Co‐clustering of Fcγ and B cell receptors induces dephosphorylation of the Grb2‐associated binder 1 docking protein

Abstract

The immunoreceptor tyrosine-based inhibitory motif (ITIM) of human type IIb Fcg receptor (FcgRIIb) is phosphorylated on its tyrosine upon co-clustering with the B cell receptor (BCR). The phosphorylated ITIM (p-ITIM) binds to the SH2 domains of polyphosphoinositol 5-phosphatase (SHIP) and the tyrosine phosphatase, SHP-2. We investigated the involvement of the molecular complex composed of the phosphorylated SHIP and FcgRIIb in the activation of SHP-2. As a model compound, we synthesized a bisphosphopeptide, combining the sequences of p-ITIM and the N-terminal tyrosine phosphorylated motif of SHIP with a flexible spacer. This compound bound to the recombinant SH2 domains of SHP-2 with high affinity and activated the phosphatase in an in vitro assay. These data suggest that the phosphorylated FcgRII-SHIP complexes formed in the intact cells may also activate SHP-2. Grb2-associated binder 1 (Gab1) is a multisite docking protein, which becomes tyrosine-phosphorylated in response to various types of signaling, including BCR. In turn it binds to the SH2 domains of SHP-2, SHIP and the p85 subunit of phosphatidyl inositol 3-kinase (PtdIns3-K) and may regulate their activity. Gab1 is a potential substrate of SHP-2, thus its binding to FcgRIIb may modify the Gab1-bound signaling complex. We show here that Gab1 is part of the multiprotein complex assembled by FcgRIIb upon its co-clustering with BCR. Gab1 may recruit SH2 domaincontaining molecules to the phosphorylated FcgRIIb. SHP-2, activated upon the binding to FcgRIIb-SHIP complex, partially dephosphorylates Gab1, resulting in the release of PtdIns3-K and ultimately in the inhibition of downstream activation pathways in BCR/FcgRIIb co-aggregated cells.

The PI3K pathway drives the maturation of mast cells via microphthalmia transcription factor

“…23 SHIP also has been shown to recruit p62Dok and to modulate Ras activity and also may induce the activation of SHP2. 24,25 Our in vitro and in vivo findings suggest that the increased rate of BMMC maturation and tissue mast cell numbers because of SHIP deficiency are predominantly a result of enhanced PI3K activity via the recruitment and activation of the p85␣-p110 PI3K complex, because deficiency of p85␣ alone in BMMCs lacking SHIP corrects the enhanced maturation defect in vitro and partially restores normal mast cell numbers in vivo. These findings are further supported by conducting experiments using WT and p85␣ Ϫ/Ϫ MCps expressing activated versions of PI3K and AKT, respectively.…”

Fc gamma receptor IIb modulates the molecular Grb2 interaction network in activated B cells

“…T cells expressing a phophataseinactive SHP-2 variant develop normally but generate enhanced immune responses. It has been shown previously that FcγRIIb co-engagement with the BCR leads to dephosphorylation of CD19 and Gab-2 [39,40]. An increased integration of SHP-2 might be the explanation for this observation.…”

Optimization of the cellular import of functionally active SH2-domain-interacting phosphopeptides

“…1c). Our earlier peptide pull-down experiments followed by Western blot revealed that the major molecule interacting both with pITIM, the chimera and GD-LD PPs was SHP-2 [15,39] indicating that these PPs may regulate the activity of SHP-2 phosphatase ex vivo.…”

“…SHP-2 is activated by the simultaneous engagement of its two SH2 domains by a bisphosphorylated activation motif [2,38]. Accordingly, we also tested the effects of a chimeric bisphosphorylated peptide consisting of pITIM and a phosphotyrosine-containing peptide of SHIP combined with a flexible linker [TEIINPNY(p)MGVG(AG) 15 AENTITY(p)SLLMHP, chimera] [39]. This chimeric peptide induced the tyrosine phosphorylation of proteins at 30, 50 and 66-70 kDa in a dose-dependent manner (Fig.…”