Cloning and structural analysis of Rho kinase

The small GTP-binding protein Rho functions as a molecular switch in the formation of focal adhesions, stress fibers, cytokinesis and transcriptional activation. The biochemical mechanism underlying these actions remains unknown. Using a ligand overlay assay, a 160 kDa platelet protein has been isolated that binds specifically to GTP-bound Rho. This protein, p160, undergoes autophosphorylation at its serine and threonine residues and shows the kinase activity to exogenous substrates. Both activities are enhanced by the addition of GTP-bound Rho. A cDNA encoding p160 codes for a 1354 amino acid protein. This protein has a Ser/Thr kinase domain at its N-terminus, followed by a coiled-coil structure approximately 600 amino acids long, and at the C-terminus, a cysteine-rich zinc finger-like motif and a pleckstrin homology region. The N-terminus region including the kinase domain and a part of the coiled-coil structure shows strong homology to myotonic dystrophy kinase over 500 residues. When co-expressed with RhoA in COS cells, p160 is co-precipitated with the expressed Rho and its kinase activity is activated, indicating that p160 can associate physically and functionally with Rho both in vitro and in vivo (Ishizaki, 1996).

ROK alpha binds RhoA only in its active GTP-bound state and introduction of RhoA cDNA into HeLa cells results in translocation of the cytoplasmic kinase to plasma membranes, consistent with ROK alpha being a target for RhoA. Analysis of the cDNA has revealed that ROK alpha contains an N-terminal region. Another cDNA has been isolated that encodes a protein (ROK beta) with 90% identity to ROK alpha in the kinase domain. Both ROK alpha and ROK beta, which each have a molecular mass of 160 kDa, contain a highly conserved cysteine/histidine-rich domain located within a putative pleckstrin homology domain. The kinases bind RhoA, RhoB, and RhoC but not Rac1 and Cdc42. The Rho-binding domain comprises about 30 amino acids. Mutations within this domain cause partial or complete loss of Rho binding. The morphological effects of ROK alpha were investigated by microinjecting HeLa cells with DNA constructs encoding various forms of ROK alpha. Full-length ROK alpha promotes formation of stress fibers and focal adhesion complexes, consistent with its being an effector of RhoA. ROK alpha truncated at the C terminus promotes this formation and also extensive condensation of actin microfilaments and nuclear disruption. The proteins exhibit protein kinase activity which is required for stress fiber formation; the kinase-dead ROK alpha K112A and N-terminally truncated mutants show no such promotion. The latter mutant instead induces disassembly of stress fibers and focal adhesion complexes, accompanied by cell spreading. These effects are mediated by the C-terminal region containing Rho-binding, cysteine/histidine-rich, and pleckstrin homology domains. Thus, the multidomained ROK alpha appears to be involved in reorganization of the cytoskeleton, with the N and C termini acting as positive and negative regulators, respectively, of the kinase domain whose activity is crucial for formation of stress fibers and focal adhesion complexes (Leung, 1996).

Rho is implicated in physiological functions associated with actin-myosin filaments such as cytokinesis, cell motility, and smooth muscle contraction. Rho-kinase stoichiometrically phosphorylates myosin light chain (MLC). Peptide mapping and phosphoamino acid analyses reveal that the primary phosphorylation site of MLC by Rho-kinase is Ser-19, which is the site phosphorylated by MLC kinase. Rho-kinase phosphorylates recombinant MLC, whereas it fails to phosphorylate recombinant MLC, which contains Ala substituted for both Thr-18 and Ser-19. The phosphorylation of MLC by Rho-kinase results in the facilitation of the actin activation of myosin ATPase. Thus, it is likely that once Rho is activated, then it can interact with Rho-kinase and activate it. The activated Rho-kinase subsequently phosphorylates MLC. This may partly account for the mechanism by which Rho regulates cytokinesis, cell motility, or smooth muscle contraction (Amano, 1996).

Two genes have been identified that are associated with the hypodermal cell shape changes that occur during elongation of the C. elegans embryo. The first gene, let-502, encodes a protein with high similarity to Rho-binding Ser/Thr kinases and to human myotonic dystrophy kinase (DM-kinase). Strong mutations in let-502 block embryonic elongation, and let-502 reporter constructs are expressed in hypodermal cells at the elongation stage of development. The second gene, mel-11, was identified by mutations that act as extragenic suppressors of let-502. mel-11 encodes a protein similar to the 110- to 133-kD regulatory subunits of vertebrate smooth muscle myosin-associated phosphatase (PP-1M). It is suggested that the LET-502 kinase and the MEL-11 phosphatase subunit act in a pathway linking a signal generated by the small GTP-binding protein Rho to a myosin-based hypodermal contractile system that drives embryonic elongation. LET-502 may directly regulate the activity of the MEL-11 containing phosphatase complex and the similarity between LET-502 and DM-kinase suggests a similar function for DM-kinase (Wissmann, 1997).

Rho kinase regulation of myosin, stress fibers and focal adhesion

Rho is implicated in myosin light chain (MLC) phosphorylation, which results in contraction of smooth muscle and interaction of actin and myosin in nonmuscle cells. The guanosine triphosphate (GTP)-bound, active form of RhoA (GTP.RhoA) specifically interacts with the myosin-binding subunit (MBS: see Drosophila Myosin binding subunit) of myosin phosphatase, which regulates the extent of phosphorylation of MLC. Rho-associated kinase (Rho-kinase), which is activated by GTP.RhoA, phosphorylates MBS and consequently inactivates myosin phosphatase. Overexpression of RhoA or activated RhoA in NIH 3T3 cells increases phosphorylation of MBS and MLC. Thus, Rho appears to inhibit myosin phosphatase through the action of Rho-kinase (Kimura, 1996).

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 induces the formation of stress fibers and focal adhesions, whereas microinjection of the inactive catalytic domain, the Rho-binding domain, or the pleckstrin-homology domain inhibits 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 (Amano, 1997).

p160ROCK is a serine/threonine protein kinase that binds selectively to GTP-Rho; when bound, it becomes active. To identify its function, HeLa cells were transfected with wild type and mutants of p160ROCK and the morphology of the transfected cells were examined. Transfection with wild type and mutants containing the kinase domain and the coiled-coil forming region induces focal adhesions and stress fibers, while no induction is observed with a kinase-defective mutant or a mutant containing only the kinase domain. Rho-induced formation of focal adhesions and stress fibers is inhibited by co-expression of a mutant defective in both kinase and Rho-binding activities. Rho, however, still induces an increase in F-actin content in these cells. These results suggest that p160ROCK works downstream of Rho to induce formation of focal adhesions, and that Rho-induced actin polymerization is mediated by other effector(s) (Ishizaki, 1997).

Rho induces the formation of actin stress fibers and mediates the formation of diverse actin structures. What remains unclear is how Rho regulates its effectors to elicit such functions. GTP-bound Rho activates its effector mDia1 by disrupting mDia1's intramolecular interactions. mDia1 protein is the first identified mammalian homolog of Drosophila Diaphanous, which is essential for cytokinesis, and belongs to a family of formin-homology (FH) proteins. FH proteins share structural features, including the tandemly aligned FH1 and FH2 domains in their carboxy-terminal halves. The FH1 domain contains repetitive polyproline sequences, which interact with an actin-monomer-binding protein, profilin. FH proteins regulate cellular morphogenic events, possibly in collaboration with profilin. Indeed, mDia1, profilin and RhoA have been found to co-localize in dynamic plasma-membrane structures such as phorbol-ester-induced membrane ruffles and phagocytic cups. Because the inactivation of Rho by microinjection of C3 exoenzyme inhibits such co-localization, endogenous Rho may regulate the subcellular assembly of filamentous actin (F-actin) through mDia1 signaling, although the type of actin structure formed under the control of Rho-mDia1 signaling has largely remained unknown. In addition, a genetic study of a Costa Rica family, members of which often develop deafness without other symptoms, has identified hDIA1 as a gene responsible for non-syndromic deafness. The protein encoded by this gene is the human counterpart of mDia1. In the affected hDIA1 protein, 21 aberrant amino acids are substituted for the C-terminal 52 amino acids. It remains unknown how this C-terminal truncation induces pathogenicity and why it is inherited as a dominant trait (Watanabe, 1999 and references therein).

