The small guanosine triphosphatase (GTPase) Rho is implicated in the formation of stress fibers and focal adhesions in fibroblasts stimulated by extracellular signals such as lysophosphatidic acid (LPA). Rho-kinase is activated by Rho and may mediate some biological effects of Rho. Microinjection of the catalytic domain of Rho-kinase into serum-starved Swiss 3T3 cells induced the formation of stress fibers and focal adhesions, whereas microinjection of the inactive catalytic domain, the Rho-binding domain, or the pleckstrin-homology domain inhibited the LPA-induced formation of stress fibers and focal adhesions. Thus, Rho-kinase appears to mediate signals from Rho and to induce the formation of stress fibers and focal adhesions.
Background: The Rho small GTPase regulates myosin II activity through the phosphorylation of the myosin light chain (MLC) by activating Rhokinase, which is a target of Rho. Several lines of evidence point to an important role of Rho in the action of lysophosphatidic acid (LPA) and thrombin in provoking neurite retraction in N1E-115 neuroblastoma cells.
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...
The small GTPase Rho is implicated in cytoskeletal rearrangements including stress fiber and focal adhesion formation and in the transcriptional activation of c-fos serum response element. In vitro, Rho-kinase, which is activated by Rho, phosphorylates not only myosin light chain (MLC) (thereby activating myosin ATPase) but also myosin phosphatase, thus inactivating myosin phosphatase. Rho-kinase is involved in the formation of stress fibers and focal adhesions in fibroblasts. Here we show that the expression of constitutively active Rho-kinase increased the level of MLC phosphorylation. The activity of Rho-kinase was necessary for maintaining the vinculin-containing focal adhesions, whereas organized actin stress fibers were not necessary for this. The microinjection of constitutively active Rho-kinase into fibroblasts induced the formation of focal adhesions to some extent under the conditions where organized actin stress fibers were disrupted. The expression of constitutively active Rhokinase also stimulated the transcriptional activity of c-fos serum response element. These results suggest that Rho-kinase has distinct roles in divergent pathways downstream of Rho, which include MLC phosphorylation leading to stress fiber formation, focal adhesion formation, and gene expression.
Background: ERM (ezrin, radixin, and moesin) proteins function as membrane-cytoskeletal linkers, and are known to be localized at ®lopodia and microvilli-like structures. We have shown that Rhoassociated kinase (Rho-kinase)/ROKa/ROCK II phosphorylates moesin at Thr-558 at the lower stream of Rho, and the phosphorylation is crucial to the formation of microvilli-like structures (Oshiro, N., Fukata, Y. & Kaibuchi, K. (1998) Phosphorylation of moesin by Rho-associated kinase (Rhokinase) plays a crucial role in the formation of microvilli-like structures. J. Biol. Chem. 273, 34663± 34666). However, the role of ERM proteins in the formation of ®lopodia is less well characterized.
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