Association of protein phosphatase 2A with its substrate vimentin intermediate filaments in 9L rat brain tumor cells

The importance of protein phosphatases in maintaining the integrity of inte rmediate filaments is supported by the fact that intermediate filaments wou ld undergo a massive reorganization in cells treated with inhibitors of pro tein phosphatases 1 and 2A. Herein we used okadaic acid to investigate the differential roles of protein phosphatases 1 and 2A in the maintenance of i ntermediate filament integrity in 9L rat brain tumor cells. Protein phospha tase 2A activity was substantially inhibited after treatment with 400 nM ok adaic acid for 2 h, whereas the activity of protein phosphatase 1 was only slightly affected. Furthermore, protein phosphatase 2A shows selective spec ificity toward phosphovimentin, which was immunologically precipitated from isotopically labeled and okadaic acid-treated cells. Further biochemical f ractionation and microscopic studies revealed that vimentin intermediate fi laments were colocalized with protein phosphatase 2A, bur not protein phosp hatase 1, in control cells. On okadaic acid treatment, vimentin filament di sassembled and protein phosphatase 2A redistributed throughout the cytoplas m, suggesting that these two proteins separate from each other, whereas pro tein phosphatase 2A was inhibited. This working hypothesis was further supp orted by treatment with a low concentration (40 nM) of okadaic acid, which causes the same phenomenon. Taken together, our results showed that protein phosphatase 2A could be assigned to the intermediate filaments to serve th e physiological role in maintaining the proper phosphorylation level of int ermediate filaments in normal cells. This finding should pave the way for t he elucidation of the regulatory mechanism of intermediate filament organiz ation governed by protein phosphorylation. (C) 2000 Wiley-Liss, Inc.