Active mDia1 induces the formation of thin actin stress fibers, which are disorganized in the absence of activity of the Rho-associated kinase ROCK. Moreover, active mDia1 transforms ROCK-induced condensed actin fibers into structures reminiscent of Rho-induced stress fibers. Thus mDia1 and ROCK work concurrently during Rho-induced stress-fiber formation. Intriguingly, mDia1 and ROCK, depending on the balance of the two activities, induce actin fibers of various thicknesses and densities. Thus Rho may induce the formation of different actin structures affected by the balance between mDia1 and ROCK signaling (Watanabe, 1999).

RhoA plays a critical role in signaling pathways activated by serum-derived factors, such as lysophosphatidic acid (LPA), including the formation of stress fibers in fibroblasts and neurite retraction and rounding of soma in neuronal cells. Ectopic expression of v-Crk (an SH2/SH3 domain, containing adapter proteins) potentiates nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells and promotes the survival of cells when NGF is withdrawn. When cultured in 15% serum or lysophosphatidic acid-containing medium, the majority of v-Crk-expressing PC12 cells (v-CrkPC12 cells) display a flattened phenotype with broad lamellipodia and are refractory to NGF-induced neurite outgrowth unless serum is withdrawn. v-Crk-mediated cell flattening is inhibited by treatment of cells with C3 toxin or by mutation in the Crk SH2 or SH3 domain. Transient cotransfection of 293T cells with expression plasmids for p160ROCK (Rho-associated coiled-coil-containing kinase) and v-Crk, but not SH2 or SH3 mutants of v-Crk, results in hyperactivation of p160ROCK. Moreover, the level of phosphatidylinositol-4,5-bisphosphate is increased in v-CrkPC12 cells, as compared to the levels in mutant v-Crk-expressing cells or wild-type cells, consistent with PI(4)P5 kinase being a downstream target for Rho. Expression of v-Crk in PC12 cells does not result in activation of Rac- or Cdc42-dependent kinases PAK and S6 kinase, demonstrating specificity for Rho. In contrast to native PC12 cells, in which focal adhesions and actin stress fibers are not observed, immunohistochemical analysis of v-CrkPC12 cells reveals focal adhesion complexes that are formed at the periphery of the cell and are connected to actin cables. The formation of focal adhesions correlates with a concomitant upregulation in the expression of focal adhesion proteins FAK, paxillin, alpha3-integrin, and a higher-molecular-weight form of beta1-integrin. These results indicate that v-Crk activates the Rho-signaling pathway and serves as a scaffolding protein during the assembly of focal adhesions in PC12 cells (Altun-Gultekin, 1998).

Phagocytosis through Fcgamma receptor (FcgammaR) or complement receptor 3 (CR) requires Arp2/3 complex-mediated actin polymerization, although each receptor uses a distinct signaling pathway. Rac and Cdc42 are required for actin and Arp2/3 complex recruitment during FcgammaR phagocytosis, while Rho controls actin assembly at complement receptor 3 (CR) phagosomes. To better understand the role of Rho in CR phagocytosis, the idea was tested that a known target of Rho, Rho-kinase (ROK), might control phagocytic cup formation and/or engulfment of particles. Inhibitors of ROK (dominant-negative ROK and Y-27632) and of the downstream target of ROK, myosin-II (ML7, BDM, and dominant-negative myosin-II), were used to test this idea. Inhibition of the Rho --> ROK --> myosin-II pathway causes a decreased accumulation of Arp2/3 complex and F-actin around bound particles, which leads to a reduction in CR-mediated phagocytic engulfment. FcgammaR-mediated phagocytosis, in contrast, is independent of Rho or ROK activity and is only dependent on myosin-II for particle internalization, not for actin cup formation. While myosins have been previously implicated in FcgammaR phagocytosis, this is the first demonstration of a role for myosin-II in CR phagocytosis (Olazabal, 2002).

alphaß1 integrins have been implicated in the survival, spreading, and migration of cells and tissues. To explore the underlying biology, conditions were identified where primary ß1 null keratinocytes adhere, proliferate, and display robust alphavß6 integrin-induced, peripheral focal contacts associated with elaborate stress fibers. Mechanistically, this appears to be due to reduced FAK and Src and elevated RhoA and Rock activities. Visualization on a genetic background of GFPactin shows that ß1 null keratinocytes spread, but do so aberrantly, and when induced to migrate from skin explants in vitro, the cells are not able to rapidly reorient their actin cytoskeleton toward the polarized movement. As judged by RFPzyxin/GFPactin videomicroscopy, the alphavß6-actin network does not undergo efficient turnover. Without the ability to remodel their integrin-actin network efficiently, alphaß1-deficient keratinocytes cannot respond dynamically to their environment and polarize movements (Raghavan, 2003).

The results underscore a novel and distinct role for alphaß1 integrins in regulating this equilibrium in focal adhesion dynamics. Not surprisingly, three well-known regulators of focal contacts, FAK, RhoA, and Rock, appear to be at the heart of this regulation. As judged by immunofluorescence with purportedly specific phospho-FAK Abs, activated FAK localizes to the focal contacts of ß1 null keratinocytes. By this criterion, the underlying defects in focal contact turnover and in overall FAK and Src activities are not attributable to a defect in targeting FAK to alphavß6 focal contacts, and indeed, ligand-engaged alphavß6 can bind and activate FAK. Rather, in the absence of ß1, alphavß6 appears unable on its own to activate FAK to the threshold levels needed to properly control focal adhesion-actin cytoskeletal dynamics. Irrespective of the precise underlying mechanism, the consequences to this imbalance are excessive adhesion and inefficient spreading (Raghavan, 2003).

Although tyrosine kinase inhibitors can block focal adhesion formation in some situations, a greater role for tyrosine phosphorylation has been found in focal adhesion turnover and cell motility. Thus, activated FAK negatively regulates RhoA activity, and FAK null fibroblasts express robust actin stress-fiber networks that can be dissipated by Rock inhibition. The ability of Rho and Rock inhibitors to disperse both stress fibers and associated focal contacts in ß1 null keratinocytes provides compelling evidence that a FAK-RhoA imbalance is at the root of the focal adhesion-cytoskeletal imbalance in these cells. Although more complicated mechanisms are possible, the data are consistent with a model whereby in the absence of alphaß1 integrins, FAK/Src activation is not fully achieved, thereby diminishing p190RhoGAP phosphorylation, and yielding elevated RhoA/Rock activities (Raghavan, 2003).

Accumulating evidence suggests that p21(Cip1) located in the cytoplasm might play a role in promoting transformation and tumor progression. Oncogenic H-RasV12 contributes to the loss of actin stress fibers by inducing cytoplasmic localization of p21(Cip1), which uncouples Rho-GTP from stress fiber formation by inhibiting Rho kinase (ROCK). Concomitant with the loss of stress fibers in Ras-transformed cells, there is a decrease in the phosphorylation level of cofilin, which is indicative of a compromised ROCK/LIMK/cofilin pathway. Inhibition of MEK in Ras-transformed NIH3T3 results in restoration of actin stress fibers accompanied by a loss of cytoplasmic p21(Cip1), and increased phosphorylation of cofilin. Ectopic expression of cytoplasmic but not nuclear p21(Cip1) in Ras-transformed cells is effective in preventing stress fibers from being restored upon MEK inhibition and inhibits phosphorylation of cofilin. p21(Cip1) forms a complex with ROCK in Ras-transformed cells in vivo. Furthermore, inhibition of the PI 3-kinase pathway results in loss of p21(Cip1) expression, accompanied by restoration of phosphocofilin, that is not accompanied by stress fiber formation. These results suggest that restoration of cofilin phosphorylation in Ras-transformed cells is necessary but not sufficient for stress fiber formation. These findings define a novel mechanism for coupling cytoplasmic p21(Cip1) to the control of actin polymerization by compromising the Rho/ROCK/LIMK/cofilin pathway by oncogenic Ras. These studies suggest that localization of p21(Cip1) to the cytoplasm in transformed cells contributes to pathways that favor not only cell proliferation, but also cell motility, thereby contributing to invasion and metastasis (Lee, 2003).

