REGULATION OF THE ASSOCIATION OF ADDUCIN WITH ACTIN-FILAMENTS BY RHO-ASSOCIATED KINASE (RHO-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. Here we purified MBS-interacting proteins, identified them as adducin, and fou nd that MBS specifically interacted with adducin in vitro and in vivo. Adducin is a membrane-skeletal protein that promotes the binding of s pectrin to actin filaments and is concentrated at the cell-cell contac t sites in epithelial cells. We also found that Rho-kinase phosphoryla ted alpha-adducin in vitro and in vivo and that the phosphorylation of alpha-adducin by Rho-kinase enhanced the interaction of alpha-adducin with actin filaments in vitro. Myosin phosphatase composed of the cat alytic subunit and MBS showed phosphatase activity toward alpha-adduci n, which was phosphorylated by Rho-kinase. This phosphatase activity w as inhibited by the phosphorylation of MBS by RHO-kinase. These result s suggest that Rho-kinase and myosin phosphatase regulate the phosphor ylation state of adducin downstream of Rho and that the increased phos phorylation of adducin by Rho-kinase causes the interaction of adducin with actin filaments.