Shc contains two Grb2 binding sites needed for efficient formation of complexes with SOS in B lymphocytes

Cross-linking of the B-cell antigen receptor (BCR) induces tyrosine phosphorylation of She, which is believed to lead to the activation of Pas, Previous work has shown that tyrosine-phosphorylated She forms complexes with another adapter protein, Grb2, and the Pas guanine nucleotide exchange factor SOS. Here, we demonstrate that phosphorylation of She by the hematopoietic cell-specific tyrosine kinase Syk induces binding of Grb2 to She, suggesting that Syk phosphorylates She in stimulated B cells, Surprisingly, Syk-phosphorylated She possesses two Grb2 binding sites rather than the one site that has been previously reported, Both of these sites are required for efficient formation of Shc-Grb2-SOS complexes in vitro and in vivo. We suggest that two Grb2 proteins anchored by a single She protein bind simultaneously to one SOS molecule, resulting in a complex that is more stable than a complex containing only a single Grb2 protein bound to one SOS molecule. This model is consistent with our observation that BCR stimulation greatly increases the amount of SOS associated with Grb2.