The transition of cell-matrix adhesions from the initial punctate focal complexes into the mature elongated form, known as focal contacts, requires GTPase Rho activity. In particular, activation of myosin II-driven contractility by a Rho target known as Rho-associated kinase (ROCK) has been shown to be essential for focal contact formation. To dissect the mechanism of Rho-dependent induction of focal contacts and to elucidate the role of cell contractility, mechanical force was applied to vinculin-containing dot-like adhesions at the cell edge using a micropipette. Local centripetal pulling leads to local assembly and elongation of these structures and to their development into streak-like focal contacts, as revealed by the dynamics of green fluorescent protein-tagged vinculin or paxillin and interference reflection microscopy. Inhibition of Rho activity by C3 transferase suppresses this force-induced focal contact formation. However, constitutively active mutants of another Rho target, the formin homology protein mDia1, are sufficient to restore force-induced focal contact formation in C3 transferase-treated cells. Force-induced formation of the focal contacts still occurs in cells subjected to myosin II and ROCK inhibition. Thus, as long as mDia1 is active, external tension force bypasses the requirement for ROCK-mediated myosin II contractility in the induction of focal contacts. These experiments show that integrin-containing focal complexes behave as individual mechanosensors exhibiting directional assembly in response to local force (Riveline, 2001).

Myosin II regulation during C. elegans embryonic elongation: LET-502/ROCK, MRCK-1 and PAK-1, three kinases with different roles

Myosin II plays a central role in epithelial morphogenesis; however, its role has mainly been examined in processes involving a single cell type. This study analyzed the structure, spatial requirement and regulation of myosin II during C. elegans embryonic elongation, a process that involves distinct epidermal cells and muscles. Novel GFP probes were developed to visualize the dynamics of actomyosin remodeling; it was found that the assembly of myosin II filaments, but not actin microfilaments, depends on the myosin regulatory light chain (MLC-4) and essential light chain (MLC-5). To determine how myosin II regulates embryonic elongation, mlc-4 mutants were rescued with various constructs and found that MLC-4 is essential in a subset of epidermal cells. Phosphorylation of two evolutionary conserved MLC-4 serine and threonine residues is important for myosin II activity and organization. In an RNAi screen for potential myosin regulatory light chain kinases, it was found that the ROCK, PAK and MRCK homologs act redundantly. The combined loss of ROCK and PAK, or ROCK and MRCK, completely prevented embryonic elongation, but a constitutively active form of MLC-4 could only rescue a lack of MRCK. This result, together with systematic genetic epistasis tests with a myosin phosphatase mutation, suggests that ROCK and MRCK regulate MLC-4 and the myosin phosphatase. Moreover, it is suggested that ROCK and PAK regulate at least one other target essential for elongation, in addition to MLC-4 (Gally, 2009).

Domain structure of Rho kinase

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. The mode of action of inhibitors of Rho-kinase has been examined. The chemical compounds such as HA1077 and Y-32885 inhibit not only the Rho-kinase activity but also the activity of protein kinase N, one of the targets of Rho, but has 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, interacts with Rho-kinase and thereby inhibits the Rho-kinase activity, whereas RB/PH (TT) has 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 blocks 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 (Amano, 1999).

Rho kinase and cell cycle

Rho kinase is required for sustained ERK signaling and the consequent mid-G(1) phase induction of cyclin D1 in fibroblasts. These Rho kinase effects are mediated by the formation of stress fibers and the consequent clustering of alpha5beta1 integrin. Mechanistically, alpha5beta1 signaling and stress fiber formation allow for the sustained activation of MEK, and this effect is mediated upstream of Ras-GTP loading. Interestingly, disruption of stress fibers with myosin light chain kinase inhibitor ML-7 leads to G(1) phase arrest while comparable disruption of stress fibers with Y27632 (an inhibitor of Rho kinase) or dominant-negative Rho kinase leads to a more rapid progression through G(1) phase. Inhibition of either MLCK or Rho kinase blocks sustained ERK signaling, but only Rho kinase inhibition allows for the induction of cyclin D1 and activation of cdk4 via Rac/Cdc42. The levels of cyclin E, cdk2, and their major inhibitors, p21(cip1) and p27(kip1), are not affected by inhibition of MLCK or Rho kinase. Overall, these results indicate that Rho kinase-dependent stress fiber formation is required for sustained activation of the MEK/ERK pathway and the mid-G(1) phase induction of cyclin D1, but not for other aspects of cdk4 or cdk2 activation. They also emphasize that G(1) phase cell cycle progression in fibroblasts does not require stress fibers if Rac/Cdc42 signaling is allowed to induce cyclin D1 (Roovers, 2003).

The Rho-Rho kinase pathway controls cyclin D1 expression by preventing its early G1 phase induction in response to Rac and/or Cdc42, thus increasing its dependence on ERK signaling and actin stress fiber formation. The Rho kinase effector LIM kinase (see Drosophila LIM-kinase1) is responsible for this effect. Surprisingly, inhibition of Rac-dependent cyclin D1 expression by LIM kinase is independent of both cofilin phosphorylation and actin polymerization. Instead, specific mutation of its nuclear localization and export sequences show that LIM kinase acts in the nucleus to suppress Rac/Cdc42-dependent cyclin D1 expression. These results therefore describe an unexpected role for LIM kinase that requires nuclear translocation. The effect of nuclear LIM kinase on cyclin D1 expression ultimately regulates the duration of G1 phase and the degree to which G1 phase progression depends on actin stress fiber formation and imposition of cellular tension (Roovers, 2003b).

G1 phase progression in mammalian cells is mediated by the activities of cyclin D-cdk4 (or cdk6) and cyclin E-cdk2. The activation of these enzymes is regulated by a complex interplay of signaling pathways that reflect conditions in the extracellular environment. For example, the induction of cyclin D1, typically the rate-limiting step in the activation of cdk4/6, involves cooperative signaling by receptor tyrosine kinases (RTKs; receptors for many mitogenic growth factors), integrins (receptors for extracellular matrix proteins), and the actin cytoskeleton. At least in most fibroblasts, cyclin D1 is induced in mid-G1 phase (9 hr after mitogen stimulation of quiescent cells), and this mid-G1 phase induction requires sustained (5-6 hr) ERK activity. RTKs, integrins, and actin stress fibers are jointly required to sustain the ERK signal long enough to induce cyclin D1 (Roovers, 2003b).

Cyclin D1 can also be induced by Rac and/or Cdc42 in an ERK-independent manner. Rac/Cdc42-dependent induction of cyclin D1 requires RTK and integrin signaling, but it is independent of stress fiber formation. In fact, if cyclin D1 is induced by Rac/Cdc42, then all of G1 phase progression in fibroblasts can occur in the absence of stress fibers and the consequent imposition of cellular tension. Rac/Cdc42 signaling also results in an early G1 phase induction of cyclin D1 (3 hr after mitogenic stimulation of quiescent cells), and this premature induction leads to a correspondingly early activation of cdk4 and cdk2, as well as a several hour decrease in the duration of G1 phase as cells leave quiescence. Thus, the choice of signaling pathways used to induce cyclin D1 (sustained ERK versus Rac/Cdc42) has at least two distinct consequences for cell cycle progression through G1 phase (Roovers, 2003b).

