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• W2052022100 abstract "Accumulating evidence suggests that p21Cip1 located in the cytoplasm might play a role in promoting transformation and tumor progression. Here we show that oncogenic H-RasV12 contributes to the loss of actin stress fibers by inducing cytoplasmic localization of p21Cip1, 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 p21Cip1, and increased phosphorylation of cofilin. Ectopic expression of cytoplasmic but not nuclear p21Cip1 in Ras-transformed cells was effective in preventing stress fibers from being restored upon MEK inhibition and inhibited phosphorylation of cofilin. p21Cip1 was also found to form a complex with ROCK in Ras-transformed cells in vivo. Furthermore, inhibition of the PI 3-kinase pathway resulted in loss of p21Cip1 expression accompanied by restoration of phosphocofilin, which was 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. Our findings define a novel mechanism for coupling cytoplasmic p21Cip1 to the control of actin polymerization by compromising the Rho/ROCK/LIMK/cofilin pathway by oncogenic Ras. These studies suggest that localization of p21Cip1 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. Accumulating evidence suggests that p21Cip1 located in the cytoplasm might play a role in promoting transformation and tumor progression. Here we show that oncogenic H-RasV12 contributes to the loss of actin stress fibers by inducing cytoplasmic localization of p21Cip1, 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 p21Cip1, and increased phosphorylation of cofilin. Ectopic expression of cytoplasmic but not nuclear p21Cip1 in Ras-transformed cells was effective in preventing stress fibers from being restored upon MEK inhibition and inhibited phosphorylation of cofilin. p21Cip1 was also found to form a complex with ROCK in Ras-transformed cells in vivo. Furthermore, inhibition of the PI 3-kinase pathway resulted in loss of p21Cip1 expression accompanied by restoration of phosphocofilin, which was 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. Our findings define a novel mechanism for coupling cytoplasmic p21Cip1 to the control of actin polymerization by compromising the Rho/ROCK/LIMK/cofilin pathway by oncogenic Ras. These studies suggest that localization of p21Cip1 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. p21Cip1/Waf1 (hereafter referred to as p21) is best known for its ability to directly block the kinase activities of a broad range of cyclin/cyclin-dependent kinase (CDK) 1The abbreviations used are: CDK, cyclin-dependent kinase; ROCK, Rho kinase; LIMK, LIM kinase; MEK, mitogen-activated protein kinase kinase; ERK, extracellular signal-regulated kinase; PI3K, phosphatidylinositol 3-kinase; NLS, nuclear localization signal; EGFP, enhanced green fluorescence protein; Cip, CDK inhibitory protein; Kip, kinase inhibitory protein; mAb, monoclonal antibody; Me2SO, dimethyl sulfoxide; DAPI, 4′,6-diamidino-2-phenylindole; TM, tropomyosin; HMW, high molecular weight.1The abbreviations used are: CDK, cyclin-dependent kinase; ROCK, Rho kinase; LIMK, LIM kinase; MEK, mitogen-activated protein kinase kinase; ERK, extracellular signal-regulated kinase; PI3K, phosphatidylinositol 3-kinase; NLS, nuclear localization signal; EGFP, enhanced green fluorescence protein; Cip, CDK inhibitory protein; Kip, kinase inhibitory protein; mAb, monoclonal antibody; Me2SO, dimethyl sulfoxide; DAPI, 4′,6-diamidino-2-phenylindole; TM, tropomyosin; HMW, high molecular weight. complexes in response to anti-mitogenic signals or DNA damage (1.Sherr C.J. Roberts J.M. Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5072) Google Scholar, 2.Roninson I.B. Cancer Letts. 