Co-clustering of Fc gamma and B cell receptors induces dephosphorylation of the Grb2-associated binder 1 docking protein

The immunoreceptor tyrosine-based inhibitory motif (ITIM) of human type Ilb Fc gamma receptor (Fc gamma RIIb) is phosphorylated on its tyrosine upon c o-clustering with the B cell receptor (BCR). The phosphorylated ITIM (p-ITI M) binds to the SH2 domains of polyphosphoinositol 5-phosphatase (SHIP) and the tyrosine phosphatase, SHP-2. We investigated the involvement of the mo lecular complex composed of the phosphorylated SHIP and Fc gamma RIIb in th e activation of SHP-2. As a model compound, we synthesized a bisphosphopept ide, combining the sequences of p-ITIM and the N-terminal tyrosine phosphor ylated motif of SHIP with a flexible spacer. This compound bound to the rec ombinant SH2 domains of SHP-2 with high affinity and activated the phosphat ase in an in vitro assay. These data suggest that the phosphorylated Fc gam ma RII-SHIP complexes formed in the intact cells may also activate SHP-2. G rb2-associated binder 1 (Gab1) is a multisite docking protein, which become s tyrosine-phosphorylated in response to various types of signaling, includ ing BCR. In turn it binds to the SH2 domains of SHP-2, SHIP and the p85 sub unit of phosphatidyl inositol 3-kinase (PtdIns3-K) and may regulate their a ctivity. Gab1 is a potential substrate of SHP-2, thus its binding to Fc gam ma RIIb may modify the Gab1-bound signaling complex. We show here that Gab1 is part of the multiprotein complex assembled by Fc gamma RIIb upon its co -clustering with BCR. Gab1 may recruit SH2 domain-containing molecules to t he phosphorylated Fc gamma RIIb. SHP-2, activated upon the binding to Fc ga mma RIIb-SHIP complex, partially dephosphorylates Gab1, resulting in the re lease of PtdIns3-K and ultimately in the inhibition of downstream activatio n pathways in BCR/Fc gamma RIIb co-aggregated cells.