Rho kinase determines whether cyclin D1 is induced by sustained ERK or Rac/Cdc42. Rho kinase is required for sustained ERK signaling because it promotes stress fiber formation and integrin clustering/signaling in growth factor-treated cells. Rho kinase also suppresses Rac/Cdc42-dependent cyclin D1 induction downstream of GTP-loading. This inhibitory effect of Rho kinase on Rac/Cdc42 signaling maintains the mid-G1 phase, ERK-dependent induction of cyclin D1 that is typically seen in fibroblastic cells (Roovers, 2003b).

Rho kinase is best known as a regulator of actin stress fibers through its stimulatory effects on contractility and actin polymerization. Rho kinase promotes contractility by inhibiting myosin light chain (MLC) phosphatase and by direct phosphorylation of MLC itself. Rho kinase promotes actin polymerization by activating LIM kinase (LIMK) and phosphatidylinositol 4-phosphate 5-kinase. Its effect on LIMK is the best understood: Rho kinase activates LIMK1 and LIMK2 by phosphorylating T508 and T505, respectively, which in turn catalyze the inactivating phosphoryation of cofilin on S3. Although exceptions exist, cofilin typically promotes actin depolymerization, so its inactivation by the Rho kinase-LIMK pathway stimulates actin polymerization. The combined effects of Rho kinase on MLC and LIMK phosphorylation result in stress fiber formation. Note, however, that mDia (a Rho kinase-independent effector of Rho) and PAK (an effector of Rac and Cdc42) also contribute to actin polymerization. Besides regulating the kinetics of ERK activation, the polymerization of actin that is associated with stress fiber formation can directly regulate gene expression because a subset of SRF-dependent genes is strongly stimulated by the consequent depletion of the g-actin pool (Roovers, 2003b and references therein).

In contrast to its well-characterized effects on stress fiber formation, the mechanism by which Rho kinase suppresses Rac/Cdc42 signaling to cyclin D1 remains completely unexplored. LIMK is the effector that suppresses Rac/Cdc42 signaling to cyclin D1. Surprisingly, the suppressive effect of LIMK on Rac/Cdc42-mediated cyclin D1 induction is independent of cofilin (its only characterized substrate) and actin polymerization (its only characterized effect). Moreover, specific mutation of its nuclear localization and export sequences show that LIM kinase acts in the nucleus to suppress Rac/Cdc42-dependent expression of cyclin D1. Thus, in addition to identifying the Rho kinase effector that suppresses Rac/Cdc42-signaling to cyclin D1, these studies reveal a specific role for LIMK in the nucleus (Roovers, 2003b).

Rho-kinase phosphorylates PAR-3 and disrupts PAR complex formation

A polarity complex of PAR-3, PAR-6, and atypical protein kinase C (aPKC) functions in various cell polarization events. PAR-3 directly interacts with Tiam1/Taim2 (STEF), Rac1-specific guanine nucleotide exchange factors, and forms a complex with aPKC-PAR-6-Cdc42•GTP, leading to Rac1 activation. RhoA antagonizes Rac1 in certain types of cells. However, the relationship between RhoA and the PAR complex remains elusive. This study found that, in mammalian cultured cells, Rho-kinase/ROCK/ROK, the effector of RhoA, phosphorylated PAR-3 at Thr833 and thereby disrupted its interaction with aPKC and PAR-6, but not with Tiam2. Phosphorylated PAR-3 was observed in the leading edge, and in central and rear portions of migrating cells having front-rear polarity. Knockdown of PAR-3 by small interfering RNA (siRNA) impaired cell migration, front-rear polarization, and PAR-3-mediated Rac1 activation, which were recovered with siRNA-resistant PAR-3, but not with the phospho-mimic PAR-3 mutant. It is proposed that RhoA/Rho-kinase inhibits PAR complex formation through PAR-3 phosphorylation, resulting in Rac1 inactivation (Nakayama, 2008).

This study found here that PAR-3 is heavily phosphorylated at Thr833 in the central and rear regions of polarized migrating cells, and this phosphorylation is diminished by treatment with kinase inhibitor Y-27632, suggesting that RhoA/Rho-kinase phosphorylates PAR-3 there. Consistently, RhoA activity is higher in the central and rear portions than in the front area in the migrating cells. Knockdown PAR-3 (PAR-3 KD) induced the cells into having multiple small leading edges without front-rear polarity, and the effect of PAR-3 KD was rescued by RNAi-resistant PAR-3 but not with mimic phosphorylation of PAR-3 (PAR-3-833D). Treatment with Y-27632 also induced the cells into having multiple small leading edges. Thus, the phosphorylation of PAR-3 by Rho-kinase may prevent Rac1 activation and lamellipodia formation at the leading edge to control front-rear polarity and directional migration (Nakayama, 2008).

It was also found that Rho-kinase phosphorylated PAR-3 in the leading edge of polarized migrating cells. This is consistent with the previous observations that RhoA and Rho-kinase are activated in both leading edge and rear regions in the migrating cell. What is the physiological significance of PAR-3 phosphorylation in the leading edge? It is speculated that spatio-temporal on-off regulation of Rac1 activity is necessary for proper cell migration. During cell migration, the adhesion signal from integrin may activate Rac1 through PAR-3 in Cdc42-dependent and -independent manners. Integrin also activates RhoA and thereby Rho-kinase. Rho-kinase phosphorylates PAR-3 and in turn disrupts the PAR complex, which can prevent overactivation of Rac1. In support of this, the treatment of HeLa cells with Rho-kinase inhibitor enhanced lamellar length and cell migration. This pathway may serve as a local negative feedback signal to control the leading edge (Nakayama, 2008).

Rho kinase and development

let-502 rho-binding kinase and mel-11 myosin phosphatase regulate Caenorhabditis elegans embryonic morphogenesis. Genetic analysis presented here establishes the following modes of let-502 action: (1) loss of only maternal let-502 results in abnormal early cleavages, (2) loss of both zygotic and maternal let-502 causes elongation defects, and (3) loss of only zygotic let-502 results in sterility. The morphogenetic function of let-502 and mel-11 is apparently redundant with another pathway since elimination of these two genes results in progeny that undergo near-normal elongation. Triple mutant analysis indicates that unc-73 (Rho/Rac guanine exchange factor) and mlc-4 (myosin light chain) act in parallel to or downstream of let-502/mel-11. In contrast mig-2 (Rho/Rac), daf-2 (insulin receptor), and age-1 (PI3 kinase) act within the let-502/mel-11 pathway. Mutations in the sex-determination gene fem-2, which encodes a PP2c phosphatase (unrelated to the MEL-11 phosphatase), enhances mutations of let-502 and suppressed those of mel-11. fem-2's elongation function appears to be independent of its role in sexual identity since the sex-determination genes fem-1, fem-3, tra-1, and tra-3 have no effect on mel-11 or let-502. By itself, fem-2 affects morphogenesis with low penetrance. fem-2 blocks the near-normal elongation of let-502; mel-11, indicating that fem-2 acts in a parallel elongation pathway. The action of two redundant pathways likely ensures accurate elongation of the C. elegans embryo (Piekny, 2000).

Rho-binding kinase and the myosin phosphatase targeting subunit regulate nonmuscle contractile events in higher eukaryotes. Genetic evidence indicates that the C. elegans homologs regulate embryonic morphogenesis by controlling the actin-mediated epidermal cell shape changes that transform the spherical embryo into a long, thin worm. LET-502/Rho-binding kinase triggers elongation while MEL-11/myosin phosphatase targeting subunit inhibits this contractile event. Mutations are described in the nonmuscle myosin heavy chain gene nmy-1 that were isolated as suppressors of the mel-11 hypercontraction phenotype. However, a nmy-1 null allele displays elongation defects less severe than mutations in let-502 or in the single nonmuscle myosin light chain gene mlc-4. This results because nmy-1 is partially redundant with another nonmuscle myosin heavy chain, nmy-2, which was previously known only for its role in anterior/posterior polarity and cytokinesis in the early embryo. At the onset of elongation, NMY-1 forms filamentous-like structures similar to actin, and LET-502 is interspersed with these structures, where it may trigger contraction. MEL-11, which inhibits elongation, is initially cytoplasmic. In response to LET-502 activity, MEL-11 becomes sequestered away from the contractile apparatus, to the plasma membrane, when elongation commences. Upon completion of morphogenesis, MEL-11 again appears in the cytoplasm where it may halt actin/myosin contraction (Piekny, 2003).

