Phosphorylation of vimentin by RHO-associated kinase at a unique amino- terminal site that is specifically phosphorylated during cytokinesis

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, Ser³⁸ and Ser⁷¹, were identified as the major phosphorylation sites for Rho-kinase, and Ser⁷¹ was the most favored and unique phosphorylation site for Rho- kinase in vitro. To analyze the vimentin phosphorylation by Rho-kinase in vivo, we prepared an antibody GK71 that specifically recognizes the phosphorylation of vimentin-Ser⁷¹. Ectopic expression of constitutively active Rho-kinase in COS-7 cells induced phosphorylation of vimentin at Ser⁷¹, followed by the reorganization of vimentin filament networks. During the cell cycle, the phosphorylation of vimentin-Ser⁷¹ 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-Ser⁷¹ was observed in the various 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.