2002; 179: 1-14Crossref PubMed Scopus (365) Google Scholar, 3.Coqueret O. Trends Cell Biol. 2003; 13: 65-70Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar). Despite its function as a cell cycle inhibitor, which implies that p21 is a tumor suppressor, elevated level of p21 in the cytoplasm has been reported to be critical for promoting cell transformation and survival (4.Alpan R.S. Pardee A.B. Cell Growth Differ. 1996; 7: 893-901PubMed Google Scholar, 5.Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5201) Google Scholar, 6.Zhou B.P. Liao Y. Xia W. Spohn B. Lee M.-H. Hung M.-C. Nat. Cell Biol. 2001; 3: 245-252Crossref PubMed Scopus (893) Google Scholar, 7.Asada M. Yamada T. Ichijo H. Delia D. Miyazono K. Fukumuro K. Mizutani S. EMBO J. 1999; 18: 1223-1234Crossref PubMed Scopus (531) Google Scholar, 8.Tanaka H. Yamashita T. Asada M. Mizutani S. Yoshikawa H. Tohyama M. J. Cell Biol. 2002; 158: 321-329Crossref PubMed Scopus (133) Google Scholar). Strikingly, the level of p21 expression is highly increased in various human cancers such as breast cancer, bladder cancer, pancreatic cancer, and glioblastoma (9.Biankin A.V. Kench J.G. Morey A.L. Lee C.-S. Biankin S.A. Head D.R. Hugh T.B. Henshall S.M. Sutherland R.L. Cancer Res. 2001; 61: 8830-8837PubMed Google Scholar, 10.Korkolopoulou P. Konstantinidou A.E. Thomas-Tsagli E. Christodoulou P. Kapralos P. Davaris P. Appl. Immunohistochem. Mol. Morphol. 2000; 8: 285-292Crossref PubMed Scopus (38) Google Scholar, 11.Winters Z.E. Hunt N.C. Bradburn M.J. Royds J.A. Turley H. Harris A.L. Norbury C.J. Eur. J. Cancer. 2001; 37: 2405-2412Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 12.Jung J.M. Bruner J.M. Ruan S. Langford L.A. Kyritsis A.P. Kobayashi T. Levin V.A. Zhang W. Oncogene. 1995; 11: 2021-2028PubMed Google Scholar). It remains unclear how elevated cytoplasmic p21 might contribute to tumorigenesis. One possibility is that p21 is sequestered away from the nucleus in transformed cells thereby preventing it from binding to nuclear cyclin/CDK complexes, thus allowing sufficient cyclin/CDK activity for cell cycle progression (13.Orend G. Hunter T. Ruoslahti E. Oncogene. 1998; 16: 2575-2583Crossref PubMed Scopus (119) Google Scholar). Alternatively, relocalization of p21 to the cytoplasm may target cytoplasmic molecules such as apoptosis signal-regulating kinase 1 (ASK1) thereby promoting cell survival (7.Asada M. Yamada T. Ichijo H. Delia D. Miyazono K. Fukumuro K. Mizutani S. EMBO J. 1999; 18: 1223-1234Crossref PubMed Scopus (531) Google Scholar). The Rho family of GTPases, Rho, Rac, and Cdc42, regulate cell morphology, cytokinesis, and cell motility through reorganization of actin filaments (14.Bar-sagi D. Hall A. Cell. 2000; 103: 227-238Abstract Full Text Full Text PDF PubMed Scopus (694) Google Scholar). The interplay between these GTPases plays a critical role in the regulation of cell morphology and motility. Activation of Rac enhances cell spreading and migration by stimulation of actin polymerization at the plasma membrane and promoting lamellipodia formation. By contrast, Rho stimulates contractility and adhesion by inducing the formation of actin stress fibers and focal adhesions. Rho cycles between GDP-bound inactive and GTP-bound active forms, and the GTP-bound form binds to specific targets to exert its biological functions. Two closely related Rho kinases, ROCK-I and -II, have been established to be key downstream effectors of Rho to form stress fibers and focal adhesions (15.Amano M. Fukata Y. Kaibuchi K. Exp. Cell Res. 2000; 261: 44-51Crossref PubMed Scopus (450) Google Scholar). Rho kinases contribute to the increased actin-myosin II-mediated contractility by directly phosphorylating myosin light chain (MLC) and negatively regulate myosin light chain phosphatase (MLCP) by phosphorylating myosin binding subunit (MBS) of MLCP (16.Amano M. Ito M. Kimura K. Fukata Y. Chihara K. Nakano T. Matsura Y. Kaibuchi K. J. Biol. Chem. 1996; 271: 20246-20249Abstract Full Text Full Text PDF PubMed Scopus (1654) Google Scholar, 17.Kimura N. Ito M. Amano M. Chihara K. Fukata Y. Nakafuku M. Yamamori B. Feng J. