THE REGULATORY SER(262) OF MICROTUBULE-ASSOCIATED PROTEIN-TAU IS PHOSPHORYLATED BY PHOSPHORYLASE-KINASE

Abnormally phosphorylated tan is the major component of paired helical filaments found in the brains of patients suffering from Alzheimer's disease. Therefore, the identification of kinases that phosphorylate t au is of considerable interest. A DEAE-Sepharose column resolved porci ne brain extract into five tau kinase activity peaks. Among these peak s, two were completely inhibited by EGTA, indicating that these two ac tivity peaks contained Ca2+-dependent tau kinases. One of the above tw o Ca2+-dependent tau kinase activity peaks also contained phosphorylas e kinase activity. The tau kinase and phosphorylase kinase activities associated with this peak could not be separated from each other by Su perose 12 gel filtration, hydroxylapatite, and calmodulin-agarose affi nity chromatographies. Phosphorylase kinase, purified from rabbit skel etal muscle, phosphorylated tau to a stoichiometry of 2.1 mol of phosp hate/mol of tau and converted tau to a species with a retarded mobilit y on SDS polyacrylamide gel electrophoresis. The apparent K-m and k(ca t) values for tau phosphorylation by muscle phosphorylase kinase were 6.9 mu M and 47.4 min(-1), respectively. As a substrate of muscle phos phorylase kinase, phosphorylase was eight times better than tau. Seque nce analyses of tryptic and thermolytic phosphopeptides derived from t au phosphorylated by muscle phosphorylase kinase revealed five phospho rylation sites, Ser(237), Ser(262), Ser(285), Ser(305), and Ser(352). Among these sites, Ser(262) was previously shown to be phosphorylated in human tau from fetal, adult, and Alzheimer's diseased brains (Seube rt, P., Mawal-Dewan, M., Barbour, R., Jakes, R., Goedert, M., Johnson, G. V. W., Litersky, J. M., Schenk, D., Lieberburg, I., Trojanowski, J . Q., and Lee, V. M. Y. (1995) J. Biol. Chem. 270, 18917-18922); and i ts phosphorylation abolished tau's binding to microtubules (Drewes, G. , Trinczek, B., Illenberger, S., Biernat, J., Schmitt-Ulms, G., Meyer, H. E., Mandelkow, E.-M., and Mandelkow, E. (1995) J. Biol. Chem. 270, 7679-7688). Slot-blot analysis using a monoclonal antibody against mu scle phosphorylase kinase and an activity assay using phosphorylase re vealed that phosphorylase kinase was present in microtubules extensive ly purified by repeated cycles of polymerization and depolymerization. Taken together, these results suggest that in neurons, phosphorylase kinase may be one of the kinases that participate in the phosphorylati on of tau.