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Mol Biol Cell. Jan 2002; 13(1): 336–347.
PMCID: PMC65092

Post-transcriptional Down-regulation of ROCKI/Rho-kinase through an MEK-dependent Pathway Leads to Cytoskeleton Disruption in Ras-transformed Fibroblasts

Jennifer Lippincott-Schwartz, Monitoring Editor

Abstract

Transformation by oncogenic Ras profoundly alters actin cytoskeleton organization. We investigated Ras-dependent signaling pathways involved in cytoskeleton disruption by transfecting normal rat kidney (NRK) cells with different Ras mutants. RasV12S35, a mutant known to activate specifically the Raf/MAPK pathway, led to stress fiber and focal contact disruption, whereas the adherens junctions remained intact. Next, we found that pharmacological inhibition of MEK was sufficient to restore the cytoskeletal defects of ras-transformed NRK cells, including assembly of stress fibers and focal contacts, but it did not induce reorganization of the cell-cell junctions. Investigating the mechanism underlying this phenotypic reversion, we found that the sustained MAPK signaling resulting from Ras-transformation down-regulated the expression of ROCKI and Rho-kinase, two-Rho effectors required for stress fiber formation, at the post-transcriptional level. On MEK inhibition, ROCKI/Rho-kinase expression and cofilin phosphorylation were increased, demonstrating that the Rho-kinase/LIM-kinase/cofilin pathway was functionally restored. Finally, using dominant negative or constitutively active mutants, we demonstrated that expression of ROCKI/Rho-kinase was both necessary and sufficient to promote cytoskeleton reorganization in NRK/ras cells. These findings further establish the Ras/MAPK pathway as the critical pathway involved in cytoskeleton disruption during Ras-transformation, and they suggest a new mechanism, involving alteration in ROCKI/Rho-kinase expression, by which oncogenic Ras can specifically target the actin-based cytoskeleton and achieve morphological transformation of the cells.

INTRODUCTION

Oncogenic transformation is characterized not only by deregulated growth control but also by pronounced morphological changes resulting from alterations in the organization of the actin cytoskeleton and adhesive interactions. Changes in the organization of actin filaments are highly correlated with anchorage-independent growth and tumorigenicity, suggesting a fundamental role for actin fibers in cell growth control (reviewed in Pawlak and Helfman, 2001 blue right-pointing triangle). Alterations in actin filament structure are associated with decreased expression of numerous cytoskeletal proteins (Button et al., 1995 blue right-pointing triangle). Forced re-expression of these proteins by transfection, including α-actinin (Gluck et al., 1993 blue right-pointing triangle; Nikolopoulos et al., 2000 blue right-pointing triangle), profilin (Janke et al., 2000 blue right-pointing triangle), vinculin (Rodriguez-Fernandez et al., 1992 blue right-pointing triangle), and tropomyosin (Prasad et al., 1993 blue right-pointing triangle, 1999 blue right-pointing triangle; Takenaga and Masuda, 1994 blue right-pointing triangle; Gimona et al., 1996 blue right-pointing triangle; Braverman et al., 1996 blue right-pointing triangle; Janssen and Mier, 1997 blue right-pointing triangle) can reduce or abrogate the transformed phenotype. Although the mechanisms by which these proteins contribute to growth control remain to be fully elucidated, these studies demonstrate that changes in the expression of specific structural components of the actin cytoskeleton can contribute to transformation.

Small GTPases of the Ras superfamily control a wide spectrum of cellular processes by switching between inactive GDP- and active GTP-bound states. When bound to GTP, these proteins regulate cell behavior by binding to effector molecules and by altering their localization, protein-protein interactions, and activity. Small GTPase proteins can be divided into subfamilies as follows: members of the Ras subfamily regulate cell proliferation and differentiation, whereas members of the Rho subfamily were first identified as regulators of the actin cytoskeleton but also affect gene expression and proliferation (Bar-Sagi and Hall, 2000 blue right-pointing triangle). The importance of the Ras subfamily of genes in the control of cell proliferation is demonstrated by the high frequency of mutations, among the highest of any gene in human cancers, that activate Ras in tumors (Hunter, 1997 blue right-pointing triangle). Ras is known to activate multiple effectors, including Raf, which in turn activates the mitogen-activated protein kinase kinase (MEK) and mitogen-activated protein kinase (MAPK) cascade, the phosphatidylinositol 3-kinase (PI 3-K), and the RalGDS family of guanine nucleotide exchange factors for Ral GTPases (Katz and McCormick, 1997 blue right-pointing triangle). A major task is to discern which effector pathway contributes to which aspect of the transformed phenotype (Shields et al., 2000 blue right-pointing triangle).

In addition to structural components such as tropomyosins (TM), organization of actin filaments is under the control of the Rho subfamily of small GTPases: Rho, Rac, and Cdc42 (Bishop and Hall, 2000 blue right-pointing triangle). Among these, Rho regulates the assembly of actin stress fibers and focal contacts through activation of the downstream effectors mDia and the closely related kinases ROCKI/Rho-kinase (Amano et al., 1997 blue right-pointing triangle; Watanabe et al., 1999 blue right-pointing triangle). Activation of Rho-kinase by Rho is implicated in stress fiber and in focal contact formation (Leung et al., 1996 blue right-pointing triangle; Amano et al., 1997 blue right-pointing triangle). Rho-kinase increases the phosphorylation of myosin light chains and thus increases acto-myosin–based contractility, by directly phosphorylating myosin light chains and by negatively regulating myosin phosphatase (Kureishi et al., 1997 blue right-pointing triangle; reviewed in Fukata et al., 2001 blue right-pointing triangle). The resulting contractile forces are thought to contribute to the formation of stress fibers and focal contacts (Burridge et al. 1997 blue right-pointing triangle, Helfman et al., 1999 blue right-pointing triangle). Rho-kinase also activates LIM-kinase, which subsequently phosphorylates cofilin and thereby inhibits its actin-depolymerizing activity, thus contributing to actin fiber stabilization (Yang et al., 1998 blue right-pointing triangle; Maekawa et al., 1999 blue right-pointing triangle; Bamburg et al., 1999 blue right-pointing triangle; Ohashi et al., 2000 blue right-pointing triangle).

Rho-dependent signaling is required for transformation by oncogenic Ras (Khosravi-Far et al., 1995 blue right-pointing triangle; Qiu et al., 1995 blue right-pointing triangle; Zhong et al., 1997 blue right-pointing triangle). Although Rho activation is known to promote the development of stress fibers and focal contacts, Ras-transformed fibroblasts generally exhibit a loss of these structural elements characteristic of Rho activity. These results have led to the interpretation that Rho activity may be reduced in Ras-transformed cells. Accordingly, activated Rho can restore stress fibers in Ras-transformed Rat1 fibroblasts, suggesting that Rho can be inactivated in these Ras-transformed cells (Izawa et al., 1998 blue right-pointing triangle). By contrast, inhibition of Rho-kinase was shown to block Ras transformation in NIH3T3 cells (Sahai et al., 1999 blue right-pointing triangle) and to attenuate invasiveness of tumor cells (Itoh et al., 1999 blue right-pointing triangle). Thus, the relationship between Ras- and Rho-dependent signaling pathways during transformation is still unclear.

