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

Aggregation of Fc epsilon RI on mast cells results in the phosphorylation o f the Fc epsilon RI gamma chain on tyrosine and threonine residues within t he immunoreceptor tyrosine-based activation motif, In the present study we sought to identify the site of threonine phosphorylation in Fc epsilon RI g amma and investigate its functional importance. We found that threonine 60 was phosphorylated in vitro and in vivo. Expression of a mutated Fc epsilon RI gamma (T60A), in either Fc epsilon RI gamma-deficient or gamma-null mas t cells, resulted in a delay of Fc epsilon RI endocytosis, inhibition of TN F-alpha mRNA production, and inhibition of degranulation but did not affect Fc epsilon RI-induced cell adhesion. Tyrosine phosphorylation of the T60A mutant gamma chain was normal, but Syk phosphorylation was dramatically red uced in these transfectents, This correlated with reduced co-immunoprecipit ation of Fc epsilon RI gamma with Syk. Substitution of an aspartic residue for threonine 60 of the Fc epsilon RI gamma reconstituted complete activati on of Syk and co-immunoprecipitation of Fc epsilon RI gamma with Syk. We co nclude that the negative charge provided by phosphorylation of threonine 60 of the Fc epsilon RI gamma is required for the appropriate interaction and activation of Syk, This is a likely requirement for immunoreceptor tyrosin e-based activation motifs involved in Syk activation.