Rho-associated kinases (Rho kinases), which are downstream effectors of RhoA GTPase, regulate diverse cellular functions including actin cytoskeletal organization. Rho kinases also direct the early stages of chick and mouse embryonic morphogenesis. Rho kinase transcripts are enriched in cardiac mesoderm, lateral plate mesoderm and the neural plate. Treatment of neurulating embryos with Y27632, a specific inhibitor of Rho kinases, blocks migration and fusion of the bilateral heart primordia, formation of the brain and neural tube, caudalward movement of Hensen's node, and establishment of normal left-right asymmetry. Moreover, Y27632 induces precocious expression of cardiac alpha-actin, an early marker of cardiomyocyte differentiation, coincident with the upregulated expression of serum response factor and GATA4. In addition, specific antisense oligonucleotides significantly diminish Rho kinase mRNA levels and replicate many of the teratologies induced by Y27632. Thus, this study reveals new biological functions for Rho kinases in regulating major morphogenetic events during early chick and mouse development (Weil, 2001).

The epidermis comprises multiple layers of specialized epithelial cells called keratinocytes. As cells are lost from the outermost epidermal layers, they are replaced through terminal differentiation, in which keratinocytes of the basal layer cease proliferating, migrate upwards, and eventually reach the outermost cornified layers. Normal homeostasis of the epidermis requires that the balance between proliferation and differentiation be tightly regulated. The GTP binding protein RhoA plays a fundamental role in the regulation of the actin cytoskeleton and in the adhesion events that are critically important to normal tissue homeostasis. Two central mediators of the signals from RhoA are the ROCK serine/threonine kinases ROCK-I and ROCK-II. ROCK's role in the regulation of epidermal keratinocyte function was examined by using a pharmacological inhibitor and expressing conditionally active or inactive forms of ROCK-II in primary human keratinocytes. vlocking ROCK function results in inhibition of keratinocyte terminal differentiation and an increase in cell proliferation. In contrast, activation of ROCK-II in keratinocytes results in cell cycle arrest and an increase in the expression of a number of genes associated with terminal differentiation. Thus, these results indicate that ROCK plays a critical role in regulating the balance between proliferation and differentiation in human keratinocytes (McMullan, 2004).

Development of the endocardial cushions in the heart involves cell migration and cell differentiation, which is known as epithelial-mesenchymal transformation (EMT). These processes are regulated by cell signaling systems. Yet, the roles of intracellular GTPases and their effectors on these cellular activities remain to be addressed. This study investigated the role of Rho GTPase-associated kinases (ROCKs) in endocardial cushion development. Using reverse transcription (RT) and polymerase chain reaction (PCR), expression of the rock1 and rock2 genes was found in the endocardial cushions during development. To investigate the role of ROCKs in development, the ROCK inhibitor Y27632 and adenoviruses containing a dominant negative form of the rock gene were used to treat cultured endocardial cushions and cells. In monolayer cell culture and three-dimensional tissue culture, blockade of ROCK inhibits EMT development. Using three-dimensional collagen gel assays and confocal microscopy, inhibition of cell migration was observed with ROCK inhibition. Examination of cell morphology and actin cytoskeleton revealed that inhibition of ROCK activity disturbed cytoskeletal organization and blocked the formation of lamellipodia and filopodia. Collectively, these data show that ROCKs play an essential role in endothelial cell differentiation and migration during endocardial cushion development (Zhao, 2004).

Embryonic morphogenesis involves the coordinate behaviour of multiple cells and requires the accurate balance of forces acting within different cells through the application of appropriate brakes and throttles. In C. elegans, embryonic elongation is driven by Rho-binding kinase (ROCK) and actomyosin contraction in the epidermis. This study identified an evolutionary conserved, actin microfilament-associated RhoGAP (RGA-2) that behaves as a negative regulator of LET-502/ROCK. The small GTPase RHO-1 is the preferred target of RGA-2 in vitro, and acts between RGA-2 and LET-502 in vivo. Two observations show that RGA-2 acts in dorsal and ventral epidermal cells to moderate actomyosin tension during the first half of elongation: (1) time-lapse microscopy shows that loss of RGA-2 induces localised circumferentially oriented pulling on junctional complexes in dorsal and ventral epidermal cells; (2) specific expression of RGA-2 in dorsal/ventral, but not lateral, cells rescues the embryonic lethality of rga-2 mutants. It is proposed that actomyosin-generated tension must be moderated in two out of the three sets of epidermal cells surrounding the C. elegans embryo to achieve morphogenesis (Diogon, 2007).

Rho kinase and transformation

RhoA controls signaling processes required for cytoskeletal reorganization, transcriptional regulation, and transformation. The ability of RhoA mutants to transform cells correlates not with transcription but with their ability to bind ROCK-I, an effector kinase involved in cytoskeletal reorganization. A recently developed specific ROCK inhibitor, Y-27632, and ROCK truncation mutants were used to investigate the role of ROCK kinases in transcriptional activation and transformation. In NIH3T3 cells, Y-27632 does not prevent the activation of serum response factor, transcription of c-fos or cell cycle re-entry following serum stimulation. Repeated treatment of NIH3T3 cells with Y-27632, however, substantially disrupts their actin fiber network but does not affect their growth rate. Y-27632 blocks focus formation by activated RhoA and its guanine-nucleotide exchange factors Dbl and mNET1. It does not affect the growth rate of cells transformed by Dbl and mNET1, but restores normal growth control at confluence and prevents their growth in soft agar. Y-27632 also significantly inhibits focus formation by Ras, but has no effect on the establishment or maintenance of transformation by Src. Furthermore, it significantly inhibits anchorage-independent growth of two out of four colorectal tumor cell lines. Consistent with these data, a truncated ROCK derivative exhibits weak ability to cooperate with activated Raf in focus formation assays. It is concluded that ROCK signaling is required for both the establishment and maintenance of transformation by constitutively activated RhoA, and that it contributes to the Ras-transformed phenotype. These observations provide a potential explanation for the requirement for Rho in Ras-mediated transformation. Moreover, the inhibition of ROCK kinases may be of therapeutic use (Sahai, 1999).

Miscellaneous Rho kinase targets

The small GTPase Rho is believed to regulate the actin cytoskeleton and cell adhesion through its specific targets. Rho targets protein kinase N, Rho-associated kinase (Rho-kinase), and the myosin binding subunit (MBS) of myosin phosphatase. This study purified MBS-interacting proteins, identified them as adducin (see Drosophila Hu-li tai shao), and found that MBS specifically interacts with adducin in vitro and in vivo. Adducin is a membrane-skeletal protein that promotes the binding of spectrin to actin filaments and is concentrated at the cell-cell contact sites in epithelial cells. It was also found that Rho-kinase phosphorylates alpha-adducin in vitro and in vivo and that the phosphorylation of alpha-adducin by Rho-kinase enhances the interaction of alpha-adducin with actin filaments in vitro. Myosin phosphatase composed of the catalytic subunit and MBS showed phosphatase activity toward alpha-adducin, which was phosphorylated by Rho-kinase. This phosphatase activity was inhibited by the phosphorylation of MBS by Rho-kinase. These results suggest that Rho-kinase and myosin phosphatase regulate the phosphorylation state of adducin downstream of Rho and that the increased phosphorylation of adducin by Rho-kinase causes the interaction of adducin with actin filaments (Kimura, 1998).

