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Rho associated kinases: General

Caenorhabditis elegans embryonic elongation is driven by cell shape changes that cause a contraction of the epidermal cell layer enclosing the embryo. This process requires a Rho-associated kinase (LET-502) and is opposed by the activity of a myosin phosphatase regulatory subunit (MEL-11). Myosin phosphatase affects the phosphorylation state of myosin regulatory light chain and thereby regulates the level of actin-activated myosin Mg-ATPase activity. Myosin phosphatase holoenzyme consists of three subunits: a type 1 catalytic subunit that belongs to the PP1c subfamily of Ser/Thr phosphatases, a combined regulatory and targeting subunit, and a smaller subunit of unknown function. The holoenzyme dephosphorylates Ser19 of myosin regulatory light chain thus counteracting phosphorylation of this residue by myosin light chain kinase. Myosin phosphatase activity itself is regulated by phosphorylation of the regulatory subunit, which decreases activity of the holoenzyme. This inhibition is linked to GTP levels and thus suggests a role for the small GTPases, especially Rho. The recently identified Rho-binding kinase is activated by RhoGTP and subsequently inactivates the myosin phosphatase holoenzyme by phosphorylation of the regulatory subunit. mel-11 activity is required both in the epidermis during embryonic elongation and in the spermatheca of the adult somatic gonad. let-502 and mel-11 reporter gene constructs show reciprocal expression patterns in the embryonic epidermis and the spermatheca, and mutations of the two genes have opposite effects in these two tissues. These results are consistent with let-502 and mel-11 mediating tissue contraction and relaxation, respectively. mel-11 embryonic inviability is genetically enhanced by mutations in a Rac signaling pathway, suggesting that Rac potentiates or acts in parallel with the activity of the myosin phosphatase complex. Since Rho has been implicated in promoting cellular contraction, these results support a mechanism by which epithelial morphogenesis is regulated by the counteracting activities of Rho and Rac (Wissmann, 1999).

Rho is implicated in physiological functions associated with actin-myosin filaments such as cytokinesis, cell motility, and smooth muscle contraction. Rho-associated serine/threonine kinase (see Drosophila Rho-associated kinase), which is activated by GTP Rho, stoichiometrically phosphorylates myosin light chain (MLC). 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 contained 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, it can then interact with and activate Rho-kinase. 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).

Platelets respond to various stimuli with rapid changes in shape followed by aggregation and secretion of their granule contents. Platelets lacking the alpha-subunit of the heterotrimeric G protein Gq do not aggregate and degranulate but still undergo shape change after activation through thromboxane-A2 (TXA2) or thrombin receptors. In contrast to thrombin, the TXA2 mimetic U46619 leads to the selective activation of G12 and G13 in Galphaq-deficient platelets, indicating that these G proteins mediate TXA2 receptor-induced shape change. TXA2 receptor-mediated activation of G12/G13 results in tyrosine phosphorylation of pp72(syk) and stimulation of pp60(c-src) as well as in phosphorylation of myosin light chain (MLC) in Galphaq-deficient platelets. Both MLC phosphorylation and shape change induced through G12/G13 in the absence of Galphaq are inhibited by the C3 exoenzyme from Clostridium botulinum, by the Rho-kinase inhibitor Y-27632 and by a cAMP-analog. These data indicate that G12/G13 couple receptors to tyrosine kinases as well as to the Rho/Rho-kinase-mediated regulation of MLC phosphorylation. Evidence is provided that G12/G13-mediated Rho/Rho-kinase-dependent regulation of MLC phosphorylation participates in receptor-induced platelet shape change (Klages, 1999).

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 (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. The expression of constitutively active Rho-kinase increases the level of MLC phosphorylation. The activity of Rho-kinase is necessary for maintaining the vinculin-containing focal adhesions. The microinjection into fibroblasts of constitutively active Rho-kinase to some extent induces the formation of focal adhesions, under conditions where organized actin stress fibers are disrupted. The expression of constitutively active Rho-kinase also stimulates 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 (Chihara, 1997).

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).

