The role of Syk phosphorylation of tubulin during B -cell activation
Abstract
Tubulin is phosphorylated on tyrosine during B-cell activation. One of the kinases that phosphorylates tubulin in activated B-cells is the protein tyrosine kinase Syk. The objective of the present study was to determine the effect of this tubulin modification on tubulin polymerization properties. To address this question, the chick B-cell line DT-40 was utilized. This cell line was advantageous to use as it has a Syk negative variant (Syk–/–) and also contains only one of the known members of the Src family of kinases. DT-40 cells were activated either using pervanadate or anti-IgM antibodies. The presence of tyrosine-phosphorylated tubulin in microtubule cytoskeleton preparations was determined with anti-phosphotyrosine and anti-tubulin antibodies. Tyrosine-phosphorylated tubulin was found to be incorporated into microtubules. However, only in pervanadate-treated cells was the presence of tyrosine-phosphorylated tubulin apparent by immunofluorescence microscopy. Immunoblotting with anti-phosphotyrosine antibodies revealed the phosphorylation of only α-tubulin in pervanadate-treated cells. In contrast, in anti-IgM treated cells a number of other proteins were also phosphorylated. The tubulin phosphorylation was Syk-dependent as no microtubule staining was observed in the Syk–/– cell line. Additionally, in pervanadate-treated cells centrosomes were identified as sites for Syk-dependent phosphorylation. To directly assay the effect of tubulin phosphorylation on its polymerization properties, an in vitro tubulin polymerization assay was utilized. Syk phosphorylated-tubulin was able to incorporate into microtubules; however, the assembled microtubules were slightly shorter than those assembled from unphosphorylated tubulin. These results suggest that Syk phosphorylation of tubulin during B-cell activation may serve to alter its polymerization properties. Additionally, the presence of tyrosine-phosphorylated tubulin in microtubules could enhance SH2 domain/microtubule interactions and lead to the formation of signalling complexes.
Degree
Ph.D.
Advisors
Asai, Purdue University.
Subject Area
Cellular biology|Molecular biology
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