grb2 binders. Vav is a putative nu cleotide exchange factor and a target for BCR-induced tyrosine phospho rylation. C3G exerts nucleotide exchange activity on the Ras-related R ap1 protein. While Sos binds to both Grb2 Src homology-3 (SH3) domains , Vav was found to associate selectively with the carboxyl-terminal SH 3 domain, while C3G bound selectively to the amino-terminal SH3 domain of bacterially expressed Grb2. Despite the association of Vav with Gr b2 in vitro, we could not demonstrate an interaction between endogenou s Vav and Grb2 molecules in primary B cells. Instead, Vav was found to inducibly associate with the Grb2-related adaptor protein Crk upon BC R stimulation. C3G did not bind to either Grb2, She, or Crk in vivo. I nstead, C3G was found in association with the Crk-L adaptor, both befo re and after BCR stimulation. We show that Crk-L also participates in BCR signaling, since it inducibly interacts with tyrosine-phosphorylat ed 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.