The actin cytoskeleton undergoes extensive remodeling during cell morphogenesis and motility. The small guanosine triphosphatase Rho regulates such remodeling, but the underlying mechanisms of this regulation remain unclear. Cofilin exhibits actin-depolymerizing activity that is inhibited as a result of its phosphorylation by LIM-kinase. Cofilin is phosphorylated in N1E-115 neuroblastoma cells during lysophosphatidic acid-induced, Rho-mediated neurite retraction. This phosphorylation is sensitive to Y-27632, a specific inhibitor of the Rho-associated kinase ROCK. ROCK, which is a downstream effector of Rho, does not phosphorylate cofilin directly but phosphorylates LIM-kinase, which in turn is activated in order to phosphorylate cofilin. Overexpression of LIM-kinase in HeLa cells induces the formation of actin stress fibers in a Y-27632-sensitive manner. These results indicate that phosphorylation of LIM-kinase by ROCK and consequently increased phosphorylation of cofilin by LIM-kinase contribute to Rho-induced reorganization of the actin cytoskeleton (Maekawa, 1999).

Previous studies have shown that LIM-kinase activation and cofilin phosphorylation occur in response to Rac, another member of the Rho family GTPases, but not to Rho. It was determined that activation of Rho results in cofilin phosphorylation by LIM-kinase in vivo by expressing constitutively active V14-Rho and tagged cofilin in COS-7 cells. This experiment was done under conditions in which the activity of endogenous Rac was inhibited by expression of the dominant negative mutant N17-Rac, and that of endogenous Rho was inhibited by culture in serum-free medium. Expression of N17-Rac almost completely abolishes the cofilin phosphorylation observed in cells expressing tagged cofilin alone, suggesting that endogenous Rac is activated under basal conditions and induces cofilin phosphorylation. In the presence of N17-Rac, V14-Rho increases cofilin phosphorylation in transfected cells, and this increase is inhibited by coexpression of a dominant negative ROCK mutant, ROCK-KDIA. These observations suggest that the Rho-ROCK pathway is linked to cofilin phosphorylation in vivo, and that the previous studies may have masked this pathway by activation of endogenous Rac and Rho. The link between Rho-ROCK signaling and the LIM-kinase-cofilin pathway was investigated at the morphological level. Overexpression of LIMK1 in HeLa cells induces the formation of thick, bundled stress fibers that resemble those induced by active Rho or ROCK. Incubation of the transfected cells with Y-27632 results in the dissolution of these fibers, indicating that LIM-kinase collaborates with the Rho-ROCK pathway to induce stress fiber formation (Maekawa, 1999 and references).

Vimentin, the most widely expressed intermediate filament protein, serves as an excellent substrate for Rho-associated kinase (Rho-kinase) and vimentin phosphorylated by Rho-kinase has lost its ability to form filaments in vitro. Two amino-terminal sites on vimentin, Ser38 and Ser71, are the major phosphorylation sites for Rho-kinase, and Ser71 is the most favored and unique phosphorylation site for Rho-kinase in vitro. To analyze the vimentin phosphorylation by Rho-kinase in vivo, an antibody (GK71) was prepared that specifically recognizes the phosphorylation of vimentin-Ser71. Ectopic expression of constitutively active Rho-kinase in COS-7 cells induces phosphorylation of vimentin at Ser71, followed by the reorganization of vimentin filament networks. During the cell cycle, the phosphorylation of vimentin-Ser71 occurs only at the cleavage furrow in late mitotic cells but not in interphase or early mitotic cells. This cleavage furrow-specific phosphorylation of vimentin-Ser71 was observed in the various types of cells that were examined. All these accumulating observations increase the possibility that Rho-kinase may have a definite role in governing regulatory processes in assembly-disassembly and turnover of vimentin filaments at the cleavage furrow during cytokinesis (Goto, 1998).

Rho-associated kinase regulates formation of stress fibers and focal adhesions, myosin fiber organization, and neurite retraction through the phosphorylation of cytoskeletal proteins, including myosin light chain, the ERM family proteins (ezrin, radixin, and moesin) and adducin. Rho-kinase phosphorylates a type III intermediate filament (IF) protein, glial fibrillary acidic protein (GFAP), exclusively at the cleavage furrow during cytokinesis. The roles of Rho-kinase in cytokinesis, in particular organization of glial filaments during cytokinesis, has been examined. Expression of the dominant-negative form of Rho-kinase inhibits the cytokinesis of Xenopus embryo and mammalian cells, the result being production of multinuclei. A series of mutant GFAPs were constructed, where Rho-kinase phosphorylation sites were variously mutated, and they were expressed in type III IF-negative cells. The mutations induce impaired segregation of glial filament (GFAP filament) into postmitotic daughter cells. As a result, an unusually long bridge-like cytoplasmic structure forms between the unseparated daughter cells. Alteration of other sites, including the cdc2 kinase phosphorylation site, leads to no remarkable defect in glial filament separation. These results suggest that Rho-kinase is essential not only for actomyosin regulation but also for segregation of glial filaments into daughter cells which in turn ensures correct cytokinetic processes (Yasui, 1998).

Rho-associated kinase phosphorylates myosin binding subunit (MBS) of myosin phosphatase and thereby inactivates the phosphatase activity in vitro. Rho-kinase is thought to regulate the phosphorylation state of the substrates including myosin light chain (MLC), ERM (ezrin/radixin/moesin) family proteins and adducin by their direct phosphorylation and by the inactivation of myosin phosphatase. The sites of phosphorylation of MBS by Rho-kinase have been identified as Thr-697 and Ser-854; antibody has been prepared that specifically recognizes MBS phosphorylated at Ser-854. It has been found by use of this antibody that the stimulation of MDCK epithelial cells with tetradecanoylphorbol-13-acetate (TPA) or hepatocyte growth factor (HGF) induces the phosphorylation of MBS at Ser-854 under the conditions in which membrane ruffling and cell migration are induced. Pretreatment of the cells with Botulinum C3 ADP-ribosyltransferase (C3), which is thought to interfere with Rho functions, or Rho-kinase inhibitors inhibit the TPA- or HGF-induced MBS phosphorylation. The TPA stimulation enhances the immunoreactivity of phosphorylated MBS in the cytoplasm and membrane ruffling area of MDCK cells. In migrating MDCK cells, phosphorylated MBS as well as phosphorylated MLC at Ser-19 are localized in the leading edge and posterior region. Phosphorylated MBS is localized on actin stress fibers in REF52 fibroblasts. The microinjection of C3 or dominant negative Rho-kinase disrupts stress fibers and weakens the accumulation of phosphorylated MBS in REF52 cells. During cytokinesis, phosphorylated MBS, MLC and ERM family proteins accumulate at the cleavage furrow, and the phosphorylation level of MBS at Ser-854 is increased. Taken together, these results indicate that MBS is phosphorylated by Rho-kinase downstream of Rho in vivo, and suggest that myosin phosphatase and Rho-kinase spatiotemporally regulate the phosphorylation state of Rho-kinase substrates, including MLC and ERM family proteins in vivo in a cooperative manner (Kawano, 1999).

Stimulation of phospholipase D (PLD) by membrane receptors is now recognized as a major signal transduction pathway involved in diverse cellular functions. Rho proteins control receptor signaling to PLD, and these GTPases have been shown to directly stimulate purified recombinant PLD1 enzymes in vitro. Stimulation of PLD activity, measured in the presence of phosphatidylinositol 4,5-bisphosphate, by RhoA in membranes of HEK-293 cells expressing the m3 muscarinic acetylcholine receptor (mAChR) is phosphorylation-dependent. Therefore, the possible involvement of the RhoA-stimulated serine/threonine kinase, Rho-kinase, was investigated. Overexpression of Rho-kinase and constitutively active Rho-kinase (Rho-kinase-CAT) but not of kinase-deficient Rho-kinase-CAT, markedly increases m3 mAChR-mediated but not protein kinase C-mediated PLD stimulation, similar to overexpression of RhoA. Expression of the Rho-inactivating C3 transferase abrogates the stimulatory effect of wild-type Rho-kinase, but not of Rho-kinase-CAT. Recombinant Rho-kinase-CAT mimics the phosphorylation-dependent PLD stimulation by RhoA in HEK-293 cell membranes. Finally, the Rho-kinase inhibitor HA-1077 largely inhibits RhoA-induced PLD stimulation in membranes as well as PLD stimulation by the m3 mAChR but not by protein kinase C in intact HEK-293 cells. It is concluded that Rho-kinase is involved in Rho-dependent PLD stimulation by the G protein-coupled m3 mAChR in HEK-293 cells. Thus, these findings identify Rho-kinase as a novel player in the receptor-controlled PLD signaling pathway (Schmidt, 1999).

