ASSOCIATION OF THE MYOSIN-BINDING SUBUNIT OF MYOSIN PHOSPHATASE AND MOESIN - DUAL REGULATION OF MOESIN PHOSPHORYLATION BY RHO-ASSOCIATED KINASE AND MYOSIN PHOSPHATASE

The small GTPase Rho is believed to regulate the actin cytoskeleton an d cell adhesion through its specific targets. We previously identified the Rho targets: protein kinase N, Rho-associated kinase (Rho-kinase) , and the myosin-binding subunit (MBS) of myosin phosphatase. We found that in MDCK epithelial cells, MBS accumulated at the tetradecanoylph orbol-13-acetate (TPA)-induced membrane ruffling area, where moesin, a member of the ERM (ezrin, radixin, and moesin) family, was localized. Neither membrane ruffling nor an accumulation of moesin and MBS at th e free-end plasma membrane was induced when MDCK cells were stimulated with TPA after the microinjection of C3, which ADP-ribosylates and in activates Rho. MBS was colocalized with moesin at the cell-cell contac t sites in MDCK cells. We also found that moesin was coimmunoprecipita ted with MBS from MDCK cells. Recombinant MBS interacted with the amin o-terminal domains of moesin and ezrin. Myosin phosphatase composed of the catalytic subunit and MBS showed phosphatase activity toward moes in, which was phosphorylated by Rho-kinase. The phosphatase activity w as inhibited when MBS was phosphorylated by Rho-kinase. These results suggest that MBS is recruited with moesin to the plasma membrane and t hat myosin phosphatase and Rho-kinase regulate the phosphorylation sta te of moesin downstream of Rho.