THE GAB1 PROTEIN IS A DOCKING SITE FOR MULTIPLE PROTEINS INVOLVED IN SIGNALING BY THE B-CELL ANTIGEN RECEPTOR

Gab1 is a member of the docking/scaffolding protein family which inclu des IRS-1, IRS-2, c-Cbl, p130(cas), and p62(dok). These proteins conta in a variety of protein-protein interaction motifs including multiple tyrosine residues that when phosphorylated can act as binding sites fo r Src homology 2 (SH2) domain-containing signaling proteins. We show i n the RAMOS human B cell line that Gab1 is tyrosine-phosphorylated in response to B cell antigen receptor (BCR) engagement. Moreover, tyrosi ne phosphorylation of Gab1 correlated with the binding of several SH2- containing signaling proteins to Gab1 including Shc, Grb2, phosphatidy linositol 3-kinase, and the SHP-2 tyrosine phosphatase, Far Western an alysis showed that the SH2 domains of Shc, SHP-2, and the p85 subunit of phosphatidylinositol 3-kinase could bind directly to tyrosine-phosp horylated Gab1 isolated from activated RAMOS cells. In contrast, the G rb2 SH2 domain did not bind directly to Gab1 but instead to the Shc an d SHP-2 associated with Gab1, We also show that Gab1 is present in the membrane-enriched particulate fraction of RAMOS cells and that Gab1/s ignaling protein complexes are found in this fraction after BCR engage ment. Thus, tyrosine-phosphorylated Gab1 may recruit cytosolic signali ng proteins to cellular membranes where they can act on membrane-bound targets. This may be a critical step in the activation of multiple BC R signaling pathways.