Application of MEK inhibitors was reported to cause morphological reversion of Ras-transformed fibroblasts (Fukazawa and Uehara, 2000 blue right-pointing triangle; Reuveni et al., 2000 blue right-pointing triangle). The signaling events associated with this phenotypic reversion, however, have not been fully investigated. In the present study, we have investigated the signaling pathways activated by Ras, as well as the mechanism by which oncogenic Ras can specifically target the actin-based cytoskeleton and thereby achieve morphological transformation of the cells.

MATERIALS AND METHODS

Antibodies and Reagents

The mouse monoclonal anti-vinculin, anti-β-actin, anti–c-myc (clone 9E10), and anti-tropomyosin (TM) (clone TM311) antibodies (mAbs), as well as the rabbit polyclonal anti-β catenin antibody, were purchased from Sigma Chemical (St. Louis, MO). The mouse anti-Rho-kinase and anti-ROCKI mAbs were obtained from Transduction Laboratories (Lexington, KY). The mouse antiphosphorylated MAPK (Tyr204) mAb and the rabbit polyclonal anti-MAPK were from Santa Cruz Biotechnology (Santa Cruz, CA). The rabbit polyclonal anticofilin was from Cytoskeleton (Denver, CO). The rabbit serum recognizing cofilin when phosphorylated by LIM-kinase was kindly provided by J. R. Bamburg (Colorado State University, Fort Collins, CO). The rabbit polyclonal antiphospho-Akt (Ser473) was from Cell Signaling Technology (Beverly, MA). The mAb 12CA5 to the HA tag was produced and purified by the Antibody Facility of Cold Spring Harbor Laboratory (Cold Spring Harbor, NY). Secondary antibodies Cy3-conjugated goat anti-mouse and goat anti-rabbit IgG were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Oregon green-conjugated phalloidin was from Molecular Probes (Eugene, OR). PD098059, U0126, and LY294002 were from Calbiochem (La Jolla, CA), and Yoshitomi Pharmaceutical Industries (Osaka, Japan) kindly provided Y27632. All chemicals and reagents were obtained from Sigma, unless otherwise indicated, and all tissue culture reagents were from Life Technologies (Gaithersburg, MD).

Cell Culture and Drug Treatments

Normal and v-Ki-ras–transformed normal rat kidney (NRK) cells were from ATCC (NRK ATCC CRL 1570 and 1569, respectively). Cells were maintained in DMEM containing 5% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin in a humidified air (5% CO2) atmosphere, at 37°C. Cells from subconfluent dishes were treated with DMSO alone or with various concentrations of PD098059, U0126, LY294002, or Y27632 (all prepared as 50 mM stock in DMSO and stored at −20°C) before being fixed for immunofluorescence or lysed for Western blotting or RNA extraction.

Expression Vectors and Transient and Stable Transfections

Plasmids pDCR-Ha-Ras (G12V, T35S), pDCR-Ha-Ras (G12V, Y40C), and pDCR-Ha-Ras (G12V, E37G), in which HA-tagged Ras proteins were expressed under the control of CMV promoter were a generous gift of M. A. White (University of Texas, Southwestern Medical Center, Dallas, TX). pEF-BOS-myc-Rho-kinase-CAT and pEF-BOS-myc-Rho-kinase-RB/PH(TT) constructs, encoding myc-tagged mutants of Rho-kinase, were kindly provided by K. Kaibuchi (Nara Institute of Science and Technology, Nara, Japan). For stable expression of the Ras effector loop mutants, NRK cells were transfected by the calcium phosphate procedure. Transfected cells were selected in G418-containing medium (500 μg/ml) for 2 weeks, and drug-resistant colonies were pooled and analyzed, as described in RESULTS. For transient transfections, cells were grown on glass coverslips in DMEM containing 5% FBS. After 1 day of culture, cells were transfected with plasmid DNA with Lipofectamine PLUS reagent (Life Technologies) according to the manufacturer's protocol. At 20 h post-transfection, cells were eventually treated with DMSO, PD098059, or U0126 for 30 h or with Y27632 for 1 h. Coverslips were then fixed and stained for immunofluorescence.

Immunofluorescence

Cells grown on glass coverslips were fixed with 3% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 in PBS for 15 min, then blocked for 30 min with 1% BSA at room temperature. Incubations with primary antibodies against vinculin (1:400), TM (1:100), β-catenin (1:500), HA-tag (1:800), or myc-tag (1:500) were conducted at room temperature for 1 h. After washing, cells were incubated with Cy3-conjugated secondary antibodies (1:500) for 45 min. To stain actin, fixed cells were incubated with Oregon green-conjugated phalloidin. Cells were finally stained with 4′6-diamidino-2-phenylindole (DAPI), and coverslips were mounted using Prolong Antifade (Molecular Probes). Samples were examined and pictures acquired on a Zeiss Axiophot microscope equipped with a Photometrics SenSys (Oberkochen, Germany) cooled CCD camera using Image 2.0.5 software (Oncor, Gaithersburg, MD). All photographs were taken at the same magnification.

Western Blot

Control and treated cells were washed with ice-cold PBS containing 1 mM sodium orthovanadate before direct extraction in 2% SDS Laemmli sample buffer. Lysates were clarified by centrifugation (16,000 g, 15 min at 4°C), and protein concentrations were measured by bicinchoninic acid protein assay (Bio-Rad, Hercules, CA). Equal amounts of proteins were resolved by SDS-PAGE and were transferred to nitrocellulose membrane (Schleicher & Schuell, Keene, NH). The membrane was blocked in 5% nonfat dried milk or 2% BSA in Tris-buffered saline plus 0.1% Tween 20 and incubated with primary antibodies for 1 h, followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Immunoreactive bands were detected by chemiluminescence (NEN, Boston, MA), according to the manufacturer's instructions.

Rho-GTP Pull-down Assay

Measurement of GTP-bound Rho was performed using the Rho Activation Assay kit (Upstate Biotechnology), following the manufacter's instructions. Briefly, the RhoA-binding domain of Rhotekin expressed as a GST-fusion protein was used to affinity precipitate GTP-bound Rho from cells lysed in 50 mM Tris, pH 7.2, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 500 mM NaCl, 10 mM MgCl2 and a cocktail of protease inhibitors (Roche). Precipitated Rho-GTP was then detected by immunoblot analysis, using a polyclonal anti-Rho (-A, -B, -C) antibody (Upstate Biotechnology).

Semiquantitative RT-PCR

Total RNAs were extracted with Trizol reagent (Life Technologies). cDNA synthesis and PCR amplification were performed with Superscript One-Step RT-PCR (Life Technologies), using 0.2 μg RNA. Rho-kinase cDNAs were amplified with sense primer (5′-ATGTCGACTGGGGACAGTTTTGAGACT) and antisense primer (5′-CTATAGATTTCTTCTTTGATTTCCCTC) for 15, 20, and 25 cycles, to remain within the exponential phase of amplification. As an internal control, rat GAPDH cDNA was also amplified with sense primer (5′-GTTCCAGTATGATTCTACCCACGG) and antisense primer (5′-ATGAGCCCTTCCACGATGCCAAAG). Ten-microliter aliquots of the PCR reaction were size-separated on a 2% agarose gel, photographed, and analyzed by densitometry using AlphaImager 2200 v5.5 software (Alpha Innotech Corp., San Leandro, CA).