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. Science. 1996; 273: 245-248Crossref PubMed Scopus (2412) Google Scholar). Rho kinases also activate LIM kinase, which subsequently phosphorylates cofilin and thereby inhibits its actin-depolymerizing activity, thus leading to stabilization of actin stress fibers (18.Maekawa M. Ishizaki T. Boku S. Watanabe N. Fujita A. Iwamatsu A. Obinata T. Ohashi K. Mizuno K. Narumiya S. Science. 1999; 285: 895-898Crossref PubMed Scopus (1257) Google Scholar). Ras (H-Ras, K-Ras, N-Ras) regulates cell growth, differentiation, and cell motility (14.Bar-sagi D. Hall A. Cell. 2000; 103: 227-238Abstract Full Text Full Text PDF PubMed Scopus (694) Google Scholar). The frequency of Ras mutations is one of the highest of any gene in human cancers (19.Hunter T. Cell. 1997; 88: 333-346Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar). Cells transformed by oncogenic Ras are characterized not only by deregulated growth control but also pronounced 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 (20.Bondy G.P. Wilson S. Chambers A.F. Cancer Res. 1985; 45: 6005-6009PubMed Google Scholar, 21.Fox P.L. Sa G. Dobrowolski S.F. Stacey D.W. Oncogene. 1994; 9: 3519-3526PubMed Google Scholar, 22.Pawlak G. Helfman D.M. Curr. Opin. Gene Dev. 2001; 11: 41-47Crossref PubMed Scopus (279) Google Scholar). We and others (23.Pawlak G. Helfman D.M. Mol. Biol. Cell. 2002; 13: 336-347Crossref PubMed Scopus (78) Google Scholar, 24.Sahai E. Olson M.F. Marshall C.J. EMBO J. 2001; 20: 755-766Crossref PubMed Scopus (327) Google Scholar) have shown that transformation of fibroblasts cells by oncogenic Ras induces constitutive activation of MEK, which causes disruption of actin cytoskeleton by inactivating the Rho-ROCK-LIM kinase pathway. The inhibition of Rho-dependent stress fiber formation contributes to the increased motility of Ras-transformed fibroblasts (24.Sahai E. Olson M.F. Marshall C.J. EMBO J. 2001; 20: 755-766Crossref PubMed Scopus (327) Google Scholar). However, the mechanism of this inactivation has not been elucidated. In this study, we show that cytoplasmic p21 plays a critical role in the morphological and cytoskeletal changes observed in Ras-transformed fibroblasts. We demonstrate that sustained activation of both MEK and PI3K effector pathways are necessary for the elevation of p21 protein in the cytoplasm of Ras-transformed NIH3T3 cells. The cytoplasmic p21 forms a physical complex with ROCK and inhibits its activity, thereby contributing to the loss of actin stress fibers by compromising the ROCK/LIMK/cofilin pathway. Our findings suggest a novel physiological role for cytoplasmic p21 in remodeling of the actin cytoskeleton by oncogenic Ras. Antibodies and Reagents—The mouse monoclonal anti-ROCK-I mAb (clone 46) and anti-ROCK-II mAb (clone 21) were purchased from Transduction Laboratories (Lexington, KY). Anti-p21Cip1 mAb and rabbit polyclonal antibody detecting cofilin phosphorylated by LIMK at Ser-3 was from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-vinculin antibody (hVIN-1) was from Sigma Chemical. The rabbit polyclonal anti-cofilin was from Cytoskeleton (Denver, CO). The rabbit anti-ROCK-I pAb was kindly provided by Dr. Jian Du in Dr. Greg Hannon's laboratory (Cold Spring Harbor Laboratory, 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 and rhodamine-conjugated phalloidins were from Molecular Probes (Eugene, OR). U0126, LY294002, and Y27632 were from Calbiochem (La Jolla, CA). All tissue