Syk, Activated by Cross-linking the B-cell Antigen Receptor, Localizes to the Cytosol Where It Interacts with and Phosphorylates -Tubulin on Tyrosine

Syk (p72 ) is a 72-kDa, nonreceptor, protein-tyrosine kinase that becomes tyrosine-phosphorylated and activated in B lymphocytes following aggregation of the B-cell antigen receptor. To explore the subcellular location of activated Syk, anti-IgM-activated B-cells were fractionated into soluble and particulate fractions by ultracentrifugation. Activated and tyrosine-phosphorylated Syk was found predominantly in the soluble fraction and was not associated with components of the antigen receptor. Similarly, the activated forms of Syk and its homolog, ZAP-70, were found in soluble fractions prepared from pervanadate-treated Jurkat T-cells. A 54-kDa protein that co-immunoprecipitated with Syk from the soluble fraction of activated B-cells was identified by peptide mapping as α-tubulin. α-Tubulin was an excellent in vitro substrate for Syk and was phosphorylated on a single tyrosine present within an acidic stretch of amino acids located near the carboxyl terminus. α-Tubulin was phosphorylated on tyrosine in intact cells following aggregation of the B-cell antigen receptor in a reaction that was inhibited by the Syk-selective inhibitor, piceatannol. Thus, once activated, Syk releases from the aggregated antigen receptor complex and is free to associate with and phosphorylate soluble proteins including α-tubulin.