Rho plays pivotal roles in the Ca2+ sensitization of smooth muscle. However, the GTP-bound active form of Rho fails to exert Ca2+-sensitizing effects in Triton X-100-permeabilized smooth muscle preparations, due to the loss of the important diffusible cofactor. The contractile effects of Rho-associated kinase (Rho-kinase), recently identified as a putative target of Rho, are demonstrated on the Triton X-100-permeabilized smooth muscle of rabbit portal vein. Introduction of the constitutively active form of Rho-kinase into the cytosol of Triton X-100-permeabilized smooth muscle provokes a contraction and a proportional increase in levels of monophosphorylation of myosin light chain in both the presence and the absence of cytosolic Ca2+. These effects of constitutively active Rho-kinase are insensitive to wortmannin (a potent myosin light chain kinase inhibitor). Immunoblot analysis reveals that the amount of native Rho-kinase is markedly lower in Triton X-100-permeabilized tissue than in intact tissue. These results demonstrate that Rho-kinase directly modulates smooth muscle contraction through myosin light chain phosphorylation, independent of the Ca2+-calmodulin-dependent myosin light chain kinase pathway (Kureishi, 1997).

The ezrin/radixin/moesin (ERM) proteins are involved in actin filament/plasma membrane interaction that is regulated by Rho. ERM proteins are directly phosphorylated by Rho-associated kinase (Rho-kinase), a direct target of Rho. Recombinant full-length and COOH-terminal half radixin were incubated with the constitutively active catalytic domain of Rho-kinase: ~30 and ~100% of these molecules, respectively, are phosphorylated mainly at the COOH-terminal threonine (T564). To detect Rho-kinase-dependent phosphorylation of ERM proteins in vivo, a mAb was raised that recognizes the T564-phosphorylated radixin as well as ezrin and moesin, phosphorylated at the corresponding threonine residue (T567 and T558, respectively). Immunoblotting of serum-starved Swiss 3T3 cells with this mAb reveals that after LPA stimulation, ERM proteins are rapidly phosphorylated at T567 (ezrin), T564 (radixin), and T558 (moesin) in a Rho-dependent manner and then dephosphorylated within 2 min. The T564 phosphorylation of recombinant COOH-terminal half radixin does not affect its ability to bind to actin filaments in vitro but significantly suppresses its direct interaction with the NH2-terminal half of radixin. These observations indicate that the Rho-kinase-dependent phosphorylation interferes with the intramolecular and/ or intermolecular head-to-tail association of ERM proteins. Such associations are important mechanisms for the regulation of the activity of ERM proteins in their role as actin filament/plasma membrane cross-linkers (Matsui, 1998).

The regulation of morphological changes in eukaryotic cells is a complex process involving major components of the cytoskeleton including actin microfilaments, microtubules, and intermediate filaments (IFs). The putative effector of RhoA, RhoA-binding kinase alpha (ROKalpha), is a serine/threonine kinase that has been implicated in the reorganization of actin filaments and in myosin contractility. ROKalpha also directly affects the structural integrity of IFs. Overexpression of active ROKalpha, like that of RhoA, causes the collapse of filamentous vimentin, a type III IF. A RhoA-binding-deficient, kinase-inactive ROKalpha inhibits the collapse of vimentin IFs induced by RhoA in HeLa cells. In vitro, ROKalpha binds and phosphorylates vimentin at its head-rod domain, thereby inhibiting the assembly of vimentin. ROKalpha colocalizes predominantly with the filamentous vimentin network, which remains intact in serum-starved cells. Treatment of cells with vinblastine, a microtubule-disrupting agent, also results in filamentous vimentin collapse and concomitant ROKalpha translocation to the cell periphery. ROKalpha translocation does not occur when the vimentin network remains intact in vinblastine-treated cells at 4 degrees C or in the presence of the dominant-negative RhoAN19 mutant. Transient translocation of ROKalpha is also observed in cells subjected to heat shock, which causes the disassembly of the vimentin network. Thus, the translocation of ROKalpha to the cell periphery upon overexpression of RhoAV14 or growth factor treatment is associated with disassembly of vimentin IFs. These results indicate that Rho effectors known to act on microfilaments may be involved in regulating the assembly of IFs. Vimentin when phosphorylated also exhibits reduced affinity for the inactive ROKalpha. The translocation of ROKalpha from IFs to the cell periphery upon action by activated RhoA and ROKalpha suggests that ROKalpha may initiate its own cascade of activation (Sin, 1998).

