Abstract
Coligation of FcgammaRIIb1 with the B cell receptor (BCR) or FcepsilonRI on mast cells inhibits B cell or mast cell activation. Activity of the inositol phosphatase SHIP is required for this negative signal. In vitro, SHIP catalyzes the conversion of the phosphoinositide 3-kinase (PI3K) product phosphatidylinositol 3,4, 5-trisphosphate (PIP3) into phosphatidylinositol 3,4-bisphosphate. Recent data demonstrate that coligation of FcgammaRIIb1 with BCR inhibits PIP3-dependent Btk (Bruton's tyrosine kinase) activation and the Btk-dependent generation of inositol trisphosphate that regulates sustained calcium influx. In this study, we provide evidence that coligation of FcgammaRIIb1 with BCR induces binding of PI3K to SHIP. This interaction is mediated by the binding of the SH2 domains of the p85 subunit of PI3K to a tyrosine-based motif in the C-terminal region of SHIP. Furthermore, the generation of phosphatidylinositol 3,4-bisphosphate was only partially reduced during coligation of BCR with FcgammaRIIb1 despite a drastic reduction in PIP3. In contrast to the complete inhibition of Tec kinase-dependent calcium signaling, activation of the serine/threonine kinase Akt was partially preserved during BCR and FcgammaRIIb1 coligation. The association of PI3K with SHIP may serve to activate PI3K and to regulate downstream events such as B cell activation-induced apoptosis.
Highlights
Coligation of Fc␥RIIb1 with the B cell receptor (BCR) or Fc⑀RI on mast cells inhibits B cell or mast cell activation
Recruitment of phosphoinositide 3-kinase (PI3K) to Tyrosine-phosphorylated SHIP upon Coligation of the B Cell Receptor with Fc␥RIIb1—A20 cells were stimulated with F(abЈ)2 or intact anti-IgG antibodies, and immunoprecipitates of PI3K were resolved by SDS-PAGE and probed for associated phosphotyrosine-containing proteins by Western blotting
To test whether SHIP was directly associated with PI3K or whether it was immunoprecipitated as part of the receptor complex by the stimulating intact anti-Ig antibody, the lysates were incubated with protein G-agarose beads alone prior to SDS-PAGE and Western blotting with anti-SHIP antibodies
Summary
7489 –7494, 1999 Printed in U.S.A. The SH2 Domain-containing Inositol 5-Phosphatase (SHIP) Recruits the p85 Subunit of Phosphoinositide 3-Kinase during Fc␥RIIb1-mediated Inhibition of B Cell Receptor Signaling*. Recent data demonstrate that coligation of Fc␥RIIb1 with BCR inhibits PIP3-dependent Btk (Bruton’s tyrosine kinase) activation and the Btk-dependent generation of inositol trisphosphate that regulates sustained calcium influx. We provide evidence that coligation of Fc␥RIIb1 with BCR induces binding of PI3K to SHIP This interaction is mediated by the binding of the SH2 domains of the p85 subunit of PI3K to a tyrosine-based motif in the C-terminal region of SHIP. Coengagement of BCR with Fc␥RIIb1 resulted in a tyrosine phosphorylation-dependent recruitment of the p85 subunit of PI3K to SHIP This interaction is mediated by direct binding of the SH2 domain of PI3K to a signature motif in the C-terminal region of SHIP. Production of PIP2 and activation of Akt ( called protein kinase B) were observed during BCR/Fc␥RIIb1 coengagement
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