culture reagents were from Invitrogen. Cell Culture and Retroviral Infection—Normal NIH3T3 cells were obtained from the Tissue Culture Facility of Cold Spring Harbor Laboratory. Cells were maintained in Dulbecco's modified Eagle's medium containing 10% BCS, 100 units/ml penicillin, and 100 μg/ml streptomycin in a humidifier air (5% CO2) atmosphere, at 37 °C. The stable cell line expressing H-RasV12 was obtained by the following protocol: pWZL-Hygro H-RasV12 and corresponding empty retroviral vector were used to transfect the ecotropic packaging cell line ϕ NX. Transfection was performed by the calcium phosphate method. At 72 h post-transfection, viral supernatants were collected, filtered, and supplemented with 4 μg/ml polybrene. The supernatant was then used to infect NIH3T3 cells. After infection, cells were selected in hygromycin (50 μg/ml) for 14 days. Plasmids and Transfection—Retroviral pWZL-Hygro H-RasV12 vector was a generous gift of Yvette Seger in the Hannon laboratory (Cold Spring Harbor Laboratory). pEGFP-full-length-p21 (amino acids 1–164) and pEGFP-ΔNLS-p21 (amino acids 1–140) were provided by Dr. Minoru Asada (International Medical Center of Japan). pEGFP-N1-XAC, expressing wild-type Xenopus cofilin tagged with EGFP at the C terminus, was kindly provided by James Bamburg (Colorado State University). For transient transfection, cells were grown at 60–70% confluency in Dulbecco's modified Eagle's medium containing 10% BCS. Cells were transfected with a total of 2 μg of expression vectors per dish, using LipofectAMINE PLUS reagent (Invitrogen) according to the manufacturer's protocol. At 24 h post-transfection, cells were split and plated onto glass coverslips followed by incubation for additional 24 h. In some cases, cells on the coverslips were treated with Me2SO, U0126, or LY294002 for various time periods, then the coverslips were harvested and processed for immunofluorescence. Immunofluorescence—Cells grown on glass coverslips were fixed with 3% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 for 5 min, then blocked for 30 min with 1% bovine serum albumin/phosphate-buffered saline at room temperature. For immunofluorescence, fixed cells were incubated for 1h with mouse monoclonal p21 antibody (Santa Cruz Biotechnology) or monoclonal vinculin antibody followed by Cy3-conjugated goat anti-mouse IgG for 1 h. To visualize F-actin, Oregon green, or rhodamine-conjugated phalloidin (1:100) was diluted in 1% bovine serum albumin/phosphate-buffered saline for staining. The coverslips were stained with 4′6-diamidino-2-phenylindole (DAPI), and then mounted using Prolong Antifade (Molecular Probes). Samples were examined, and pictures were acquired on a Zeiss Axiophot microscope equipped with a Photometrics Sensys (Oberkochen, Germany) cooled CCD camera using Openlab 3.1.1 software. All photographs were taken at the same magnification. Immunoprecipitation and Western Blot Analysis—Cell lysates were prepared as previously described in 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 10% glycerol, 0.5% Nonidet-P40 including protease inhibitor mixture tablets (Roche Applied Science) (8.Tanaka H. Yamashita T. Asada M. Mizutani S. Yoshikawa H. Tohyama M. J. Cell Biol. 2002; 158: 321-329Crossref PubMed Scopus (133) Google Scholar). The cell lysates were centrifuged at 13,000 × g for 20 min, and the supernatant was collected. Immunoprecipitations were performed for 1 h at 4 °C using polyclonal anti-ROCK-I antibody. The immunocomplexes were collected with protein G-Sepharose (Amersham Biosciences) slurry (50% v/v), washed four times with lysis buffer, and subjected to SDS-PAGE. For Western blot, control and treated cells were washed with ice-cold phosphate-buffered saline containing 1 mm sodium orthovanadate before direct extraction in 2× SDS Laemmli Sample Buffer. Lysates were clarified by centrifugation (16,000 × g for 15 min at 4 °C), and protein concentrations were measured by Bradford assay (Bio-Rad, Hercules, CA). 