SummaryAggregation of the high affinity receptor for immunoglobulin E (FceI~I) on mast cells results in rapid tyrosine phosphorylation and activation ofSyk, a cytoplasmic protein tyrosine kinase. To examine the role of Syk in the Fcel~I signaling pathway, we identified a variant of RBL-2H3 cells that has no detectable Syk by immunoblotting and by in vitro kinase reactions. In these Syk-deficient TBIA2 cells, aggregation of FceRI induced no histamine release and no detectable increase in total cellular protein tyrosine phosphorylation. However, stimulation of these cells with the calcium ionophore did induce degranulation. FcetkI aggregation induced tyrosine phosphorylation of the 13 and ~/subunits of the receptor, but no increase in the tyrosine phosphorylation ofphospholipase C-~/1 and phosphohpase C-~/2 and no detectable increase in intracellular free Ca 2+ concentration. By transfection, cloned lines were established with stable expression of Syk. In these reconstituted cells, FcdkI aggregation induced tyrosine phosphorylation of phosphohpase C-~/1 and phospholipase C-'y2, an increase in intracellular free Ca 2+ and histamine release. These results demonstrate that Syk plays a critical role in the early Fcd~l-mediated signaling events. It further demonstrates that Syk activation occurs downstream of receptor phosphorylation, but upstream of most of the FcdkI-mediated protein tyrosine phosphorylations.
The linker region of Syk and ZAP70 tyrosine kinases plays an important role in regulating their function. There are three conserved tyrosines in this linker region; Tyr317 of Syk and its equivalent residue in ZAP70 were previously shown to negatively regulate the function of Syk and ZAP70. Here we studied the roles of the other two tyrosines, Tyr342 and Tyr346 of Syk, in Fc epsilon RI-mediated signaling. Antigen stimulation resulted in Tyr342 phosphorylation in mast cells. Syk with Y342F mutation failed to reconstitute Fc epsilon RI-initiated histamine release. In the Syk Y342F-expressing cells there was dramatically impaired receptor-induced phosphorylation of multiple signaling molecules, including LAT, SLP-76, phospholipase C-gamma2, but not Vav. Compared to wild-type Syk, Y342F Syk had decreased binding to phosphorylated immunoreceptor tyrosine-based activation motifs and reduced kinase activity. Surprisingly, mutation of Tyr346 had much less effect on Fc epsilon RI-dependent mast cell degranulation. An anti-Syk-phospho-346 tyrosine antibody indicated that antigen stimulation induced only a very minor increase in the phosphorylation of this tyrosine. Therefore, Tyr342, but not Tyr346, is critical for regulating Syk in mast cells and the function of these tyrosines in immune receptor signaling appears to be different from what has been previously reported for the equivalent residues of ZAP70.
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