There is considerable evidence that mammalian β-tubulin is phosphorylated. Specifically, of the seven β isotypes, the phosphorylated one is βIII, the isotype found almost entirely in neurons. The phosphate is added at a serine and perhaps a tyrosine near the C-terminus. All the evidence to date has been gathered by growth of cells and tissues in the presence of radioactive inorganic phosphate followed by tubulin isolation and determination of the labeled tubulin; thus, the actual extent of phosphorylation of βIII is unknown. Nor is it known if α-tubulin and the other β isotypes are phosphorylated by a mechanism which would not be revealed by previous experiments. In addition, the role of tubulin phosphorylation is unknown. We have purified the αβII-, αβIII-, and αβIV-tubulin dimers from bovine brain and have determined their phosphate content chemically. We have found that α-tubulin is not phosphorylated and neither are the βII or αIV isotypes. However, βIII is phosphorylated with a stoichiometry of about 1.52 mol/mol. We have found that the phosphate on βIII is resistant to a wide variety of phospnatases except for human erythrocyte phosphatase 2A and that removal of the phosphate inhibits microtubule assembly in vitro stimulated by microtubule-associated protein 2 (MAP 2). However such an inhibition was not evident when microtubule assembly was induced in the absence of microtubule-associated proteins. Our results suggest the possibility that βIII phosphorylation may play a role in regulating microtubule assembly in vivo.
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