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 kinaserelated Cdc42-binding kinase ␤ (MRCK␤). The COOHterminal 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 MRCK␤, 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 MRCK␤ or myosin light chain. Thus, RB/PH (TT) appears to specifically inhibit Rho-kinase in vivo.There is mounting evidence that the small GTPase Rho plays crucial roles in the rearrangements of cytoskeleton and cell adhesion (1-3). Rho cycles between GDP-bound inactive and GTPbound active forms, and the GTP-bound form binds to specific effectors and then exerts its biological functions. Numerous putative Rho effectors have been identified; PKN 1 (4, 5), Rho-kinase/ROK␣/ROCK II (6 -8), myosin-binding subunit of myosin phosphatase (9), mDia1 (10), citron (11), citron kinase (12), rhophilin, rhotekin (11), Kv1.2 (13), and phospholipase D (14). ROCK I/ROK␤ is an isoform of Rho-kinase (7,8). Rho-kinase is implicated in many processes downstream of Rho; stress fiber and focal adhesion formation (15-17), smooth muscle contraction (18), intermediate filament disassembly (19,20), neurite retraction (21, 22), microvilli formation (23), cytokinesis (24), and cell migration (25). Rho-kinase regulates the phosphorylation of MLC by the direct phosphorylation of MLC and by the inactivation of myosin phosphatase through the phosphorylation of myosin-binding subunit (9,26). In addition to MLC and myosinbinding subunit, Rho-kinase phosphorylates the ezrin/radixin/ moesin family proteins and adducin in vitro (27,28). To unravel in vivo functions of Rho-kinase, it is necessary to develop specific probes for Rho-kinase. Recently, chemical compounds such as Y-27632, Y-32885, and HA1077 have been shown to inhibit the Rho-kinase activity in a manner competitive with ATP (29), and to suppress hypertension in model animals. However, the modes of action and specificity of these chemical compounds have not yet been elucidated.Rho-kinase is composed of NH 2 -terminal catalytic, coiledcoil, Rho-binding, and COOH-terminal PH domains (6). When the COOH-terminal portion of...

Rho-kinase and myosin phosphatase are implicated in the phosphorylation-state of myosin light chain downstream of Rho, which is thought to induce smooth muscle contraction and stress fibre formation in non-muscle cells. Here, we found that microtubule-associated proteins, Tau and MAP2, interacted with the myosin-binding subunit (MBS) of myosin phosphatase, and were the possible substrates of both Rhokinase and myosin phosphatase. We determined the phosphorylation sites of Tau (Thr245, Thr377, Ser409) and MAP2 (Ser1796) by Rho-kinase. We also found that Rho-kinase phosphorylated Tau at Ser262 to some extent. Phosphorylation by Rho-kinase decreased the activity of Tau to promote microtubule assembly in vitro. Substitutions of Ala for Ser/Thr at the phosphorylation sites of Tau (Tau-AAA) did not affect the activity to promote microtubule assembly, while substitutions of Asp for Ser/Thr (Tau-DDD), which are expected to mimic the phosphorylation-state of Tau, slightly reduced the activity. When Tau, or mutated forms of Tau, were expressed in PC12 cells, followed by treatment with cytochalasin D, they promoted extension of the cell process in a cytochalasindependent manner. However, Tau-DDD showed the weaker activity in this capacity than wild-type Tau or Tau-AAA. These results suggest that the phosphorylation-state of these residues of Tau affects its activity both in vitro and in vivo. Thus, it is likely that the Rho-kinase/MBS pathway regulates not only the actin-myosin system but also microtubule dynamics.

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