Protein Interactions of Src Homology 2 (SH2) Domain-Containing Inositol Phosphatase (SHIP): Association with Shc Displaces SHIP from FcgRIIb in B Cells1

Our recent studies revealed that the inositol phosphatase Src homology 2 (SH2) domain-containing inositol phosphatase (SHIP) is phosphorylated and associated with Shc exclusively under negative signaling conditions in B cells, which is due to recruitment of the SHIP SH2 domain to the FcgRIIb. In addition, we reported that SHIP-Shc interaction involves both SHIP SH2 and Shc phosphotyrosine binding domains. These findings reveal a paradox in which the single SH2 domain of SHIP is simultaneously engaged to two different proteins: Shc and FcgRIIb. To resolve this paradox, we examined the protein interactions of SHIP. Our results demonstrated that isolated FcgRIIb contains SHIP but not Shc; likewise, Shc isolates contain SHIP but not FcgRIIb. In contrast, SHIP isolates contain both proteins, revealing two separate pools of SHIP: one bound to FcgRIIb and one bound to Shc. Kinetic studies reveal rapid SHIP association with FcgRIIb but slower and more transient association with Shc. Affinity mea-surements using a recombinant SHIP SH2 domain and phosphopeptides derived from FcgRIIb (corresponding to Y273) and Shc (corresponding to Y317) revealed an approximately equal rate of binding but a 10-fold faster dissociation rate for FcgRIIb compared with Shc phosphopeptide and yielding in an affinity of 2.1 mM for FcgRIIb and 0.26 mM for Shc. These findings are consistent with a model in which SHIP transiently associates with FcgRIIb to promote SHIP phosphorylation, whereupon SHIP binds to Shc and dissociates from FcgRIIb. The Journal of Immunology, 1999, 162: 1408–1414. T he positive signal transduction process triggered by theB cell Ag receptor, surface Ig (sIg),4 features three in-dependent biochemical pathways including activation of the g isoform of phospholipase C, stimulation of phospha