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

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 FcγRIIb. 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 FcγRIIb. To resolve this paradox, we examined the protein interactions of SHIP. Our results demonstrated that isolated FcγRIIb contains SHIP but not Shc; likewise, Shc isolates contain SHIP but not FcγRIIb. In contrast, SHIP isolates contain both proteins, revealing two separate pools of SHIP: one bound to FcγRIIb and one bound to Shc. Kinetic studies reveal rapid SHIP association with FcγRIIb but slower and more transient association with Shc. Affinity measurements using a recombinant SHIP SH2 domain and phosphopeptides derived from FcγRIIb (corresponding to Y273) and Shc (corresponding to Y317) revealed an approximately equal rate of binding but a 10-fold faster dissociation rate for FcγRIIb compared with Shc phosphopeptide and yielding in an affinity of 2.1 μM for FcγRIIb and 0.26 μM for Shc. These findings are consistent with a model in which SHIP transiently associates with FcγRIIb to promote SHIP phosphorylation, whereupon SHIP binds to Shc and dissociates from FcγRIIb.