Induction of vimentin modification and vimentin-HSP72 association by withangulatin A in 9L rat brain tumor cells.

Withangulatin A induced cell rounding up and the morphological alteration resulted from the reorganization of all of the major cytoskeletal components, i.e., vimentin, tubulin, and actin, as revealed by immunofluorescence techniques. When the withangulatin A-treated cells changed to a round-up morphology, vimentin intermediate filaments were found to be collapsed and clustered around the nucleus. The alteration was accompanied by characteristic changes of vimentin molecules, including augmentation of phosphorylation, retardation of electrophoretic mobility, and decrease in detergent extractability. The levels of vimentin phosphorylation were augmented by 2.5- and 1.8-fold in cells incubated with 50 microM withangulatin A for 1 and 3 h, respectively. The electrophoretic mobility of vimentin was partially retarded in cells treated with withangulatin A for 1 h at 10 microM and a completely upshift mobility was observed after 5 h treatment at 50 microM. In addition, vimentin molecules became less extractable by nonident P-40 after the cells were treated with withangulatin A and this effect was dose dependent. The decrease in solubility of vimentin was accompanied by the redistribution of HSP72 into the detergent nonextractable fraction and these two events were well correlated. Our results suggest that withangulatin A induced the modification of vimentin, which resulted in the alteration of cell morphology and redistribution of intracellular HSP72, an event that may play an important role in the induction of heat-shock response.