Protein interactions of Src homology 2 (SH2) domain-containing inositol phosphatase (SHIP): Association with Shc displaces SHIP from Fc gamma RIIb inB cells

Our recent studies revealed that the inositol phosphatase Src homology 2 (S H2) domain-containing inositol phosphatase (SHIP) is phosphorylated and ass ociated with Shc exclusively under negative signaling conditions in B cells , which is due to recruitment of the SHIP SH2 domain to the Fc gamma RIIb. In addition, we reported that SHIP-Shc interaction involves both SHIP SH2 a nd Shc phosphotyrosine binding domains. These findings reveal a parades in which the single SH2 domain of SHIP is simultaneously engaged to two differ ent proteins: Shc and Fc gamma RIIb, To resolve this paradox, we examined t he protein interactions of SHIP. Our results demonstrated that isolated Fc gamma RIIb contains SHIP but not Shc; likewise, Shc isolates contain SHIP b ut not Fc gamma RIIb. In contrast, SHIP isolates contain both proteins, rev ealing two separate pools of SHIP: one bound to Fc gamma RIIb and one bound to Shc. Kinetic studies reveal rapid SHIP association with Fc gamma RIIb b ut slower and more transient association with Shc. Affinity measurements us ing a recombinant SHIP SH2 domain and phosphopeptides derived from Fc gamma RIIb (corresponding to Y273) and Shc (corresponding to Y317) revealed an a pproximately equal rate of binding but a 10-fold faster dissociation rate f or Fc gamma RIIb compared with Shc phosphopeptide and yielding in an affini ty of 2.1 mu M for Fc gamma RIIb and 0.26 mu M for Shc. These findings are consistent with a model in which SHIP transiently associates with Fc gamma RIIb to promote SHIP phosphorylation, whereupon SHIP binds to Shc and disso ciates from Fc gamma RIIb.