A NOVEL TAXOL-INDUCED VIMENTIN PHOSPHORYLATION AND STABILIZATION REVEALED BY STUDIES ON STABLE MICROTUBULES AND VIMENTIN INTERMEDIATE FILAMENTS

To understand how protein phosphorylation modulates cytoskeletal organ ization, we used immunofluorescence microscopy to examine the effects of okadaic acid, a serine/threonine protein phosphatase inhibitor, and taxol, a microtubule-stabilizing agent, on stable (acetylated and det yrosinated) microtubules, vimentin intermediate filaments and other cy toskeletal elements in CV-1 cells. Okadaic acid caused major changes:i n both stable microtubules and vimentin intermediate filaments, but th rough independent mechanisms. At 300 nM, okadaic acid caused apparent fragmentation and loss of stable microtubules which was not prevented by prior exposure to K252a, In contrast, major reorganization of vimen tin intermediate filaments elicited at 750 nM okadaic acid was prevent ed by prior exposure to K252a, Taxol pretreatment blocked the effects of okadaic acid on stable microtubules and vimentin intermediate filam ents. Recent reports have revealed that taxol can activate cellular si gnal transduction pathways in addition to its known ability to promote microtubule stabilization, so the possibility that taxol-induced resi stance of vimentin intermediate filaments to okadaic acid was through a microtubule-independent mechanism involving direct phosphorylation o f intermediate filament proteins was explored. Vimentin immunoprecipit ation from cytoskeletal extracts from P-32-labeled cells revealed that taxol (4 mu M, 1 or 2 hours) caused about a 2-fold increase in viment in phosphorylation, This phosphorylation was recovered exclusively in cytoskeletal vimentin, in contrast to the increased phosphorylation of soluble and cytoskeletal vimentin caused by exposure to 750 nM okadai c acid, Phosphorylation of soluble and cytoskeletal vimentin from cell s exposed to taxol (4 mu M, 1 hour) then okadaic acid (750 nM, 1 hour) was comparable to taxol-treatment alone, These findings demonstrate a novel new activity of taxol, induction of vimentin phosphorylation, t hat may impact organization and stability.