INFLUENCE OF TYROSINE PHOSPHORYLATION ON PROTEIN-INTERACTION WITH FC-GAMMA-RIIA

The cytoplasmic tail of Fc gamma RIIa present on human neutrophils sha res with other antigen receptors a common amino acid sequence called I TAM (Immunoreceptor Tyrosine-based Activation Motif). After receptor l igation. the tyrosine residues within this motif become phosphorylated . We prepared a recombinant fusion protein of the cytoplasmic tail of Fc gamma RIIa (containing the ITAM) with glutathione-S-Transferase (GS T-CT) to characterize the phosphorylation of Fc gamma RIIa and its abi lity to interact with other proteins involved in signal transduction. The GST-CT became phosphorylated in the presence of Lyn, Hck and Syk ( immunoprecipitated from human neutrophils), but not in the presence of Fgr. Of the active kinases, only Lyn (mainly present in the membrane fraction) was found to associate with the GST-CT in the absence of ATP . This association was also observed in immunoprecipitates of Fc gamma RIIa from resting neutrophils, suggesting that Lyn might be the kinas e responsible for the initial Fc gamma RIIa phosphorylation. Moreover, we observed specific association of Syk and the p85 subunit of PI 3-k inase after incubation of the CST-CT with neutrophil cytosol. This int eraction was dependent on tyrosine phosphorylation of the GST-CT. Subs titution of 269Tyr by Phe almost completely abolished tyrosine phospho rylation of the fusion protein. Substitution of either 253Tyr or 269Ty r eliminated Syk binding, but only 253Tyr appeared to be essential for p85 binding. We hypothesize that, upon activation, the membrane-assoc iated Lyn is responsible for the initial tyrosine phosphorylation of F c gamma RIIa, thus creating a docking site for Syk and PI 3-kinase. (C ) 1997 Elsevier Science B.V.