The COOH Terminus of Rho-kinase Negatively Regulates Rho-kinase Activity

Abstract Rho- kinaseis implicated in the phosphorylation of myosin light chain downstream of Rho, which is thought to induce smooth muscle contraction and stress fiberformation in non-muscle cells. Here, we examined the mode of action of inhibitors of Rho- kinase. The chemical compounds such as HA1077 and Y-32885 inhibited not only the Rho- kinaseactivity but also the activity of protein kinaseN, one of the targets of Rho, but had less of an effect on the activity of myotonic dystrophy kinase-related Cdc42-binding kinaseβ (MRCKβ). The COOH-terminal portion of Rho- kinasecontaining Rho-binding (RB) and pleckstrin homology (PH) domains (RB/PH (TT)), in which point mutations were introduced to abolish the Rho binding activity, interacted with Rho- kinaseand thereby inhibited the Rho- kinaseactivity, whereas RB/PH (TT) had no effect on the activity of protein kinaseN or MRCKβ, suggesting that the COOH-terminal region of Rho- kinaseis a possible negative regulatory region of Rho- kinase. The expression of RB/PH (TT) specifically blocked the stress fiberand focal adhesionformation induced by the active form of Rho or Rho- kinasein NIH 3T3 cells, but not that induced by the active form of MRCKβ or myosin light chain. Thus, RB/PH (TT) appears to specifically inhibit Rho- kinasein vivo.