Requirement for a negative charge at threonine 60 of the FcRγ for complete activation of Syk

Abstract

Aggregation of FcεRI on mast cells results in the phosphorylation of the FcεRIγ chain on tyrosine and threonine residues within the immunoreceptor tyrosine-based activation motif. In the present study we sought to identify the site of threonine phosphorylation in FcεRIγ and investigate its functional importance. We found that threonine 60 was phosphorylated in vitro and in vivo. Expression of a mutated FcεRIγ (T60A), in either FcεRIγ-deficient or γ-null mast cells, resulted in a delay of FcεRI endocytosis, inhibition of TNF-α mRNA production, and inhibition of degranulation but did not affect FcεRI-induced cell adhesion. Tyrosine phosphorylation of the T60A mutant γ chain was normal, but Syk phosphorylation was dramatically reduced in these transfectants. This correlated with reduced co-immunoprecipitation of FcεRIγ with Syk. Substitution of an aspartic residue for threonine 60 of the FcεRIγ reconstituted complete activation of Syk and co-immunoprecipitation of FcεRIγ with Syk. We conclude that the negative charge provided by phosphorylation of threonine 60 of the FcεRIγ is required for the appropriate interaction and activation of Syk. This is a likely requirement for immunoreceptor tyrosine-based activation motifs involved in Syk activation.