Phosphorylation and constitutive association of the tyrosine kinase Syk and the proto -oncogene products Cbl and Vav with tubulin in B cells: Potential involvement of microtubules in the formation of multimolecular signaling complexes
Abstract
Aggregation of the B cell antigen receptor leads to the activation of the 72 kDa Syk protein-tyrosine kinase and the phosphorylation of tubulin on tyrosine. To explore the requirement of Syk catalytic activity for tubulin phosphorylation, tubulin was isolated from the cytosolic fraction of anti-IgM-activated B cells (DT40) that lacked endogenous Syk. Immunoblotting with anti-phosphotyrosine antibodies revealed that in Syk– B cells, tubulin did not become tyrosine-phosphorylated. Phosphorylation could be restored by the expression of wild-type, but not catalytically inactive, Syk, confirming that Syk catalytic activity was required for the tyrosine phosphorylation of tubulin. However, both catalytically inactive and wild-type Syk were capable of constitutive association with tubulin, indicating that tubulin phosphorylation is not required for this interaction. In addition to Syk, anti-phosphotyrosine antibody immunoblotting of proteins adsorbed to colchicine-sepharose revealed the presence of three major tubulin-associated phosphoproteins of 110, 90 and 74 kDa, whose phosphorylation was dependent on Syk expression. All three proteins were observed in avian, human and murine cells, suggesting that their appearance was not an artifact of a particular cell line or species. The proteins of 110 and 90 kDa were identified as Cb1 and Vav, two proto-oncogene products known to become prominently phosphorylated following receptor engagement. Both proteins were shown to be constitutively associated with tubulin. When cytosolic fractions prepared from cells overexpressing wild-type Syk were analyzed by gel filtration chromatography, anti-Syk immunoblotting of column fractions revealed the presence of two populations of Syk: monomer Syk and a Syk-containing complex of greater than 200 kDa that also contained tubulin. Together, these results suggest dim Syk and tubulin may participate in the formation of signaling complexes in B cells.
Degree
Ph.D.
Advisors
Harrison, Purdue University.
Subject Area
Biochemistry|Immunology|Cellular biology
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.