RESULTS

Constitutive Activation of the Ras/MAPK Pathway Is Sufficient to Disrupt the Actin Cytoskeleton in NRK Cells

Whereas untransformed NRK cells have a flat morphology and well-developed stress fibers, NRK/ras cells (rat fibroblasts transformed by v-Ki-ras) are round and are devoid of stress fibers and focal contacts. To assess the relative contribution of the various downstream effectors of Ras, we analyzed different Ras effector loop mutants for their ability to induce cytoskeleton disruption in NRK cells. Untransformed NRK cells were transfected with three different Ras effector loop mutants: RasV12S35, which activates almost exclusively the Raf pathway; RasV12C40, which activates exclusively the PI 3-K pathway; and RasV12G37, which activates RalGDS (White et al., 1995 blue right-pointing triangle). Stable cell lines were established for each construct. Expression and specificity of the transfected constructs were confirmed by Western-blot analysis using antibodies to the HA epitope tag, active phospho-MAPK, which is downstream of Raf, and phospho-Akt, which is downstream of PI 3-K. Figure Figure1A1A shows that RasV12S35 increased only MAPK phosphorylation, whereas RasV12C40 increased only Akt phosphorylation. RasV12G37 had no activatory effect on either kinase, confirming that this mutant does not activate either pathway.

Figure 1
Activation of the Ras/MAPK pathway disrupts the actin cytoskeleton and focal contacts in NRK cells. NRK cells were transfected with cDNAs encoding various mutant forms of Ras or empty vector (pDCR). G418-resistant clones were pooled and grown onto coverslips ...

Among the different cell lines, the NRK/RasV12S35 cells exhibited spindle-shape morphology and grew in multiple layers. The distribution of actin filaments and vinculin was examined by indirect immunofluorescence (Figure (Figure1B).1B). This analysis showed that the actin cytoskeleton and focal contacts were disrupted in NRK/RasV12S35 cells. In contrast, both the NRK/RasV12C40 and NRK/RasV12G37 cell lines retained organized stress fibers and abundant cell-matrix contacts characteristic of control NRK cells (Figure (Figure1B).1B). To further demonstrate that activation of a MEK-dependent pathway was responsible for the observed loss of actin fibers and focal contact, NRK/RasV12S35 cells were treated with 50 μM of the MEK inhibitor PD098059 (Dudley et al., 1995 blue right-pointing triangle). This treatment leads to restoration of stress fibers and focal contacts (Figure (Figure1C).1C). These results demonstrate that activation of the Ras/MAPK pathway is both necessary and sufficient to disrupt actin stress fibers and focal contacts in NRK cells.

Inhibition of MEK Restores Actin Cytoskeleton Organization and Focal Contact Assembly in Ras-transformed NRK Cells

We next tested whether inhibition of the Ras/MAPK pathway could reverse the phenotype of NRK cells transformed by a fully activated oncogenic Ras (NRK/ras cells). We found that NRK/ras cells treated for 24 h with 50 μM PD098059 reacquired a fibroblastic morphology and formed a flat, nonoverlapping monolayer. To examine cytoskeletal changes associated with this morphological reversion, actin was stained with fluorescence-labeled phalloidin. Stress fibers are noticeably absent from NRK/ras cells compared with the well-developed actin bundles observed in untransformed NRK cells (Figure (Figure2a,2a, compared with 2f). On treatment with PD098059, actin organization changed from patches in rounded cells (Figure (Figure2a)2a) to a diffuse network in flat cells (10 h, Figure Figure2b)2b) and then to cortical fibers surrounding cells with a more polarized morphology (24 h, Figure Figure2c).2c). After 48 h of PD098059 treatment, actin filaments were reorganized into stress fibers extending through the entire cellular body (Figure (Figure2d).2d). Bundling of actin eventually increased further at 72 h, thus resembling stress fibers of untransformed NRK cells (Figure (Figure2e,2e, compared with 2f).

Figure 2
Reorganization of the actin cytoskeleton in NRK/ras cells upon treatment with PD098059. NRK/ras cells were treated with 0.1% DMSO (a) or 50 μM PD098059 for 10 h (b), 24 h (c), 48 h (d), or 72 h (e), then fixed and stained with Oregon green-conjugated ...

Ras-transformed NRK cells also exhibit disrupted focal contacts. Immunofluorescence labeling with anti-vinculin antibodies showed that NRK cells contained well-developed focal contacts, whereas the vinculin staining in NRK/ras cells exhibited a diffuse cytoplasmic pattern (Figure (Figure3).3). A Western-blot analysis showed that the level of expression of vinculin was not affected in NRK/ras cells. Treatment of NRK/ras cells with 50 μM PD098059 for 48 h led to the assembly of vinculin-containing focal contacts (Figure (Figure3).3). These focal contacts were almost indistinguishable from those of control NRK cells. This result suggests that constitutive activation of MEK prevents the assembly of focal contacts and that inhibition of MEK is sufficient to reverse this effect.

Figure 3
Assembly of focal contacts in NRK/ras cells treated with PD098059. NRK and NRK/ras cells are compared with NRK/ras cells treated with 50 μM PD098059 for 48 h. Fixed cells were stained with anti-vinculin mAb, followed by Cy3-conjugated antimouse antibody. ...

To eliminate the possibility that PD098059 may restore actin stress fibers and focal contacts by acting on targets other than MEK1/2, we used the unrelated MEK inhibitor U0126 (Favata et al., 1998 blue right-pointing triangle). Consistent with the effects being MEK-dependent, treatment of NRK/ras cells with 25 μM of U0126 led to effects similar to those of PD098059.

Alterations in actin filament structure are correlated with decreased expression of numerous cytoskeletal proteins (Button et al., 1995 blue right-pointing triangle). It was previously shown that Ras-transformed NRK cells have substantially reduced levels of high-molecular-weight TM isoforms (Matsumura et al., 1983 blue right-pointing triangle). Because these actin-associated proteins are involved in stabilization of the actin cytoskeleton, we asked whether restoration of stress fibers in cells treated with MEK inhibitors was associated with changes in the level of TM expression level. Treatment of NRK/ras cells by either 50 μM PD098059 or 25 μM U0126 for 48 h specifically enhanced TM-1 expression (Figure (Figure4).4). The expression level of TM-2 was not affected. Immunofluorescence staining showed that the TM-1 was incorporated into the newly assembled stress fibers (our unpublished results). Taken together, these results demonstrate that the Ras/MAPK pathway plays a pivotal role in the regulation of actin cytoskeleton organization in Ras-transformed NRK cells.

Figure 4
MEK inhibition causes elevation of TM-1 expression level. Whole cell extracts of NRK and NRK/ras cells maintained in the absence (−) or presence (+) of 50 μM PD098059 or 25 μM U0126 for 48 h were immunoblotted using anti-TM ...

