Distinct roles for the linker region tyrosines of Syk in FcepsilonRI signaling in primary mast cells.

Stimulation of FcepsilonRI, the high affinity IgE receptor of mast cells results in the rapid binding of the Syk tyrosine kinase to cytoplasmic domains of FcepsilonRI and to its subsequent activation. Syk plays an essential role in signal transduction from FcepsilonRI as shown by Syk-deficient mast cells, which are defective in receptor-induced degranulation, cytokine synthesis, and intracellular pathways. However the mechanism by which Syk activates these pathways remains unclear. Activation of Syk is associated with its phosphorylation on several tyrosine residues, including the linker tyrosines Tyr317, Tyr342, and Tyr346. These residues have been proposed to play important roles in the transduction of signals by binding to other signaling proteins. To test these hypotheses in primary murine mast cells, we used retroviral infection of Syk-deficient mast cells to generate cells expressing Syk proteins bearing mutations in the linker tyrosines. We show that Tyr342 and Tyr346 contribute positively to the function of Syk and have both overlapping as well as distinct functions. Mutations in either Tyr342 or Tyr346 alone had no effect on FcepsilonRI-induced degranulation or calcium flux, whereas mutation of both residues caused a significant reduction in both pathways. In contrast, phosphorylation of PLCgamma1, PLCgamma2, and Vav1 was strongly decreased by a mutation in Tyr342 alone, whereas phosphorylation of ERK and Akt was more dependent on Tyr346. Finally we show that Tyr317 functions as a negative regulatory site and that its mutation can partially compensate for the loss of both Tyr342 and Tyr346.