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 a kinase domain; 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).

Rho cycles between the active guanosine triphosphate (GTP)-bound form and the inactive guanosine diphosphate-bound form. It regulates cell adhesion and cytokinesis, but how it exerts these actions is unknown. The yeast two-hybrid system was used to clone a complementary DNA for a protein (designated Rhophilin) that specifically binds to GTP-Rho. The Rho-binding domain of this protein has 40 percent identity with a putative regulatory domain of a protein kinase, PKN. PKN itself binds to GTP-Rho and is activated by this binding both in vitro and in vivo. This study indicates that a serine-threonine protein kinase is a Rho effector and presents an amino acid sequence motif for binding to GTP-Rho that may be shared by a family of Rho target proteins (Watanabe, 1996).

The NCK adapter protein (Drosophila homolog: Dreadlocks) is comprised of three consecutive Src homology 3 (SH3) protein-protein interaction domains and a C-terminal SH2 domain. Although the association of NCK with activated receptor protein-tyrosine kinases, via its SH2 domain, implicates NCK as a mediator of growth factor-induced signal transduction, little is known about the pathway(s) downstream of NCK recruitment. To identify potential downstream effectors of NCK, a bacterial expression library was screened to isolate proteins that bind its SH3 domains. Two molecules were isolated: the Wiskott-Aldrich syndrome protein (WASP, a putative CDC42 effector) and a serine/threonine protein kinase (PRK2, closely related to the putative Rho effector PKN). Using interspecific backcross analysis the Prk2 gene was mapped to mouse chromosome 3. Unlike WASP, which binds the SH3 domains of several signaling proteins, PRK2 specifically binds to the middle SH3 domain of NCK and (weakly) to the SH3 domain of phospholipase Cgamma. PRK2 also specifically binds to Rho in a GTP-dependent manner and cooperates with Rho family proteins to induce transcriptional activation via the serum response factor. These data suggest that PRK2 may coordinately mediate signal transduction from both the activated receptor protein-tyrosine kinases and Rho and that NCK may function as an adapter to connect receptor-mediated events to Rho protein signaling (Quilliam, 1996).

Dramatic transient changes resulting in a stellate morphology are induced in many cell types on treatment with agents that enhance intracellular cAMP levels. Thrombin fully protects cells from this inductive effect of cAMP through the thrombin receptor. The protective effect of thrombin is shown to be Rho-dependent. Clostridium botulinum C3 exoenzyme, which inactivates RhoA functions, abolishes the ability of thrombin to protect cells from responding to increased cAMP levels. A constitutively activated RhoAV14 mutant protein also prevents cells from responding to cAMP. RhoA can be specifically phosphorylated at Ser-188 by the cAMP-activated protein kinase A (PKA). RhoAV14A188, which cannot be phosphorylated by PKA in vitro, is more effective than RhoAV14 in preventing cells from responding to cAMP and in inducing actin stress fiber formation. This suggests that PKA phosphorylation of RhoA impairs its biological activity in vivo. ROKalpha, a RhoA-associated serine/threonine kinase can also prevent cells from responding to cAMP with shape changes. Phosphorylation of RhoA by PKA in vitro decreases the binding of RhoA to ROKalpha. These results indicate that RhoA and cAMP have antagonistic roles in regulating cellular morphology and suggest that cAMP-mediated down-regulation of RhoA binding to its effector ROKalpha may be involved in this antagonism (Dong, 1998).

Rho-kinase, which is activated by the small GTPase Rho, 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, Ser-854. Antibody has been prepared that specifically recognized MBS phosphorylated at Ser-854. 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 inhibits 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 localize 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).