The Ras/MAPK Pathway Is Not Implicated in Cell-Cell Contact Disruption

Transformation of NRK cells by Ras also resulted in the disruption of β-catenin containing cell-cell contacts. Immunofluorescence labeling with anti-β-catenin showed that NRK cells formed strong cell-cell junctions, whereas NRK/ras cells showed only a faint and diffuse staining (Figure (Figure5A).5A). The addition of PD098059 for 48 h did not restore β-catenin distribution in NRK/ras cells (Figure (Figure5A).5A). Further treatments with PD098059 or U0126 at various concentrations, for various times, failed to restore β-catenin–containing cell-cell contacts, suggesting that MEK does not play a role in the distribution of β-catenin in NRK/ras cells. Consistently, NRK/RasV12S35 cells still exhibited β-catenin–containing cell-cell contacts (Figure (Figure5A)5A) despite a strong activation of MEK in these cells, as shown by the constitutive phosphorylation of MAPK (see Figure Figure1A).1A). In addition to monitoring the localization and assembly of β-catenin into cell-cell contacts, we measured its expression level by immunoblotting. Consistent with the immunofluorescence data, Figure Figure5B5B shows a down-regulation of β-catenin expression in NRK/ras cells that was not restored upon MEK inhibition. The expression level of β-catenin in NRK/RasV12S35 cells was shown to be normal compared with that of NRK cells (Figure (Figure5B).5B). These results suggest that the Ras/MAPK pathway specifically targets stress fibers and focal contacts during transformation of NRK cells by Ras but is not implicated or not sufficient to disrupt cell-cell junctions.

Figure 5Figure 5
Activation of the Ras/MAPK pathway is not implicated in β-catenin–containing cell-cell junction disruption. NRK, NRK/ras, and NRK/RasV12S35 cells were treated with DMSO, PD098059, or U0126, then fixed and stained for immunofluorescence ...

ROCKI/Rho-Kinase Signaling Is Compromised in NRK/ras Cells and Is Restored Upon MEK Inhibition by a Posttranscriptional Mechanism

The Rho family of small GTPases, Rho, Rac, and Cdc42, plays a central role in regulating actin organization through downstream effectors, which activities are controlled by interactions with active GTP-bound forms of the Rho family (Bishop and Hall, 2000 blue right-pointing triangle). ROCKI/Rho-kinase are serine/threonine kinases involved in Rho-mediated actin reorganization, i.e., formation of stress fibers and focal contacts (Leung et al., 1996 blue right-pointing triangle; Amano et al., 1997 blue right-pointing triangle). To examine whether the morphological changes induced by MEK inhibitors were accompanied by modifications in Rho activation, the activity of endogenous RhoA was measured in NRK and NRK/ras cells, using a pull-down assay that only capture the active GTP-bound form of the GTPase (Ren et al., 1999 blue right-pointing triangle). There was little difference between Rho-GTP levels in parental versus Ras-transformed cells, as well as between untreated versus MEK inhibitor-treated cells (Figure (Figure6A),6A), demonstrating that neither the loss of stress fibers in Ras-transformed cells nor the restoration of stress fibers induced by MEK inhibitors were associated with changes in Rho-GTP levels.

Figure 6
Restoration of ROCKI/Rho-kinase expression and activity upon MEK inhibitor treatment of NRK/ras cells. (A) NRK and NRK/ras cells were maintained in the absence (−) or presence (+) of 50 μM PD098059 or 25 μM U0126 for 48 ...

Despite normal levels of Rho-GTP, NRK/ras cells are devoid of stress fibers, suggesting that Rho-GTP was no longer coupled to formation of actin stress fibers in these cells. Interestingly, we found that expression of both ROCKI and Rho-kinase was greatly decreased in NRK/ras cells compared with untransformed NRK cells (Figure (Figure6B).6B). On treatment with PD098059 or U0126, Rho-kinase expression returned to levels similar to that seen in NRK cells (Figure (Figure6B).6B). The levels of ROCKI and Rho-kinase were also examined in the Ras-effector mutant expressing cells, especially the NRK/Ras V12S35 cell line. No changes were observed compared with the control parental cells, suggesting that activation of MEK is required but not sufficient to induce down-regulation of ROCKI/Rho-kinase expression. ROCKI and Rho-kinase activates LIM-kinase, which subsequently phosphorylates cofilin and thereby inhibits its actin-depolymerizing activity (Yang et al., 1998 blue right-pointing triangle; Maekawa et al., 1999 blue right-pointing triangle; Bamburg et al., 1999 blue right-pointing triangle; Ohashi et al., 2000 blue right-pointing triangle). Concomitant with the restoration of Rho-kinase expression, we found that cofilin phosphorylation was induced in NRK/ras cells upon treatment with either MEK inhibitor, suggesting that the Rho-kinase/LIM-kinase/cofilin pathway was functionally restored (Figure (Figure6B).6B). Phosphorylation of the myosin light chain, another target of ROCKI/Rho-kinase (Kureishi et al., 1997 blue right-pointing triangle; Fukata et al., 2001 blue right-pointing triangle), was also increased upon ROCKI/Rho-kinase expression up-regulation (our unpublished results).

We next examined whether down-regulation of Rho-kinase in NRK/ras cells was controlled at the transcriptional level. Rho-kinase mRNA levels in NRK and NRK/ras cells were examined by semiquantitative RT-PCR analysis. As shown in Figure Figure6C,6C, steady-state levels of Rho-kinase mRNA were similar in NRK and NRK/ras cells, suggesting that the down-regulation of Rho-kinase expression in NRK/ras cells is controlled at the protein level. Up-regulation of Rho-kinase expression in NRK/ras cells treated with PD098059 also appeared to be regulated at the protein level, because the Rho-kinase mRNA level did not change significantly upon treatment with PD098059 (Figure (Figure66C).

ROCKI/Rho-Kinase Is Both Necessary and Sufficient to Promote Assembly of Stress Fibers in NRK/ras Cells

To determine whether ROCKI/Rho-kinase was involved in the morphological changes induced by inhibition of MEK in NRK/ras cells, we used the specific ROCKI/Rho-kinase inhibitor Y27632 (Kuwahara et al., 1999 blue right-pointing triangle). Treatment of NRK/ras cells with Y27632 had no effect on cell morphology (Figure (Figure7),7), suggesting that the low level of ROCKI/Rho-kinase expressed in NRK/ras cells has no apparent role in maintaining their transformed morphology. Stress fibers were induced in NRK/ras cells by treatment with U0126 for 48 h, then cells were further incubated with 10 μM Y27632 for 1 h. Inhibition of ROCKI/Rho-kinase resulted in disassembly of stress fibers and focal contacts (Figure (Figure7),7), suggesting that formation of these structures was under the control of a ROCKI/Rho-kinase–dependent pathway that was restored upon MEK inhibition.

Figure 7
Assembly of stress fibers in NRK/ras cells requires Rho-kinase activity. NRK/ras cells were treated with 0.1% DMSO or 25 μM U0126 for 48 h. Cells were then further incubated in the absence (−) or presence (+) of 10 μM ...

To further demonstrate the requirement for ROCKI/Rho-kinase activity in the morphological reversion induced by MEK inhibitors, we expressed a dominant-negative form of Rho-kinase, Rho-kinase-RB/PH(TT) (Amano et al., 1997 blue right-pointing triangle). Consistent with the decreased levels of Rho-kinase expression, NRK/ras cells transfected with Rho-kinase-RB/PH(TT) in the absence of MEK inhibitors showed essentially no change in morphology. NRK/ras cells transfected with Rho-kinase-RB/PH(TT) were then treated with PD098059 or U0126. This treatment failed to restore stress fibers or focal contacts in the cells expressing the dominant negative form of ROCKI/Rho-kinase (Figure (Figure8).8). This result demonstrates that elevation of Rho-kinase expression is absolutely required for the restoration of actin fibers in NRK/ras cells treated with MEK inhibitors.