10 μg of proteins were resolved by SDS-PAGE then transferred to nitrocellulose membrane (Schleicher & Schuell, Keene, NH). The membrane was blocked for 30 min in 5% nonfat dried milk or 1% bovine serum albumin in phosphate-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 (PerkinElmer Life Sciences). Rho-GTP Pull-down Assay—Measurement of GTP-bound Rho was performed using the Rho Activation Assay kit (Upstate Biotechnology), following the manufacturer'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 protease inhibitor mixture (Roche Applied Science). Precipitated Rho-GTP was then detected by immunoblot analysis, using a monoclonal anti-RhoA antibody (Santa Cruz Biotechnology). Ras Uncouples Rho-GTP from the ROCK/LIMK/Cofilin Pathway—NIH3T3 fibroblasts have a flat morphology and welldeveloped stress fibers, whereas cells transformed by H-RasV12 are rounded and lack stress fibers (Fig. 1A). RasV12 is known to activate several effector pathways including Raf/MEK/MAP, phosphatidylinositol 3-kinase (PI3K), and RalGDS (25.Wood K.W. Sarnecki C. Roberts T.M. Blenis J. Cell. 1992; 92: 1041-1050Abstract Full Text PDF Scopus (656) Google Scholar, 26.Rodriguez-Viciana P. Warme P.H. Khwaja A. Marte B.M. Pappin D. Das P. Waterfield M.D. Downward J. Cell. 1997; 89: 457-467Abstract Full Text Full Text PDF PubMed Scopus (954) Google Scholar, 27.Kikuchi A. Demo S.D. Ye Z.H. Chen Y.W. Williams L.T. Mol. Cell. Biol. 1994; 14: 7483-7491Crossref PubMed Scopus (244) Google Scholar). To dissect which signaling pathway is necessary for the prevention of stress fiber formation in Ras-transformed NIH3T3 cells, we treated Ras-transformed cells with U0126 or LY294002, which specifically inhibit MEK or PI3K, respectively. We found that the disruption of stress fibers and focal adhesions observed in Ras-transformed cells were reversed by treatment with U0126 but not by LY294002 (Fig. 1A). These observations are in agreement with previous studies that showed that activation of the MEK-dependent pathway, but not PI3K, is necessary for disruption of the actin stress fibers and focal adhesions by oncogenic Ras (23.Pawlak G. Helfman D.M. Mol. Biol. Cell. 2002; 13: 336-347Crossref PubMed Scopus (78) Google Scholar, 24.Sahai E. Olson M.F. Marshall C.J. EMBO J. 2001; 20: 755-766Crossref PubMed Scopus (327) Google Scholar). We also observed that the restoration of stress fibers and focal adhesions by U0126 requires ROCK activity, because either dominant negative ROCK-I or the ROCK inhibitor, Y27632, were able to prevent stress fiber formation caused by U0126 (data not shown). The small GTPase, RhoA, is known to act upstream of ROCK and induce the formation of stress fibers and focal adhesions (14.Bar-sagi D. Hall A. Cell. 2000; 103: 227-238Abstract Full Text Full Text PDF PubMed Scopus (694) Google Scholar). To examine whether the cytoskeletal changes induced by Ras oncogene were elicited by regulation of RhoA activity, we measured the levels of Rho-GTP, using an assay that only captures the active GTP-bound form of the GTPase (28.Ren X. