PHOSPHORYLATION OF BETA(III)-TUBULIN

There is considerable evidence that mammalian beta-tubulin is phosphor ylated. Specifically, of the seven beta isotypes, the phosphorylated o ne is beta(III), the isotype found almost entirely in neurons. The pho sphate 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 ti ssues in the presence of radioactive inorganic phosphate followed by t ubulin isolation and determination of the labeled tubulin; thus, the a ctual extent of phosphorylation of beta(III) is unknown. Nor is it kno wn if alpha-tubulin and the other beta isotypes are phosphorylated by a mechanism which would not be revealed by previous experiments. In ad dition, the role of tubulin phosphorylation is unknown. We have purifi ed the alpha beta(II)-, alpha beta(III)-, and alpha beta(IV)-tubulin d imers from bovine brain and have determined their phosphate content ch emically. We have found that alpha-tubulin is not phosphorylated and n either are the beta(II) or beta(IV) isotypes. However, beta(III) is ph osphorylated with a stoichiometry of about 1.52 mol/mol. We have found that the phosphate on beta(III) is resistant to a wide variety of pho sphatases 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 o f microtubule-associated proteins. Our results suggest the possibility that beta(III) phosphorylation may play a role in regulating microtub ule assembly in vivo.