SYK, ACTIVATED BY CROSS-LINKING THE B-CELL ANTIGEN RECEPTOR, LOCALIZES TO THE CYTOSOL WHERE IT INTERACTS WITH AND PHOSPHORYLATES ALPHA-TUBULIN ON TYROSINE

Syk (p72(syk)) is a 72-kDa, nonreceptor, protein-tyrosine kinase that becomes tyrosine-phosphorylated and activated in B lymphocytes followi ng aggregation of the B-cell. antigen receptor. To explore the subcell ular location of activated Syk, anti-IgM-activated B-cells were fracti onated into soluble and particulate fractions by ultracentrifugation. Activated and tyrosine-phosphorylated Syk was found predominantly in t he soluble fraction and was not associated with components of the anti gen receptor. Similarly, the activated forms of Syk and its homolog, Z AP-70, were found in soluble fractions prepared from pervanadate-treat ed Jurkat T-cells. A 54-kDa protein that co-immunoprecipitated with Sy k from the soluble fraction of activated B-cells was identified by pep tide mapping as alpha-tubulin. alpha-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 ter minus. alpha-Tubulin was phosphorylated on tyrosine in intact cells fo llowing 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 a nd is free to associate with and phosphorylate soluble proteins includ ing alpha-tubulin.