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 small GTPase RhoA controls activity of serum response factor (SRF) by inducing changes in actin dynamics. In PC12 cells, activation of SRF after serum stimulation is RhoA dependent, requiring both actin polymerization and the Rho kinase (ROCK)-LIM kinase (LIMK)-cofilin signaling pathway, previously shown to control F-actin turnover. Activation of SRF by overexpression of wild-type LIMK or ROCK-insensitive LIMK mutants also requires functional RhoA, indicating that a second RhoA-dependent signal is involved. This is provided by the RhoA effector mDia: dominant interfering mDia1 derivatives inhibit both serum- and LIMK-induced SRF activation and reduce the ability of LIMK to induce F-actin accumulation. These results demonstrate a role for LIMK in SRF activation, and functional cooperation between RhoA-controlled LIMK and mDia effector pathways (Geneste, 2002).

Rho associated kinases: Phosphatidylinositol 4-phosphate 5-kinase

Post-translationally modified Rho in its GTP-bound state stimulates phosphatidylinositol 4-phosphate 5-kinase (PIP5K) activity in mouse fibroblast lysates. To investigate whether Rho physically interacts with PIP5K (Drosophila homolog: skittles), immobilized Rho-GST was incubated with Swiss 3T3 cell lysates and tested for retained PIP5K activity. Rho-GST, but not Ras-GST or GST alone, binds significant PIP5K activity. The binding of PIP5K is independent of whether Rho is in a GTP- or GDP-bound state. An antibody against a 68-kDa human erythrocyte type I PIP5K recognizes a single 68-kDa protein eluted from Rho-GST column. The Rho-associated PIP5K responds to phosphatidic acid differentially from the erythrocyte type I PIP5K, suggesting that it could be a distinct isoform not reported previously. Rho co-immunoprecipitates with the 68-kDa PIP5K from Swiss 3T3 lysates, demonstrating that endogenous Rho also interacts with PIP5K. ADP-ribosylation of Rho with C3 exoenzyme enhances PIP5K binding by approximately eightfold, consistent with the ADP-ribosylated Rho functioning as a dominant negative inhibitor. These results demonstrate that Rho physically interacts with a 68-kDa PIP5K, although whether the association is direct or indirect is unknown (Ren, 1996).

Integrin-mediated adhesion is known to stimulate production of phosphatidylinositol 4,5-bisphosphate (4,5-PIP2) and increase 4,5-PIP2 hydrolysis in response to platelet-derived growth factor (PDGF). Treatment of cells with lovastatin, which inhibits modification of small GTP-binding proteins, reduced PIP2 levels and decreases calcium mobilization in response to PDGF and thrombin. In cell lysates, GTP gamma S stimulates PIP 5-kinase activity, and this effect is blocked by botulinum C3 exoenzyme, suggesting that Rho is responsible. GTP-bound recombinant Rho stimulates PIP 5-kinase activity, whereas GDP-Rho is much less potent and GTP-bound Rac is ineffective. Microinjected botulinum C3 exoenzyme causes diminished calcium mobilization in response to PDGF or thrombin. Conversely, microinjection of activated Rho reverses the decrease in calcium mobilization normally seen in nonadherent cells. These data demonstrate that Rho regulates 4,5-PIP2 synthesis and, indirectly, 4,5-PIP2 hydrolysis. They also raise the possibility that PIP2 synthesis mediates the effects of Rho on the actin cytoskeleton (Chong, 1994).

The Rho family GTP-binding proteins have been known to mediate extracellular signals to the actin cytoskeleton. Although several Rho interacting proteins have been found, downstream signals have yet to be determined. Many actin-binding proteins are known to be regulated by phosphatidylinositol 4,5-bisphosphate in vitro. Rho has been shown to enhance the activity of phosphatidylinositol-4-phosphate 5-kinase (PI4P5K), the phosphatidylinositol 4,5-bisphosphate synthesizing enzyme. Several isoforms of type I PI4P5K have been isolated. PI4P5K Ialpha induces massive actin polymerization resembling 'pine needles' in COS-7 cells in vivo. When truncated from the C terminus to amino acid 308 of PI4P5K Ialpha, both kinase activity and actin polymerizing activity are lost. Although the dominant negative form of Rho, RhoN19, alone decreases actin fibers, those induced by PI4P5K are not affected by the coexpression of RhoN19. These results suggest that PI4P5K is located downstream from Rho and mediates signals for actin polymerization through its phosphatidylinositol-4-phosphate 5-kinase activity (Shibasaki, 1997).

Table of contents

Rho1: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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