Binding of pp170 to microtubules is regulated by phosphorylation.
We have used a monoclonal antibody affinity column to purify from HeLa cells a protein of molecular weight 170,000 (designated pp170) which we previously identified as a nucleotide-sensitive microtubule-binding protein (Rickard, J. E., and Kreis, T. E. (1990) J. Cell Biol. 110, 1623-1633). We show here that the affinity-purified pp170 binds directly to taxol-polymerized tubulin. This association is not affected directly by MgATP. Addition of MgATP can, however, inhibit binding of pp170 to microtubules in the presence of microtubule-binding proteins from HeLa cells. This effect of MgATP correlates with phosphorylation of pp170 by a microtubule-associated kinase. Potato acid phosphatase dephosphorylates the pp170 and restores the ability of pp170 to bind to microtubules. Furthermore, binding of pp170 to microtubules in a high speed supernatant extract is inhibited by the phosphatase inhibitor okadaic acid, consistent with an inhibitory effect of pp170 phosphorylation on microtubule binding. In vivo, pp170 is phosphorylated on serine residues, with a half-life for the phosphate groups of approximately 2 h. Depolymerization of microtubules with nocodazole abolishes incorporation of 32P into the protein, apparently by increasing the rate of its dephosphorylation. Stabilization of microtubules with taxol reduces the rate of 32P incorporation into pp170 by approximately 50%, but has no significant effect on phosphate loss. These data establish that pp170 is a microtubule-binding protein, and that the microtubule interaction is inhibited by phosphorylation of pp170. The sensitivity of the in vivo phosphorylation state of pp170 to microtubule-active drugs suggests that this posttranslational modification may be an important regulator of the interaction of pp170 with microtubules in cells.
European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany.
J E
JE
Rickard
T E
TE
Kreis
D000255:Adenosine Triphosphate / Q000378:metabolism; D000470:Alkaloids / Q000494:pharmacology; D002846:Chromatography, Affinity; D006367:HeLa Cells; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism*; D015739:Nocodazole / Q000494:pharmacology; D017239:Paclitaxel; D010766:Phosphorylation; D011108:Polymers; D011233:Precipitin Tests; D014404:Tubulin / Q000378:metabolism
D000470:Alkaloids; D008869:Microtubule-Associated Proteins; D011108:Polymers; D014404:Tubulin; D000255:Adenosine Triphosphate; D017239:Paclitaxel; D015739:Nocodazole