Phosphorylation of MBS and inactivation of myosin phosphatase by Rho-kinase. (A) Coimmunoprecipitation of RhoA and Rho-kinase with platelet MBS. Coprecipitated RhoA (left) and Rho-kinase (right) were determined by probing immunoblots with antibodies against respective proteins. (B) Coprecipitation of MBS with Rho-kinase from human platelets. Immunoprecipitation with anti–Rho-kinase antibody and immunoblot analysis of MBS were described in the Materials and Methods. (C) In vitro phosphorylation of platelet MBS by GST-Rho-kinase and its inhibition by staurosporine. MBS immunoprecipitates of platelet lysates were incubated with Rho-kinase in the presence of staurosporine, as described under Materials and Methods. Protein phosphorylation was analyzed by SDS-PAGE, followed by autoradiography. Lane 1, no incubation; Lane 2, 15 minutes of incubation without GST-Rho-kinase; Lane 3, 15 minutes of incubation with GST-Rho-kinase, Lane 4; 15 minutes of incubation with GST-Rho-kinase in the presence of 1 μmol/L staurosporine. The results are representative of three independent experiments. (D) Effect of MBS phosphorylation on the activity of myosin phosphatase. MBS immunoprecipitates were incubated without GST-Rho-kinase (▵, ▴), with GST-Rho-kinase (□, ▪) or with GST-Rho-kinase and 1 μmol/L staurosporine (○, •), as described in the Materials and Methods. At the indicated times, aliquots of the reaction mixture were quenched by addition of a solution containing final 10 mmol/L EGTA to stop the reaction and were kept on ice. Activity of myosin phosphatase (open symbol) was determined immediately. Phosphorylation (solid symbol) of MBS was analyzed by SDS-PAGE and autoradiography. Points, means of three separate experiments (SD < 10%).