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] has been implicated in the regulation of actin polymerization. Since the synthesis of PI(4,5)P(2) has been suggested to be affected by Rho proteins, whether Rho-kinase is involved in the control of PI(4,5)P(2) levels was investigated. Overexpression of RhoA in HEK-293 cells increases phosphatidylinositol 4-phosphate (PI4P) 5-kinase activity and concomitantly enhances cellular PI(4,5)P(2) levels, whereas overexpression of the Rho-inactivating C3 transferase decreases both PI4P 5-kinase activity and PI(4,5)P(2) levels. These effects of RhoA can be mimicked by overexpression of wild-type Rho-kinase and of the constitutively active catalytic domain of Rho-kinase, Rho-kinase-CAT. In contrast, a kinase-deficient mutant of Rho-kinase has no effect on PI4P 5-kinase activity. Importantly, the increase in PI4P 5-kinase activity and PI(4,5)P(2) levels by wild-type Rho-kinase, but not by Rho-kinase-CAT, is completely prevented by coexpression of C3 transferase, indicating that the effect of Rho-kinase is under the control of endogenous Rho. In cell lysates, addition of recombinant RhoA and Rho-kinase-CAT stimulates PI4P 5-kinase activity. Finally, the increase in PI(4,5)P(2) levels induced by both Rho-kinase-CAT and RhoA is reversed by the Rho-kinase inhibitor HA-1077. These data suggest that Rho-kinase is involved in the Rho-controlled synthesis of PI(4,5)P(2) by PI4P 5-kinase (Weernink, 2000).

This study investigated the regulation of T-type channels by Rho-associated kinase (ROCK). Activation of ROCK via the endogenous ligand lysophosphatidic acid (LPA) reversibly inhibits the peak current amplitudes of rat Cav3.1 and Cav3.3 channels without affecting the voltage dependence of activation or inactivation, whereas Cav3.2 currents showed depolarizing shifts in these parameters. LPA-induced inhibition of Cav3.1 is dependent on intracellular GTP, and is antagonized by treatment with ROCK and RhoA inhibitors, LPA receptor antagonists or GDPssS. Site-directed mutagenesis of the Cav3.1 alpha1 subunit revealed that the ROCK-mediated effects involve two distinct phosphorylation consensus sites in the domain II-III linker. ROCK activation by LPA reduces native T-type currents in Y79 retinoblastoma and in lateral habenular neurons, and upregulates native Cav3.2 current in dorsal root ganglion neurons. These data suggest that ROCK is an important regulator of T-type calcium channels, with potentially far-reaching implications for multiple cell functions modulated by LPA (Iftinca, 2007).

Rho kinase function in convergence extension during gastrulation

During vertebrate gastrulation convergence and extension (CE), movements narrow and lengthen embryonic tissues. In Xenopus and zebrafish, a noncanonical Wnt signaling pathway constitutes the vertebrate counterpart to the Drosophila planar cell polarity pathway and regulates mediolateral cell polarization underlying CE. Despite the identification of several signaling molecules required for normal CE, the downstream transducers regulating individual cell behaviors driving CE are only beginning to be elucidated. Moreover, how defective mediolateral cell polarity impacts CE is not understood. Overexpression of zebrafish dominant-negative Rho kinase 2 (dnRok2) disrupts CE without altering cell fates, phenocopying noncanonical Wnt signaling mutants. Moreover, Rho kinase 2 (Rok2) overexpression partially suppresses the slb/wnt11 gastrulation phenotype, and ectopic expression of noncanonical Wnts modulates Rok2 intracellular distribution. In addition, time-lapse analyses associate defective dorsal convergence movements with impaired cell elongation, mediolateral orientation, and consequently failure to migrate along straight paths. Transplantation experiments reveal that dnRok2 cells in wild-type hosts neither elongate nor orient their axes. In contrast, wild-type cells are able to elongate their cell bodies in dnRok2 hosts, even though they fail to orient their axes. It is concluded that during zebrafish gastrulation Rok2 acts downstream of noncanonical Wnt11 signaling to mediate mediolateral cell elongation required for dorsal cell movement along straight paths. Furthermore, elongation and orientation of the cell body are independent properties that require both cell-autonomous and nonautonomous Rok2 function (Marlow, 2002).

Rho kinase and cell migration

All vertebrates contain two nonmuscle myosin II heavy chains, A and B, which differ in tissue expression and subcellular distributions. To understand how these distinct distributions are controlled and what role they play in cell migration, myosin IIA and IIB were examined during wound healing by bovine aortic endothelial cells. Immunofluorescence has shown that myosin IIA skews toward the front of migrating cells, coincident with actin assembly at the leading edge, whereas myosin IIB accumulates in the rear 15-30 min later. Inhibition of myosin light-chain kinase, protein kinases A, C, and G, tyrosine kinase, MAP kinase, and PIP3 kinase does not affect this asymmetric redistribution of myosin isoforms. However, posterior accumulation of myosin IIB, but not anterior distribution of myosin IIA, is inhibited by dominant-negative rhoA and by the rho-kinase inhibitor, Y-27632, which also inhibits myosin light-chain phosphorylation. This inhibition is overcome by transfecting cells with constitutively active myosin light-chain kinase. These observations indicate that asymmetry of myosin IIB, but not IIA, is regulated by light-chain phosphorylation mediated by rho-dependent kinase. Blocking this pathway inhibits tail constriction and retraction, but does not affect protrusion, suggesting that myosin IIB functions in pulling the rear of the cell forward (Kolega, 2003).

Rock2 controls TGFbeta signaling and inhibits mesoderm induction in zebrafish embryos

The Rho-associated serine/threonine kinases Rock1 and Rock2 play important roles in cell contraction, adhesion, migration, proliferation and apoptosis. Mammalian Rock2 acts as a negative regulator of the TGFbeta signaling pathway. Mechanistically, Rock2 binds to and accelerates the lysosomal degradation of TGFbeta type I receptors internalized from the cell surface in mammalian cells. The inhibitory effect of Rock2 on TGFbeta signaling requires its kinase activity. In zebrafish embryos, injection of rock2a mRNA attenuates the expression of mesodermal markers during late blastulation and blocks the induction of mesoderm by ectopic Nodal signals. By contrast, overexpression of a dominant negative form of zebrafish rock2a has an opposite effect on mesoderm induction, suggesting that Rock2 proteins are endogenous inhibitors for mesoderm induction. Thus, these data have unraveled previously unidentified functions of Rock2, in controlling TGFbeta signaling as well as in regulating embryonic patterning (Zhang, 2009).

Rho kinase and growth cone dynamics

The contributions of Rho and its downstream target p160ROCK during the early stages of axon formation were tested in cultured cerebellar granule neurons. p160ROCK inhibition (by use of a drug targeting ROCK or by use of dominant negative ROCK), triggers immediate outgrowth of membrane ruffles and filopodia (presumably by reducing the stability of the cortical actin network), followed by the generation of initial growth cone-like membrane domains from which axonal processes arise. Furthermore, a potentiation in both the size and the motility of growth cones is evident, though the overall axon elongation rate remains stable. Conversely, it is suggested that overexpression of dominant active forms of Rho or ROCK prevents initiation of axon outgrowth. Taken together, these data indicate a novel role for the Rho/ROCK pathway as a gate critical for the initiation of axon outgrowth and the control of growth cone dynamics (Bito, 2000).

