We found that vimentin, the most widely expressed intermediate filament protein, served as an excellent substrate for Rho-associated kinase (Rho-kinase) and that vimentin phosphorylated by Rho-kinase lost its ability to form filaments in vitro. Two amino-terminal sites on vimentin, Ser 38 and Ser 71 , were identified as the major phosphorylation sites for Rho-kinase, and Ser 71 was the most favored and unique phosphorylation site for Rho-kinase in vitro. To analyze the vimentin phosphorylation

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... y Rho-kinase in vivo, we prepared an antibody GK71 that specifically recognizes the phosphorylation of vimentin-Ser 71 . Ectopic expression of constitutively active Rho-kinase in COS-7 cells induced phosphorylation of vimentin at Ser 71 , followed by the reorganization of vimentin filament networks. During the cell cycle, the phosphorylation of vimentin-Ser 71 occurred only at the cleavage furrow in late mitotic cells but not in interphase or early mitotic cells. This cleavage furrow-specific phosphorylation of vimentin-Ser 71 was observed in the var- ious types of cells we examined. All these accumulating observations increase the possibility that Rho-kinase may have a definite role in governing regulatory processes in assembly-disassembly and turnover of vimentin filaments at the cleavage furrow during cytokinesis.