Figure 8
Overexpression of a dominant-negative Rho-kinase prevents assembly of stress fibers in NRK/ras cells treated with MEK inhibitors. NRK/ras cells were transiently transfected with pEF-Bos-myc-Rho-kinase-RB/PH(TT). After 24 h, cells were treated with 25 ...

Finally, we determined whether expression of Rho-kinase was sufficient to reverse the Ras-transformed phenotype. Transfection of NRK cells with a wild-type Rho-kinase produced thick stress fibers but had no effect in NRK/ras cells. Expression of a constitutively active form of Rho-kinase, Rho-kinase-CAT (Amano et al., 1997 blue right-pointing triangle) in NRK/ras cells restored the formation of stress fibers, unlike neighboring cells that do not express the construct (Figure (Figure9).9). The extent of the reversion was only partial, however, because stress fibers were very thick and cells did not spread, suggesting that other pathways are cooperating with Rho-kinase to correctly assemble actin fibers when NRK/ras cells are treated with MEK inhibitors. Taken together, these results demonstrate that Rho-kinase activity is both necessary and sufficient to promote assembly of stress fibers and focal contacts in NRK/ras cells.

Figure 9
Expression of constitutively active Rho-kinase induces stress fibers in NRK/ras cells. NRK/ras cells were transfected with pEF-Bos-myc-Rho-kinase-CAT plasmid (Rho-kinase-CAT). Cells were fixed 24 h after transfection and stained with phalloidin or myc antibodies ...

DISCUSSION

Among the most striking manifestations of the transformed phenotype are the severe disruption of the actin cytoskeleton and the loss of cell-matrix and cell-cell contacts frequently associated with altered patterns of cytoskeletal protein expression. These cellular changes contribute directly to the poor adhesiveness of transformed cells, their enhanced motility, and their ability to grow in an anchorage-independent manner (Hunter, 1997 blue right-pointing triangle; Pawlak and Helfman, 2001 blue right-pointing triangle). It is therefore important to determine the molecular mechanisms responsible for these cellular changes.

Oncogenic Ras has been shown to dramatically affect actin organization, assembly of focal contacts, and the formation of adherens junctions and tight junctions (Izawa et al., 1998 blue right-pointing triangle; Potempa and Ridley, 1998 blue right-pointing triangle; Chen et al., 2000 blue right-pointing triangle; Reuveni et al., 2000 blue right-pointing triangle). Ras is known to activate several signaling pathways and could, in principle, exert its cytoskeletal effects via several distinct pathways. PI 3-K was reported to be a Ras effector (Rodriguez-Viciana et al., 1994 blue right-pointing triangle), mediating signals from Ras to the actin cytoskeleton (Rodriguez-Viciana et al., 1997 blue right-pointing triangle) and to adherens junctions (Potempa and Ridley, 1998 blue right-pointing triangle). In this study, we show that expression of the Ras mutant RasV12C40, which strongly activates the PI 3-K pathway, was not sufficient to disrupt stress fibers and focal contacts in NRK cells. In addition, inhibition of PI 3-K by LY294002 or wortmannin failed to restore actin microfilaments or focal contacts in NRK/ras cells, further suggesting that activation of PI 3-K is not required for Ras-induced disruption of the cytoskeleton in NRK cells.

The present study rather points to the MEK/MAPK pathway as the main route mediating oncogenic Ras effects on stress fibers and focal contacts. Several lines of evidence support this conclusion. First, transfection of the Ras mutant RasV12S35, which selectively and strongly activates the Ras/MAPK pathway, induces disruption of stress fibers and focal contacts in untransformed NRK cells. Second, we showed that application of two structurally unrelated MEK inhibitors, namely PD098059 and U0126, induced complete recovery of stress fibers in NRK/ras cells, with a concomitant assembly of vinculin-containing focal contacts. Taken together, these results strongly support the notion that the Ras/MAPK pathway is a major pathway through which morphological transformation occurs in these cells. Further support comes from a recent report showing that the phenotypic reversion of Ras-transformed Rat1 fibroblasts induced by the Ras farnesylation inhibitor HR12 is mediated by the Ras/MAPK pathway (Reuveni et al., 2000 blue right-pointing triangle).

Formation of adherens junctions is also dramatically affected by oncogenic Ras, thus allowing transformed cells to migrate (Potempa and Ridley, 1998 blue right-pointing triangle; Reuveni et al., 2000 blue right-pointing triangle). Reuveni et al. (2000) blue right-pointing triangle showed that assembly of cell-cell junctions is restored in Rat1/ras cells upon HR12 treatment, most likely through inhibition of MEK. In our system, however, we demonstrated that the Ras/MAPK pathway is not implicated in regulation of this cellular structure. Application of MEK inhibitors did not restore β-catenin–containing cell-cell contacts or β-catenin expression in NRK/ras cells. In addition, untransformed NRK cells transfected with the RasV12S35 mutant retained expression of β-catenin and correct localization at cell-cell junctions. The divergent results between our study and the work of Reuveni and colleagues (2000) blue right-pointing triangle may be due to differences in cell types used in these studies (Rat1 vs. NRK) or type of transformation (Ha-ras vs. Ki-ras). Another possibility is that the ras-farnesylation inhibitor HR12 used by Reuveni and colleagues (2000) blue right-pointing triangle has a yet uncharacterized effect on another pathway implicated in cell-cell junction regulation in addition to its demonstrated effect as a MEK inhibitor. In epithelial MDCK cells, activation of both MAPK and PI 3-K pathways by Ras is required for cell-cell junction disassembly (Potempa and Ridley, 1998 blue right-pointing triangle). We tested this hypothesis in our model by treating NRK/ras cells with PD098059 together with LY294002. This treatment failed to restore cell-cell junctions (our unpublished results), strengthening the fact that epithelial and fibroblastic cells exhibit quite different phenotypes upon transformation by Ras.

Altogether, our results demonstrate that in NRK/Ras cells, organization of the actin-based cytoskeleton is mainly under the control of the Ras/MAPK pathway. Although the mechanism by which MEK or MAPK delivers the signal to the cytoskeleton is not fully understood, our results provide important new insights. We found that activation of MEK in NRK/ras cells induces alterations in ROCKI/Rho-kinase–dependent pathways, which are implicated in actin stress fiber organization without changes in the activation status of Rho. During the preparation of this manuscript, a similar conclusion was reached by Sahai et al. (2001) blue right-pointing triangle in Ras-transformed Swiss-3T3 cells. However, although Sahai et al. reported a modification in the subcellular localization of ROCKI/Rho-kinase that is thought to bring ROCKI/Rho-kinase away from its substrates, we observed that NRK/ras cells have a greatly reduced level of both ROCKI and Rho-kinase. This decreased expression was a direct consequence of sustained MAPK signaling, because inhibition of MEK restored the expression of ROCKI/Rho-kinase. We further demonstrated that ROCKI/Rho-kinase expression is controlled in NRK/ras cells at the protein level (Figure (Figure6).6). Although the most prominent role assigned to MEK/MAPK activity is related to transcriptional regulation, recent reports have suggested a role for this pathway in post-transcriptional events related to cell adhesion. MEK was found to localize to the cell periphery (Kranenburg et al., 1999 blue right-pointing triangle), while MAPK was shown to be targeted to focal contacts upon activation (Fincham et al., 2000 blue right-pointing triangle). The translocation of MEK or MAPK to focal contacts may then serve to direct specificity toward appropriate downstream targets at cellular adhesion (Fincham et al., 2000 blue right-pointing triangle). Changes in the phosphorylation state of cytoskeleton components may provide a regulatory mechanism through which MAPK could affect cytoskeleton organization. To date, however, there are no reports of direct phosphorylation of ROCK/Rho-kinase by MEK/MAPK. Interestingly, induction of calpain activity downstream of MEK/MAPK was recently demonstrated in EGFR signaling and this activation was linked to focal contacts disassembly (Glading et al., 2000 blue right-pointing triangle). Calpains also degrade Focal Adhesion Kinase (FAK) upon transformation by v-Src (Carragher et al., 2001 blue right-pointing triangle). Based on these experiments, one could hypothesized that MEK-induced activation of proteases such as calpains could be implicated in ROCKI/Rho-kinase degradation during Ras transformation.