Transfection experiments using wild-type and a kinase-dead mutant LIM kinase-1 (LIMK-1) suggest an involvement of LIMK-1 as a downstream target of ROCK in the regulation of axon formation. It is speculated that during the initial stages of axon outgrowth, the activation of the Rho/ROCK/LIMK-dependent phosphorylation cascade strongly contributes to maintaining an inactivation state of cofilin and preventing it from being activated by dephosphorylation. When this phosphorylation is counteracted, such as by use of ROCK inhibitor Y-27632 in vitro, or by Rho inhibition in vivo, local accumulation of activated cofilin may indeed trigger a rapid morphological change and generation of neuronal polarity in a Rho-regulated manner (Bito, 2000).

Collapsin response mediator protein-2 (CRMP-2) is a novel Rho-kinase substrate in the brain. CRMP-2 is a neuronal protein whose expression is up-regulated during development. Rho-kinase phosphorylates CRMP-2 at Thr-555 in vitro. An antibody that specifically recognizes CRMP-2 phosphorylated at Thr-555 has been produced. Using this antibody, it was found that Rho-kinase phosphorylates CRMP-2 downstream of Rho in COS7 cells. Phosphorylation of CRMP-2 is observed in chick dorsal root ganglion neurons during lysophosphatidic acid (LPA)-induced growth cone collapse, whereas the phosphorylation is not detected during semaphorin-3A-induced growth cone collapse. Both LPA-induced CRMP-2 phosphorylation and LPA-induced growth cone collapse are inhibited by Rho-kinase inhibitor HA1077 or Y-32885. LPA-induced growth cone collapse is also blocked by a dominant negative form of Rho-kinase. In contrast, semaphorin-3A-induced growth cone collapse is not inhibited by a dominant negative form of Rho-kinase. Furthermore, overexpression of a mutant CRMP-2, in which Thr-555 is replaced by Ala, significantly inhibited LPA-induced growth cone collapse. These results demonstrate the existence of Rho-kinase-dependent and -independent pathways for growth cone collapse and suggest that CRMP-2 phosphorylation by Rho-kinase is involved in the former pathway (Arimura, 2000).

Rho family GTPases have been implicated in neuronal growth cone guidance; however, the underlying cytoskeletal mechanisms are unclear. Multimode fluorescent speckle microscopy (FSM) was used to directly address this problem. Actin arcs that form in the transition zone are incorporated into central actin bundles in the C domain. These actin structures are Rho/Rho Kinase (ROCK) effectors. Specifically, Lysophosphatidic acid (LPA) mediates growth cone retraction by ROCK-dependent increases in actin arc and central actin bundle contractility and stability. In addition, these treatments had marked effects on MT organization as a consequence of strong MT-actin arc interactions. In contrast, LPA or constitutively active Rho had no effect on P domain retrograde actin flow or filopodium bundle number. This study reveals a novel mechanism for domain-specific spatial control of actin-based motility in the growth cone with implications for understanding chemorepellant growth cone responses and nerve regeneration (Zhang, 2003).

Two actin-based structures, filopodia and actin arcs, had profound effects on MT organization and dynamics in the growth cone. Polarized actin bundles in filopodia guide MT growth into the peripheral (P) domain and simultaneously transport MTs rearward by retrograde actin flow. The second structure, actin arcs, appear in the T zone and move into the C domain, where they contribute to central actin bundle structure. Interestingly, MTs associated with actin arcs are less dynamic than those in the P domain, exhibiting prolonged periods of slow growth due to dramatically reduced catastrophe frequencies. Arcs have been identified as a novel motile actin structure in growth cones (Zhang, 2003).

FSM was used to investigate effects of altering Rho GTPase activity on cytoskeletal dynamics in growth cones. Actin arcs and central actin bundles derived from arcs are key cytoskeletal effectors of Rho and ROCK. Rho activity affects both the stability and contractility of actin arcs and strongly affects MT behavior as a consequence of arc-MT interactions. When cells were plated on laminin substrates, growth cone retractions were observed in response to Rho activation by LPA. Interestingly, these well-described chemorepellant responses do not involve changes in peripheral retrograde actin flow or filopodium number, but rather are driven by the contraction of more central actin structures (Zhang, 2003).

p21(Cip1/WAF1) has cell cycle inhibitory activity by binding to and inhibiting both cyclin/Cdk kinases and proliferating cell nuclear antigen. p21(Cip1/WAF1) is induced in the cytoplasm during the course of differentiation of chick retinal precursor cells and N1E-115 cells. Ectopic expression of p21(Cip1/WAF1) lacking the nuclear localization signal in N1E-115 cells and NIH3T3 cells affects the formation of actin structures, characteristic of inactivation of Rho. p21(Cip1/WAF1) forms a complex with Rho-kinase and inhibits its activity in vitro and in vivo. Neurite outgrowth and branching from the hippocampal neurons are promoted if p21(Cip1/WAF1) is expressed abundantly in the cytoplasm. These results suggest that cytoplasmic p21(Cip1/WAF1) may contribute to the developmental process of the newborn neurons that extend axons and dendrites into target regions (Tanaka, 2002).

During embryonic development, tangentially migrating precerebellar neurons emit a leading process and then translocate their nuclei inside it (nucleokinesis). Netrin 1 (also known as netrin-1) acts as a chemoattractant factor for neurophilic migration of precerebellar neurons (PCN) both in vivo and in vitro. In the present work, Rho GTPases that could direct axon outgrowth and/or nuclear migration were analyzed. The expression pattern of Rho GTPases in developing PCN is consistent with their involvement in the migration of PCN from the rhombic lips. Pharmacological inhibition of Rho enhances axon outgrowth of PCN and prevents nuclei migration toward a netrin 1 source, whereas inhibition of Rac and Cdc42 sub-families impairs neurite outgrowth of PCN without affecting migration. Through pharmacological inhibition, it has been shown that Rho signaling directs neurophilic migration through Rock activation. Altogether, these results indicate that Rho/Rock acts on signaling pathways favoring nuclear translocation during tangential migration of PCN. Thus, axon extension and nuclear migration of PCN in response to netrin 1 are not strictly dependent processes because: (1) distinct small GTPases are involved; (2) axon extension can occur when migration is blocked, and (3) migration can occur when axon outgrowth is impaired (Causeret, 2004).

RhoE is a pro-survival p53 target gene that inhibits ROCK I-mediated apoptosis in response to genotoxic stress

The Rho family of GTPases regulates many aspects of cellular behavior through alterations to the actin cytoskeleton. The majority of the Rho family proteins function as molecular switches cycling between the active, GTP-bound and the inactive, GDP-bound conformations. Unlike typical Rho-family proteins, the Rnd subfamily members, including Rnd1, Rnd2, RhoE (also known as Rnd3), and RhoH, are GTPase deficient and are thus expected to be constitutively active. An unexpected role has been identified for RhoE/Rnd3 in the regulation of the p53-mediated stress response. This study demonstrates that RhoE is a transcriptional p53 target gene and that genotoxic stress triggers actin depolymerization, resulting in actin-stress-fiber disassembly through p53-dependent RhoE induction. Silencing of RhoE induction in response to genotoxic stress maintains stress fiber formation and strikingly increases apoptosis, implying an antagonistic role for RhoE in p53-dependent apoptosis. It was found that RhoE inhibits ROCK I (Rho-associated kinase I) activity during genotoxic stress and thereby suppresses apoptosis. The p53-mediated induction of RhoE in response to DNA damage favors cell survival partly through inhibition of ROCK I-mediated apoptosis. Thus, RhoE is thought to function by regulating ROCK I signaling to control the balance between cell survival and cell death in response to genotoxic stress (Ongusaha, 2006).

Rho-kinase: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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