Influence of tyrosine phosphorylation on protein interaction with FcgammaRIIa.
The cytoplasmic tail of Fc(gamma)RIIa present on human neutrophils shares with other antigen receptors a common amino acid sequence called ITAM (Immunoreceptor Tyrosine-based Activation Motif). After receptor ligation, 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 (GST-CT) to characterize the phosphorylation of Fc(gamma)RIIa and its ability 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 kinase responsible for the initial Fc(gamma)RIIa phosphorylation. Moreover, we observed specific association of Syk and the p85 subunit of PI 3-kinase after incubation of the GST-CT with neutrophil cytosol. This interaction was dependent on tyrosine phosphorylation of the GST-CT. Substitution of 269Tyr by Phe almost completely abolished tyrosine phosphorylation of the fusion protein. Substitution of either 253Tyr or 269Tyr eliminated Syk binding, but only 253Tyr appeared to be essential for p85 binding. We hypothesize that, upon activation, the membrane-associated Lyn is responsible for the initial tyrosine phosphorylation of Fc(gamma)RIIa, thus creating a docking site for Syk and PI 3-kinase.
Ibarrola
I
I
Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, University of Amsterdam.
Vossebeld
P J
PJ
Homburg
C H
CH
Thelen
M
M
Roos
D
D
Verhoeven
A J
AJ
D015703:Antigens, CD / Q000378:metabolism*; D006801:Humans; D009504:Neutrophils / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D017452:Receptors, IgG / Q000378:metabolism*; D014443:Tyrosine / Q000502:physiology*; D019061:src-Family Kinases / Q000378:metabolism*
D015703:Antigens, CD; C080568:Fc gamma receptor IIA; D010750:Phosphoproteins; D017452:Receptors, IgG; D014443:Tyrosine; D019061:src-Family Kinases