Differential Dephosphorylation of the FcRγ Immunoreceptor Tyrosine-based Activation Motif Tyrosines with Dissimilar Potential for Activating Syk

Toshiyuki Yamashita,Ryo Suzuki,Peter S Backlund,Yumi Yamashita,Alfred L Yergey,Juan Rivera

doi:10.1074/jbc.m802679200

Abstract

The cell surface-expressed gamma chain of the high affinity receptor for IgE (FcepsilonRI) can be phosphorylated on two tyrosine residues of the immunoreceptor tyrosine-based activation motif (ITAM), leading to recruitment and activation of spleen tyrosine kinase (Syk), a kinase that is essential for mast cell signaling and allergic responses. However, it is not known whether preferential phosphorylation or dephosphorylation of the two individual FcRgamma tyrosines (the N-terminal Tyr47 and C-terminal Tyr58) could regulate Syk activation. Herein we report that phosphorylation of only Tyr58 was able to elicit Syk phosphorylation and a weak rise in intracellular calcium, suggesting that Tyr58 phosphorylation may be distinctively important for Syk activation. In vitro and in vivo studies revealed that both Tyr47 and Tyr58 could be similarly phosphorylated. However, mass spectrometric analysis of the phosphorylated FcepsilonRgamma from bone marrow-derived mast cells showed that phosphorylation at Tyr47 was at least 2-fold greater than at Tyr58. This suggested that, once phosphorylated, Tyr58 is preferentially dephosphorylated. In vitro studies demonstrated more efficient dephosphorylation of Tyr58 (by the receptor-associated phosphatases SHP-1 and SHP-2) than of Tyr47. Analysis of Syk binding to wild type and mutant phosphorylated FcepsilonRI revealed that mutation at Tyr58 almost completely ablated Syk binding, whereas mutation at Tyr47 moderately reduced Syk binding. The findings argue for a novel regulatory mechanism, where dephosphorylation of phospho-Tyr58 is likely to promote the down-regulation of Syk activation and suppression of mast cell responses.

Highlights

Phosphorylation and dephosphorylation are key events in the initiation and regulation of cell signaling and responses
Prior studies already demonstrated that the Fc⑀RI␤ immunoreceptor tyrosine-based activation motif (ITAM) tyrosines differentially bind Lyn [12, 14], but to date no studies address whether differential phosphorylation/dephosphorylation might regulate such binding
Because nonreducing conditions resolved the largest number of phosphorylated FcR␥ species and might reflect multiple species capable of binding spleen tyrosine kinase (Syk), we used these conditions in far Western analysis of Syk-Src homology 2 (SH2) domain binding to phosphorylated FcR␥

Summary

EXPERIMENTAL PROCEDURES

Antibodies—The cytokinergic [15] monoclonal anti-DNP IgE was prepared from the culture supernatants of the H1 DNP⑀-26.82 hybridoma [16]. 125I-Labeled IgE was prepared as described [17]. For immunoprecipitation of IgE-bound Fc⑀RI, postnuclear supernatants were incubated with goat anti-mouse IgE (10 ␮l/ml lysate) for 1 h at 4 °C followed by incubation with protein A-Sepharose beads (30 ␮l/ml lysate; GE Healthcare) for 1 h at 4 °C. Postnuclear supernatants from 1 ϫ 109 cells were precleared by passing through a 0.5-ml Protein A-Sepharose column, and IgE-bound Fc⑀RI was purified by affinity chromatography using 0.5 ml of Protein A-Sepharose beads that were covalently bound to a rabbit antimouse IgG, F(abЈ) fragment-specific, using dimethyl pimelimidate (Pierce). LC/MS/MS Analysis of the FcR␥—In order to map which sites on the FcR␥ are phosphorylated following Fc⑀RI stimulation, protein bands from the SDS-polyacrylamide gels were digested with trypsin to produce peptides that could be analyzed by LC/MS/MS.

RESULTS

Mass error

DISCUSSION