Phosphorylation of Syk activation loop tyrosines is essential for Syk function - An in vivo study using a specific anti-Syk activation loop phosphotyrosine antibody

Syk is an important protein-tyrosine kinase in immunoreceptor signaling. Fc epsilon RI aggregation in mast cells induces tyrosine phosphorylation and increased enzymatic activity of Syk. The two adjacent tyrosines in the Syk activation loop are thought to be important for the propagation of Fc epsil on RI signaling. To evaluate the phosphorylation of these tyrosines in vivo and further understand the relationship of Syk tyrosine phosphorylation wi th its function, an antibody was developed specific for phosphorylated tyro sines in the activation loop of Syk. Fc epsilon RI aggregation on mast cell s induced the phosphorylation of both tyrosine residues of the activation l oop. The kinase activity of Syk played the major role in phosphorylating it s activation loop tyrosines both in vivo and in vitro. In Fc epsilon RI-sti mulated mast cells, the total Syk tyrosine phosphorylation paralleled the p hosphorylation of its activation loop tyrosines and downstream propagation of signals for histamine release. In contrast,:the cell surface binding of anti-ganglioside monoclonal antibody AA4 induced only strong general tyrosi ne: phosphorylation of Syk and minimal histamine release and weak phosphory lation of activation loop tyrosines. These results demonstrate that phospho rylation:of the activation loop tyrosines is important for mediating recept or signaling and is a better marker of Syk function than is total Syk tyros ine phosphorylation.