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1350) Google Scholar). Although three isoforms of Rho protein were described, we measured only RhoA-GTP, because neither RhoB nor RhoC were detectable in NIH3T3 (data not shown). The level of RhoA-GTP in Ras-transformed cells was higher than that in parental NIH3T3 (Fig. 1B), but the difference was abolished by U0126, consistent with the results obtained by others (29.Chen J.C. Zhuang S. Nguyen T.H. Boss G.R. Pilz R.B. J. Biol. Chem. 2003; 278: 2807-2818Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) that sustained activation of Raf/MEK/ERK pathway is required for the activation of RhoA protein. More importantly, these data demonstrate that active RhoA-GTP, despite its increased level, is no longer coupled via its interaction with ROCK to the formation of stress fibers in Ras-transformed cells. It was previously reported that a MEK-dependent pathway leads to disorganization of the actin cytoskeleton in Ras-transformed fibroblasts by down-regulation of ROCK-I/II protein expression (23.Pawlak G. Helfman D.M. Mol. Biol. Cell. 2002; 13: 336-347Crossref PubMed Scopus (78) Google Scholar, 24.Sahai E. Olson M.F. Marshall C.J. EMBO J. 2001; 20: 755-766Crossref PubMed Scopus (327) Google Scholar). In these studies, they showed that the amount of Rho-GTP in Ras-transformed cells was comparable to or higher than that of the untransformed control cells. Therefore, we also compared the levels of ROCK-I/II proteins between untransformed and Ras-transformed NIH3T3 cells. No changes in the levels of either ROCK-I or -II proteins were detected (Fig. 1C). ROCK-I/II can activate LIM kinase, which in turn phosphorylates cofilin and thereby inhibits its actin-depolymerizing activity, thus leading to stabilization of stress fibers (30.Bamburg J.R. Mcgough A. Ono S. Trends Cell Biol. 1999; 9: 364-370Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, 31.Ohashi K. Nagata K. Maekawa M. Ishizaki T. Narumiya S. Mizuno K. J. Biol. Chem. 2000; 275: 3577-3582Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar). To determine if Ras causes any decrease in phosphorylated cofilin in NIH3T3 cells, we used an antibody that detects cofilin phosphorylated by LIM kinase at the Ser-3 residue. We found that cofilin phosphorylation was significantly decreased by expression of Ras (Fig. 1C). Furthermore, the level of phosphorylated cofilin was restored following treatment of Ras-transformed cells with the MEK inhibitor (Fig. 1C). These results indicate an alternative mechanism exists, other than a decrease in ROCK-I/II proteins as previously reported in Ras-transformed NRK and Swiss 3T3 cells (23.Pawlak G. Helfman D.M. Mol. Biol. Cell. 2002; 13: 336-347Crossref PubMed Scopus (78) Google Scholar, 24.Sahai E. Olson M.F. Marshall C.J. EMBO J. 2001; 20: 755-766Crossref PubMed Scopus (327) Google Scholar), for uncoupling Rho-GTP from activating Rho kinases. Elevated Levels of Cytoplasmic p21 Correlates with the Disruption of Stress Fibers in Ras-transformed Cells—Despite its implied role as a tumor suppressor, the level of p21 protein is significantly elevated in many types of human cancer (2.Roninson I.B. Cancer Letts. 2002; 179: 1-14Crossref PubMed Scopus (365) Google Scholar). In agreement with these observations, we found that p21 protein expression is induced by Ras in NIH3T3 cells (Fig. 2A). This is consistent with recent studies showing that Ras activates p21 transcription and promotes p21 protein stability via blocking proteasome-mediated degradation (32.Coleman M.L. Marshall C.J. Olson M.F. EMBO J. 2003; 22: 2036-2046Crossref PubMed Scopus (138) Google Scholar, 33.Gartel A.L. Najmabadi F. Goufman E. Tyner A.L. Oncogene. 2000; 19: 961-964Crossref PubMed Scopus (46) Google Scholar). The p21 protein was decreased in Ras-transformed cells by MEK inhibition in a time-dependent manner (Fig. 2A). It was completely undetectable at 24 h post-treatment of U0126. This demonstrates that the increase in expression of p21 protein in Ras-transformed cells was dependent on sustained activation of the Raf/MEK/ERK pathway. Interestingly, the decrease of p21 following U0126 treatment was temporally associated with the restoration of stress fibers and increase in cofilin phosphorylation (Fig. 2, A and B). These observations