Rho-kinase is implicated in the phosphorylation of
myosin light chain downstream of Rho, which is thought to induce smooth muscle contraction and stress fiber formation 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-kinase activity but also the activity of
protein kinase N, one of the targets of Rho, but had less of an effect on the activity of
myotonic dystrophy kinase-related Cdc42-binding
kinase beta (MRCKbeta). The COOH-terminal portion of
Rho-kinase containing 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-kinase and thereby inhibited the
Rho-kinase activity, whereas RB/PH (TT) had no effect on the activity of
protein kinase N or MRCKbeta, suggesting that the COOH-terminal region of
Rho-kinase is a possible negative regulatory region of
Rho-kinase. The expression of RB/PH (TT) specifically blocked the stress fiber and focal adhesion formation induced by the active form of Rho or
Rho-kinase in NIH 3T3 cells, but not that induced by the active form of MRCKbeta or
myosin light chain. Thus, RB/PH (TT) appears to specifically inhibit
Rho-kinase in vivo.