The binding of a ciliary microtubule plus-end binding protein complex to microtubules is regulated by ciliary protein kinase and phosphatase activities.
Using a human autoimmune CREST antiserum, we identified a 97-kDa polypeptide at the plus ends of Tetrahymena ciliary microtubules and an antigen associated with mammalian kinetochores (Miller, J.M., Wang, W., Balczon, R., and Dentler, W.L. (1990) J. Cell. Biol. 110, 703-714). The ciliary protein is part of a 1,500-2,000-kDa complex that can be released from ciliary microtubules and from in vitro assembled brain microtubules with ATP and ATP gamma S (Wang, W., Suprenant, K.A., and Dentler, 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 inhibited, in a concentration-dependent manner, by calf intestine phosphatase and by the protein kinase inhibitor 6-dimethylaminopurine. Sodium orthovanadate, a phosphotyrosine phosphatase inhibitor, stimulated the ATP-dependent release of the 97-kDa protein from microtubules. Therefore, the ciliary fraction contains both protein kinase and phosphatase activities, and selective inhibition of these activities is necessary for the ATP-dependent binding and release of the 97-kDa protein from microtubules. When incubated with [gamma-32P]ATP, a portion of the 97-kDa protein is phosphorylated as are several other polypeptides associated 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 protein phosphorylation by axoneme-associated kinases and phosphatases.
Wang
W
W
Department of Physiology and Cell Biology, University of Kansas, Lawrence 66045-2106.
Himes
R H
RH
Dentler
W L
WL
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002923:Cilia / Q000201:enzymology / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011485:Protein Binding; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D013768:Tetrahymena / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
D008869:Microtubule-Associated Proteins; D047428:Protein Kinase Inhibitors; D014638:Vanadates; C001020:N(6),N(6)-dimethyladenine; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases; D000225:Adenine