Importantly, we demonstrated that not only expression of ROCKI/Rho-kinase was restored but also its activity, because we showed that phosphorylation of cofilin by LIM-kinase and phosphorylation of myosin light chain were both induced upon MEK inhibition. Phosphorylation of cofilin by LIMK induces a stabilization of actin filaments through inhibition of its actin-depolymerizing activity (Yang et al., 1998 blue right-pointing triangle; Maekawa et al., 1999 blue right-pointing triangle; Bamburg et al., 1999 blue right-pointing triangle; Ohashi et al., 2000 blue right-pointing triangle), whereas the phosphorylation of myosin light chains increases myosin activity (Amano et al., 1996 blue right-pointing triangle). Together, these events directly contribute to the assembly of stress fibers (Bishop and Hall, 2000 blue right-pointing triangle). Conversely, suppression of these signaling pathways most likely accounts for the loss of stress fibers in Ras-transformed cells. We have also demonstrated that up-regulation of ROCKI/Rho-kinase expression induced by MEK inhibition is critical for the concomitant restoration of the actin cytoskeleton. Both the ROCKI/Rho-kinase inhibitor Y27632, and a dominant negative mutant of Rho-kinase, Rho-kinase-RB/PH(TT) (Amano et al., 1997 blue right-pointing triangle) abolished the effects of the PD098059 treatment, demonstrating that Rho-kinase is indeed required for the process of actin stress fiber assembly and suggesting that no other pathway can compensate for the defect in Rho-kinase expression. Although previous studies have reported a phenotypic reversion of ras-transformed cells upon PD098059 treatment, our study provides a molecular mechanism through which this inhibitor acts.

These experiments prompted us to check whether overexpression of Rho-kinase in NRK/ras cells could be sufficient to reverse their transformed phenotype. Expression of a wild-type Rho-kinase failed to induce stress fibers, possibly because the protein expressed by this exogenous construct is prone to degradation like the endogenous Rho-kinase. A constitutively active mutant of Rho-kinase was able to induce stress fibers in NRK/ras cells, demonstrating that down-regulation of Rho-kinase expression is a critical step for the acquisition of a transformed phenotype during Ras transformation of NRK cells. However, compared with the effect of MEK inhibitors, this mutant fails to completely reverse cells to a normal morphology, suggesting that other proteins cooperate with Rho-kinase during PD098059-induced reversion. Rho-kinase is thought to cooperate with an additional Rho effector, mDia, in the control of stress fiber formation (Nakano et al., 1999 blue right-pointing triangle; Watanabe et al., 1999 blue right-pointing triangle). We do not know at this point, however, whether mDia is affected by Ras-transformation of NRK cells.

Cellular transformation by Ras is associated with a marked decrease in the expression of several actin-binding proteins (Button et al., 1995 blue right-pointing triangle), including high-molecular-weight TMs. Alterations in TM expression seem to be central to the transformed phenotype, because reexpression of these proteins can reverse several aspects of the transformed phenotype, including actin microfilament assembly, contact inhibition, and anchorage-dependent cell growth, but the underlying mechanisms are incompletely understood (reviewed in Pawlak and Helfman, 2001 blue right-pointing triangle). Treatment of c-Jun–transformed FR3T3 rat cells with PD098059 reversed down-regulation of TM-2 (Ljundahl et al., 1998 blue right-pointing triangle). By contrast, the ras-induced down-regulation of TMs in NIH3T3-ras cells was found to be MEK independent, because treatment with PD098059 had little effect on TM levels (Janssen et al., 1998 blue right-pointing triangle). Interestingly, we report here that inhibition of MEK in NRK/ras cells totally alleviated repression of TM-1 expression, suggesting that the decreased TM-1 level observed in NRK/ras cells is a direct result of Ras signaling pathway. TMs are actin-binding proteins know to protect actin stress fibers from the action of severing proteins, such as gelsolin (Ishikawa et al., 1989 blue right-pointing triangle; Pittenger et al., 1994 blue right-pointing triangle). It is thus likely that TMs will cooperate with ROCKI/Rho-kinase to properly assemble and stabilize the actin cytoskeleton during PD098059 treatment of NRK/ras cells. Obviously, the involvement of other proteins that were not investigated in this study is not excluded.

In summary, our results demonstrate that in NRK/Ras cells, organization of the actin cytoskeleton is mainly under the control of the Ras/MAPK pathway and that chronic activation of this pathway during Ras transformation specifically targets formation of stress fibers and focal contacts but is not implicated in cell-cell junction regulation. Our study also provides a mechanism, involving down-regulation of structural components of the cytoskeleton and inhibition of ROCKI/Rho-kinase–dependent pathways, by which oncogenic Ras can achieve morphological transformation of the cells. Although several studies have reported that Rho can cooperate with Ras to transform cells (Khosravi-Far et al., 1995 blue right-pointing triangle; Qiu et al., 1995 blue right-pointing triangle; Zhong et al., 1997 blue right-pointing triangle), down-regulation of ROCKI/Rho-kinase expression may represent a way to uncouple Rho from stress fiber formation without affecting its other biological functions, such as regulation of proliferation and migration (Olson et al., 1998 blue right-pointing triangle; Banyard et al., 2000 blue right-pointing triangle). This would explain why Ras-transformed cells generally exhibit a loss of stress fibers and focal contacts, which are characteristic of Rho activity in normal cells. Whether MAPK signals to the cytoskeleton directly by phosphorylating cytoskeletal components and/or cytoskeleton regulators, such as ROCKI/Rho-kinase, or indirectly by controlling their protein levels through transcription, translation, or degradation is under investigation.

ACKNOWLEDGMENTS

We are grateful to James R. Bamburg for the phospho-specific cofilin antibody and Kozo Kaibuchi for the Rho-kinase expression plasmids. We thank Esteban Araya and Alyssa Carlberg for excellent technical support. The assistance of Eric Julien in many aspects of our work is especially acknowledged. D.M.H. was supported by a grant from the National Cancer Institute (CA-83182).

