Phosphorylation- and activation-independent association of the tyrosine kinase Syk and the tyrosine kinase substrates Cbl and Vav with tubulin in B-cells

Aggregation of the B-cell antigen receptor leads to the activation of the 7 2-kDa Syk protein-tyrosine kinase and the phosphorylation of tubulin on tyr osine, To explore the requirement of Syk catalytic activity for tubulin pho sphorylation, tubulin was isolated from cytosolic fractions from anti-IgM-a ctivated B-cells (DT40) that lacked endogenous Syk and immunoblotted with a ntiphosphotyrosine antibodies. Tubulin was not tyrosine-phosphorylated in S yk(-) B-cells, Phosphorylation could be restored by the expression of wild- type, but not catalytically inactive, Syk. However, both catalytically inac tive and wild-type Syk were capable of constitutive association with tubuli n, indicating that tubulin phosphorylation is not required for this interac tion. Anti-phosphotyrosine antibody immunoblotting of proteins adsorbed to colchicine-agarose revealed the presence of three major tubulin-associated phosphoproteins of 110, 90, and 74 kDa, the phosphorylation of which was de pendent on Syk expression. The proteins of 110 and 90 kDa were identified a s Cbl and Vav, two proto-oncogene products known to become prominently phos phorylated following receptor engagement. Both proteins were shown to be co nstitutively associated with tubulin.