I. Ibarrola et al., INFLUENCE OF TYROSINE PHOSPHORYLATION ON PROTEIN-INTERACTION WITH FC-GAMMA-RIIA, Biochimica et biophysica acta. Molecular cell research, 1357(3), 1997, pp. 348-358
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.