Abbreviations used:

MAPK
mitogen-activated protein kinase
MEK
mitogen-activated protein kinase kinase
NRK
normal rat kidney
PI 3-K
phosphatidylinositol 3-kinase
TM
tropomyosin

Footnotes

Article published online ahead of print. Mol. Biol. Cell 10.1091/mbc.01–02-0302. Article and publication date are at www.molbiolcell.org/cgi/doi/10.1091/mbc.01–02-0302

REFERENCES

  • Amano M, Ito M, Kimura K, Fukata Y, Chihara K, Nakano T, Matsuura Y, Kaibuchi K. Phosphorylation and activation of myosin by Rho-associated kinase. J Biol Chem. 1996;271:20246–20249. [PubMed]
  • Amano M, Chihara K, Kimura K, Fukata Y, Nakamura N, Matsuura Y, Kaibuchi K. Formation of actin stress fibers and focal adhesions enhanced by Rho-kinase. Science. 1997;275:1308–1311. [PubMed]
  • Bamburg JR, McGough A, Ono S. Putting a new twist on actin: ADF/cofilins modulate actin dynamics. Trends Cell Biol. 1999;9:364–370. [PubMed]
  • Banyard J, Anand-Apte B, Symons M, Zetter BR. Motility and invasion are differentially modulated by Rho family GTPases. Oncogene. 2000;19:580–591. [PubMed]
  • Bar-Sagi D, Hall A. Ras and Rho GTPases : a family reunion. Cell. 2000;103:227–238. [PubMed]
  • Bishop AL, Hall A. Rho GTPases and their effector proteins. Biochem J. 2000;348:241–255. [PMC free article] [PubMed]
  • Braverman RH, Cooper HL, Lee HS, Prasad GL. Anti-oncogenic effects of tropomyosin: isoform specificity and importance of protein coding sequences. Oncogene. 1996;13:537–545. [PubMed]
  • Burridge K, Chrzanowska-Wodnicka M, Zhong C. Focal adhesion assembly. Trends Cell Biol. 1997;7:342–347. [PubMed]
  • Button E, Shapland C, Lawson D. Actin, its associated proteins and metastasis. Cell Motil Cytoskeleton. 1995;30:247–251. [PubMed]
  • Carragher NO, Fincham VJ, Riley D, Frame MC. Cleavage of focal adhesion kinase by different proteases during Src-regulated transformation and apoptosis. J Biol Chem. 2001;276:4270–4275. [PubMed]
  • Chen Y, Lu G, Schneeberger EE, Goodenough DA. Restoration of tight junction structure and barrier function by down-regulation of the mitogen-activated protein kinase pathway in ras-transformed Madin-Darby canine kidney cells. Mol Biol Cell. 2000;11:849–862. [PMC free article] [PubMed]
  • Dudley DT, Pang L, Decker SJ, Bridges AJ, Saltiel AR. A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc Natl Acad Sci USA. 1995;92:7686–7689. [PMC free article] [PubMed]
  • Favata MF, Horiuchi KY, Manos EJ, Daulerio AJ, Stradley DA, Feeser WS, Van Dyk DE, Pitts WJ, Earl RA, Hobbs F, Copeland RA, Magolda RL, Scherle PA, Trzaskos JM. Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J Biol Chem. 1998;273:18623–18632. [PubMed]
  • Fincham VJ, James M, Frame MC, Winder SJ. Active ERK/MAP kinase is targeted to newly forming cell-matrix adhesions by integrin engagement and v-Src. EMBO J. 2000;19:2911–2923. [PMC free article] [PubMed]
  • Fukata Y, Amano M, Kaibuchi K. Rho-Rho-kinase pathway in smooth muscle contraction and cytoskeletal reorganization of non-muscle cells. Trends in Pharmacol Sci. 2001;22:32–39. [PubMed]
  • Fukazawa H, Uehara Y. U0126 reverses Ki-ras-mediated transformation by blocking both mitogen-activated protein kinase and p70 S6 kinase pathways. Cancer Res. 2000;60:2104–2107. [PubMed]
  • Gimona M, Kazzaz J, Helfman DM. Forced expression of tropomyosin 2 or 3 in v-Ki-ras-transformed fibroblasts results in distinct phenotypic effects. Proc Natl Acad Sci USA. 1996;93:9618–9623. [PMC free article] [PubMed]
  • Glading A, Chang P, Lauffenburger DA, Wells A. Epidermal growth factor receptor activation of calpain is required for fibroblast motility and occurs via an ERK/MAP kinase signaling pathway. J Biol Chem. 2000;275:2390–2398. [PubMed]
  • Gluck U, Kwiatkowski, Ben-Ze'ev A. Suppression of tumorigenicity in simian virus 40-transformed 3T3 cells transfected with α-actinin. Proc Natl Acad Sci USA. 1993;90:383–387. [PMC free article] [PubMed]
  • Helfman DM, Levy ET, Berthier C, Shtutman M, Riveline D, Grosheva I, Lachish-Zalait A, Elbaum M, Bershadsky AD. Caldesmon inhibits nonmuscle cell contractility and interferes with the formation of focal adhesions. Mol Biol Cell. 1999;10:3097–3112. [PMC free article] [PubMed]
  • Hunter T. Oncoprotein networks. Cell. 1997;88:333–346. [PubMed]
  • Ishikawa R, Yamashiro S, Matsumura F. Differential modulation of actin-severing activity of gelsolin by multiple isoforms of cultured rat cell tropomyosin: potentiation of protective ability of tropomyosins by 83-kDa nonmuscle caldesmon. J Biol Chem. 1989;264:7490–7497. [PubMed]
  • Itoh K, Yoshioka K, Akedo H, Uehata M, Ishizaki T, Narumiya S. An essential part for Rho-associated kinase in the transcellular invasion of tumor cells. Nature Med. 1999;5:221–225. [PubMed]
  • Izawa I, Amano M, Chihara K, Yamamoto T, Kaibuchi K. Possible involvement of the inactivation of the Rho-Rho-kinase pathway in oncogenic Ras-induced transformation. Oncogene. 1998;17:2863–2871. [PubMed]
  • Janke J, Schluter K, Jandrig B, Theile M, Kolbe K, Arnold W, Grinstein E, Schwartz A, Estevez-Schwarz L, Schlag PM, Jockusch BM, Scerneck S. Suppression of tumorigenicity in breast cancer cells by the microfilament protein profilin 1. J Exp Med. 2000;191:1675–1685. [PMC free article] [PubMed]
  • Janssen RAJ, Mier JW. Tropomyosin-2 cDNA lacking the 3′ untranslated region riboregulator induces growth inhibition of v-Ki-ras-transformed fibroblasts. Mol Biol Cell. 1997;8:897–908. [PMC free article] [PubMed]
  • Janssen RAJ, Veenstra KG, Jonasch P, Mier JW. Ras- and Raf-induced down-modulation of non-muscle tropomyosin are MEK-independent. J Biol Chem. 1998;273:32182–32186. [PubMed]
  • Katz ME, McCormick F. Signal transduction from multiple Ras effectors. Curr Opin Genet Dev. 1997;7:75–79. [PubMed]
  • Khosravi-Far R, Solski PA, Clark GJ, Kinch MS, Der CJ. Activation of Rac1, RhoA and mitogen-activated protein kinases is required for ras transformation. Mol Cell Biol. 1995;15:6443–6453. [PMC free article] [PubMed]
  • Kranenburg O, Verlan I, Moolenaar WH. Dynamin is required for the activation of mitogen-activated protein (MAP) kinase by MAP kinase kinase. J Biol Chem. 1999;274:35301–35304. [PubMed]
