THE BINDING OF A CILIARY MICROTUBULE PLUS-END BINDING-PROTEIN COMPLEXTO MICROTUBULES IS REGULATED BY CILIARY PROTEIN-KINASE AND PHOSPHATASE-ACTIVITIES

Using a human autoimmune CREST antiserum, we identified a 97-kDa polyp eptide at the plus ends of Tetrahymena ciliary microtubules and an ant igen associated with mammalian kinetochores (Miller, J. M., Wang W., B alczon, R., and Dentler, W. L. (1990) J. Cell. Biol. 110, 703-714). Th e ciliary protein is part of a 1,500-2,000-kDa complex that can be rel eased from ciliary microtubules and from in vitro assembled brain micr otubules with ATP and ATP gamma S (Wang, W., Suprenant, K. A., and Den tler, W.L. (1993) J. Biol. Chem. 268, 24796-24807). Here we show that the ATP-dependent release of the 97-kDa protein from microtubules is i nhibited, in a concentration-dependent manner by calf intestine phosph atase and by the protein kinase inhibitor 6-dimethylaminopurine. Sodiu m orthovanadate, a phosphotyrosine phosphatase inhibitor, stimulated t he ATP-dependent release of the 97-kDa protein from microtubules. Ther efore, the ciliary fraction contains both protein kinase and phosphata se activities, and selective inhibition of these activities is necessa ry for the ATP-dependent binding and release of the 97-kDa protein fro m microtubules. When incubated with [gamma-P-32]ATP, a portion of the 97-kDa protein is phosphorylated as are several other polypeptides ass ociated with it. ATP sensitivity requires a low molecular weight heat- stable factor associated with axonemes. These results suggest that the association of the 97-kDa protein with microtubules is regulated by p rotein phosphorylation by axoneme-associated kinases and phosphatases.