INTERACTION OF P72(SYK) WITH THE GAMMA-SUBUNITS AND BETA-SUBUNITS OF THE HIGH-AFFINITY RECEPTOR FOR IMMUNOGLOBULIN-E, IMMUNOGLOBULIN-FC-EPSILON-RI

Activation of protein tyrosine kinases is one of the initial events fo llowing aggregation of the high-affinity receptor for immunoglobulin E (Fc epsilon RI) on RBL-2H3 cells, a model mast cell line. The protein tyrosine kinase p72(syk) (Syk), which contains two Src homology 2 (SH 2) domains, is activated and associates with phosphorylated Fc epsilon RI subunits after receptor aggregation. In this report, we used Syk S H2 domains, expressed in tandem or individually, as fusion proteins to identify Syk-binding proteins in RBL-2H3 lysates. We show that the ta ndem Syk SH2 domains selectively associate with tyrosine-phosphorylate d forms of the gamma and beta subunits of Fc epsilon RI. The isolated carboxy-proximal SH2 domain exhibited a significantly higher affinity for the Fc epsilon RI subunits than did the amino-proximal domain. Whe n in tandem, the Syk SH2 domains showed enhanced binding to phosphoryl ated gamma and beta subunits. The conserved tyrosine-based activation motifs contained in the cytoplasmic domains of the gamma and beta subu nits, characterized by two YXXL/I sequences in tandem, represent poten tial high-affinity binding sites for the dual SH2 domains of Syk. Pept ide competition studies indicated that Syk exhibits a higher affinity for the phosphorylated tyrosine activation motif of the gamma subunit than for that of the beta subunit. In addition, we show that Syk is th e major protein in RBL-2H3 cells that is affinity isolated with phosph orylated peptides corresponding to the phosphorylated gamma subunit mo tif. These data suggest that Syk associates with the gamma 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 gamma subunit and that Syk may be the major s ignaling protein that binds to Fc epsilon RI tyrosine activation motif s in RBL-2H3 cells.