Characterization of a Phospho-Specific Antibody to the Fcε Receptor γ Chain, Reveals Differences in the Regulation of Syk and Akt Phosphorylation.

We previously demonstrated that the Fc receptor γ-chain Y⁵⁸(C-terminal tyrosine) is highly susceptible to dephosphorylation; a mechanism that controls the extent of Syk activation and the downstream signaling in mast cells. Here, we explored the importance of the γ-chain Y⁴⁷ (N-terminal tyrosine) in mast cell signaling. We generated a highly sensitive and versatile phospho-specific antibody that recognized the phosphorylated Y⁴⁷ in various species. Using this antibody, we found that mutation of the FcεRIβ Y219 to phenylalanine caused a loss in the phosphorylation of the γ-chain Y⁴⁷, consistent with the previously described role of Y²¹⁹ in Lyn association with FcεRIβ and subsequent FcεRIγ phosphorylation. These conditions also diminished the tyrosine phosphorylation of Syk and LAT1 but, surprisingly, not the phosphorylation of Akt at T³⁰⁸. Mutation of Y⁴⁷ or Y⁵⁸ of the γ-chain also caused a marked inhibition of Syk and LAT1 phosphorylation, but only the latter mutant showed a reduction in Akt phosphorylation. These findings show that the full phosphorylation of Syk and LAT1 requires the FcεRIβ Y²¹⁹ and both Y⁴⁷ and Y⁵⁸ of the γ-chain. However, T³⁰⁸ phosphorylation of Akt is largely independent of FcεRIγ Y⁴⁷ phosphorylation and of the Lyn-binding site (Y²¹⁹) on the FcεRIβ. [ABSTRACT FROM AUTHOR]