PHOSPHORYLATION OF GLIAL FIBRILLARY ACIDIC PROTEIN AT THE SAME SITES BY CLEAVAGE FURROW KINASE AND RHO-ASSOCIATED KINASE

Site- and phosphorylation state-specific antibodies are useful to anal yze spatiotemporal distribution of site-specific phosphorylation of ta rget proteins in vivo. Using several polyclonal and monoclonal antibod ies that can specifically recognize four phosphorylated sites on glial fibrillary acidic protein (GFAP), we have previously reported that Th r-7, Ser-13, and Ser-34 on this intermediate filament protein are phos phorylated at the cleavage furrow during cytokinesis. This observation suggests that there exists a protein kinase named cleavage furrow kin ase specifically activated at metaphase-anaphase transition (Matsuoka, Y., Nishizawa, K., Yano, T., Shibata, M., Ando, S., Takahashi, T., an d Inagaki, M. (1992) EMBO J. 11, 2895-2902; Sekimata, M., Tsujimura, a , Tanaka, J., Takeuchi, Y., Inagaki, N., and Inagaki, M. (1996) J. Cel l Biol; 132, 635-641). Here we report that GFAP is phosphorylated spec ifically at Thr-7, Ser-13, and Ser-34 by Rho-associated kinase (Rho-ki nase), which binds to the small GTPase Rho in its GTP-bound active for m. The kinase activity of Rho-kinase toward GFAP is dramatically stimu lated by guanosine 5'-(3-O-thio)-triphosphate-bound RhoA. Furthermore, the phosphorylation of GFAP by Rho-kinase results in a nearly complet e inhibition of its filament formation in vitro. The possibility that Rho-kinase is a candidate for cleavage furrow kinase is discussed.