Differential effects of B cell receptor and B cell receptor-Fc gamma RIIB1 engagement on docking of Csk to GTPase-activating protein (GAP)-associated p62

The stimulatory and inhibitory pathways initiated by engagement of stimulatory receptors such as the B cell receptor for antigen (BCR) and inhibitory receptors such as Fc gamma receptors of the IIB1 type (Fc gamma RIIB1) intersect in ways that art: poorly understood at the molecular level. Because the tyrosine kinase Csk is a potential negative regulator of lymphocyte activation, we examined the effects of BCR and Fc gamma RIIB1 engagement on the binding of Csk to phosphotyrosine-containing proteins. Stimulation of a B lymphoma cell Line, A20, with intact anti-IgG antibody induced a direct, SH2-mediated association between Csk and a 62-kD phosphotyrosine-containing protein that was identified as RasGTPase-activating protein-associated p62 (GAP-A.p62). In contrast, stimulation of A20 cells with anti-IgG F(ab')(2) resulted in little increase in the association of Csk with GAP-A.p62. The effect of Fc gamma RIIB1 engagement on this association was abolished by blockade of Fc gamma RIIB1 with the monoclonal antibody 2.4G2. Furthermore, the increased association between Csk and GAP-A.p62 seen upon stimulation with intact anti-Ig was abrogated in the Fc gamma RIIB1-defcient cell line IIA1.6 and recovered when Fc gamma RIIB1 expression was restored by transfection. The differential effects of BCR and BCR-Fc gamma RIIB1-mediated signaling on the phosphorylation of GAP-A.p62 and its association with Csk suggest that docking of Csk to GAP-A.p62 may function in the negative regulation of antigen receptor-mediated signals in B cells.