  • Kureishi Y, Kobayashi S, Amano M, Kimura K, Kanaide H, Nakano T, Kaibuchi K, Itoh M. Rho-associated kinase directly induces smooth muscle contraction through myosin light chain phosphorylation. J Biol Chem. 1997;272:12257–12260. [PubMed]
  • Kuwahara K, Saito Y, Nakagawa O, Kishimoto I, Harada M, Ogawa E, Miyamoto Y, Hamanaka I, Kajiyama N, Takahashi N, Izumi T, Kawakami R, Tamura N, Ogawa Y, Nakao K. The effects of the selective ROCK inhibitor, Y27632, on ET-1-induced hypertrophic response in neonatal rat cardiac myocytes: possible involvement of Rho/ROCK pathway in cardiac muscle cell hypertrophy. FEBS Lett. 1999;452:314–318. [PubMed]
  • Leung T, Chen X, Manser E, Lim L. The p160 RhoA-binding kinase ROKα is a member of a kinase family and is involved in the reorganization of the cytoskeleton. Mol Cell Biol. 1996;16:5313–5327. [PMC free article] [PubMed]
  • Ljundahl S, Linger S, Franzen B, Binetruy B, Auer G, Shoshan MC. Down-regulation of tropomyosin-2 expression in c-jun-transformed rat fibroblasts involves induction of a MEK1-dependent autocrine loop. Cell Growth Diff. 1998;9:565–573. [PubMed]
  • Maekawa M, Ishizaki T, Boku S, Watanabe N, Fujita A, Iwamatsu A, Obinata T, Ohashi K, Mizuno K, Narumyia S. Signaling from Rho to the actin cytoskeleton through protein kinases ROCK and LIM-kinase. Science. 1999;285:895–898. [PubMed]
  • Matsumura F, Lin JJ-C, Yamashiro-Matsumura S, Thomas GP, Topp WC. Differential expression of tropomyosin forms in the microfilaments isolated from normal and transformed rat cultured cells. J Biol Chem. 1983;258:13954–13964. [PubMed]
  • Nakano K, Takaishi K, Kodama A, Mammoto A, Shiozaki H, Monden M, Takai Y. Distinct actions and cooperative roles of ROCK and mDia in Rho small G protein-induced reorganization of the actin cytoskeleton in Madin-Darby canine kidney cells. Mol Biol Cell. 1999;10:2481–2491. [PMC free article] [PubMed]
  • Nikolopoulos SN, Spengler BA, Kisselbach K, Evans AE, Biedler JL, Ross RA. The human non-muscle α-actinin protein encoded by the ACTN4 gene suppresses tumorigenicity of human neuroblastoma cells. Oncogene. 2000;19:380–386. [PubMed]
  • Ohashi K, Nagata K, Maekawa M, Ishizaki T, Narumyia S, Mizuno K. Rho-associated kinase ROCK activates LIM-kinase 1 by phosphorylation at threonine 508 within the activation loop. J Biol Chem. 2000;275:3577–3582. [PubMed]
  • Olson MF, Paterson HF, Marshall CJ. Signals from Ras and Rho GTPases interact to regulate expression of p21Waf1/Cip1. Nature. 1998;394:295–299. [PubMed]
  • Pawlak G, Helfman DM. Cytoskeletal changes in cell transformation and tumorigenesis. Curr Opin Genet Dev. 2001;11:41–47. [PubMed]
  • Pittenger MF, Kazzaz JA, Helfman DM. Functional properties of non-muscle tropomyosin isoforms. Curr Opin Cell Biol. 1994;6:96–104. [PubMed]
  • Potempa S, Ridley AJ. Activation of both MAP kinase and phosphatidylinositide 3-kinase by ras is required for hepatocyte growth factor/scatter factor-induced adherens junctions disassembly. Mol Biol Cell. 1998;9:2185–2200. [PMC free article] [PubMed]
  • Prasad GL, Fuldner RA, Cooper HL. Expression of transduced tropomyosin 1 cDNA suppresses neoplastic growth of cells transformed by the ras oncogene. Proc Natl Acad Sci USA. 1993;90:7039–7043. [PMC free article] [PubMed]
  • Prasad GL, Masuelli L, Raj MHG, Harindranath N. Suppression of src-induced transformed phenotype by expression of tropomyosin-1. Oncogene. 1999;18:2027–2031. [PubMed]
  • Qiu RG, Chen J, McCormick F, Symons M. A role for Rho in Ras transformation. Proc Natl Acad Sci USA. 1995;92:11781–11785. [PMC free article] [PubMed]
  • Ren X, Kiosses WB, Schwartz MA. Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton. EMBO J. 1999;18:578–585. [PMC free article] [PubMed]
  • Reuveni H, Geiger T, Geiger B, Levitzki A. Reversal of the ras-induced transformed phenotype by HR12, a novel ras farnesylation inhibitor, is mediated by the Mek/Erk pathway. J Cell Biol. 2000;151:1179–1192. [PMC free article] [PubMed]
  • Rodriguez-Fernandez JL, Geiger B, Salomon D, Sabanay I, Zoller M, Ben-Ze'ev A. Suppression of tumorigenicity in transformed cells after transfection with vinculin. J Cell Biol. 1992;119:427–438. [PMC free article] [PubMed]
  • Rodriguez-Viciana P, Warne PH, Dhand R, Vanhaesebroek B, Gout I, Fry MJ, Waterfield MD, Downward J. Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature. 1994;370:527–532. [PubMed]
  • Rodriguez-Viciana P, Warme PH, Khwaja A, Marte BM, Pappin D, Das P, Waterfield MD, Ridley A, Downward J. Role of phosphoinositide 3-OH kinase in cell transformation and control of the actin cytoskeleton by ras. Cell. 1997;89:457–467. [PubMed]
  • Sahai E, Ishizaki T, Narumiya S, Treisman R. Transformation mediated by RhoA requires activity of ROCK kinases. Curr Biol. 1999;9:136–145. [PubMed]
  • Sahai E, Olson MF, Marshall CJ. Cross-talk between ras and rho signaling pathways in transformation favors proliferation and increased motility. EMBO J. 2001;20:755–766. [PMC free article] [PubMed]
  • Shields JM, Pruit K, McFall A, Shaub A, Der CJ. Understanding Ras: ‘it ain’t over 'til it's over.'. Trends Cell Biol. 2000;10:147–154. [PubMed]
  • Takenaga K, Masuda A. Restoration of microfilament bundle organization in v-raf-transformed NRK cells after transduction with tropomyosin 2 cDNA. Cancer Lett. 1994;87:47–53. [PubMed]
  • Watanabe N, Kato T, Fujita A, Ishizaki T, Narumyia S. Cooperation between mDia1 and ROCK in Rho-induced actin reorganization. Nat Cell Biol. 1999;1:136–143. [PubMed]
  • White MA, Nicolette C, Minden A, Polverino A, Van Aelst L, Karin M, Wigler MH. Multiple Ras functions can contribute to mammalian cell transformation. Cell. 1995;80:533–541. [PubMed]
  • Yang N, Higuchi O, Ohashi K, Nagata K, Wada A, Kangawa K, Nishida E, Mizuno K. Cofilin phosphorylation by LIM-kinase 1 and its role in Rac-mediated actin reorganization. Nature. 1998;393:809–812. [PubMed]
  • Zhong C, Kinch MS, Burridge K. Rho-stimulated contractility contributes to the fibroblastic phenotype of Ras-transformed epithelial cells. Mol Biol Cell. 1997;8:2329–2344. [PMC free article] [PubMed]

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