Interaction of p72^syk with the γ and β Subunits of the High-Affinity Receptor for Immunoglobulin E, FcεRI

Abstract

Activation of protein tyrosine kinases is one of the initial events following aggregation of the high-affinity receptor for immunoglobulin E (Fc«RI) on RBL-2H3 cells, a model mast cell line. The protein tyrosine kinase p72^syk (Syk), which contains two Src homology 2 (SH2) domains, is activated and associates with phosphor-ylated Fc«RI subunits after receptor aggregation. In this report, we used Syk SH2 domains, expressed in tandem or individually, as fusion proteins to identify Syk-binding proteins in RBL-2H3 lysates. We show that the tandem Syk SH2 domains selectively associate with tyrosine-phosphorylated forms of the γ and β subunits of Fc«RI. The isolated carboxy-proximal SH2 domain exhibited a significantly higher affinity for the Fc«RI subunits than did the amino-proximal domain. When in tandem, the Syk SH2 domains showed enhanced binding to phosphorylated γ and β subunits. The conserved tyrosine-based activation motifs contained in the cytoplasmic domains of the γ and β subunits, characterized by two YXXL/I sequences in tandem, represent potential high-affinity binding sites for the dual SH2 domains of Syk. Peptide competition studies indicated that Syk exhibits a higher affinity for the phosphorylated tyrosine activation motif of the γ subunit than for that of the β subunit. In addition, we show that Syk is the major protein in RBL-2H3 cells that is affinity isolated with phosphorylated peptides corresponding to the phosphorylated γ subunit motif. These data suggest that Syk associates with the γ subunit of the high-affinity receptor for immunoglobulin E through an interaction between the tandem SH2 domains of Syk and the phosphorylated tyrosine activation motif of the γ subunit and that Syk may be the major signaling protein that binds to Fc«RI tyrosine activation motifs in RBL-2H3 cells.