suggested an inverse relationship between the concentration of p21 protein and the formation of stress fibers. Recently, it was reported that p21 is induced in the cytoplasm during neuronal differentiation and is involved in blocking Rho-induced actin remodeling leading to neurite outgrowth (8.Tanaka H. Yamashita T. Asada M. Mizutani S. Yoshikawa H. Tohyama M. J. Cell Biol. 2002; 158: 321-329Crossref PubMed Scopus (133) Google Scholar). To determine if cytoplasmic p21 was involved in remodeling of the actin cytoskeleton in Ras-transformed NIH3T3 cells, we performed immunofluorescence staining to determine the localization of p21. We found that in 90% of Ras-transformed cells p21 exists abundantly in the cytoplasm as well as in the nucleus (Fig. 2C), whereas p21 was not detectable in the majority of asynchronous NIH3T3 cells (data not shown), which is consistent with our Western blot data (Fig. 2A). However, the p21 protein became barely detectable in the cytoplasm of 70% Ras-transformed cells if treated with the MEK inhibitor for 5 h (Fig. 2C). Concomitant with a decrease in cytoplasmic p21, at 5 h post-treatment of U0126, there was an increase in the levels of phosphorylated cofilin, although it was too early to monitor obvious reassembly of stress fibers (Fig. 2, A and B). These results suggest that the loss of p21 from the cytoplasm and restoration of cofilin phosphorylation might be associated with MEK inhibition-mediated reassembly of stress fibers. Cytoplasmic p21 Is Sufficient for the Maintenance of Disorganized Stress Fibers in Ras-transformed Cells—To directly determine whether the expression of cytoplasmic p21 is sufficient to block stress fiber formation in Ras-transformed cells, we transfected Ras-transformed cells with either a full-length p21 or p21 with a deletion in the nuclear localization signal (NLS). Following 48 h post-transfection, we treated the cells with U0126 for an additional 24 h, which caused complete loss in the expression of endogenous p21 but not in that of exogenous p21 (data not shown). Therefore, we could determine if ectopic expression of ΔNLS-p21 would affect the restoration of stress fibers by the MEK inhibitor. Ectopic expression of either ΔNLS-p21 or full-length p21 had no effect on the morphology of Ras-transformed cells in the absence of U0126 (Fig. 3A). However, 88% (44/50) of cells expressing the EGFP-ΔNLS-p21 in the cytoplasm were refractory to the restoration of stress fibers caused by MEK inhibition, whereas most of cells expressing the EGFP control or EGFP-full-length p21 in the nucleus failed to show such a resistance (Fig. 3B). We also observed that exogenous p21, but not ΔNLS-p21, relocalized to the nucleus similarly as endogenous p21 following MEK inhibition (Figs. 2C and 3B), confirming that cytoplasmic p21, but not nuclear, conferred such a refractory effect. Furthermore, ectopic expression of ΔNLS-p21, but not full-length p21, was sufficient to cause disruption of the actin cytoskeleton in 47% (39/83) of untransformed NIH3T3 cells (Fig. 3C). It is noteworthy that full-length p21 showed nuclear-specific pattern of expression in 90% of transfectants, implying that p21 is normally localized in the nucleus under the absence of oncogenic Ras activation. Collectively, these results suggest that localization of p21 to the cytoplasm by Ras is involved in reorganization of the actin cytoskeleton. In Vivo Interaction of p21Cip1 with ROCK—ROCK is known to act downstream of Rho to induce stress fiber formation in fibroblasts (15.Amano M. Fukata Y. Kaibuchi K. Exp. Cell Res. 2000; 261: 44-51Crossref PubMed Scopus (450) Google Scholar). In one report, the expression of cytoplasmic p21 promotes neurite outgrowth in hippocampal neurons by forming complex with ROCK and inhibiting its activity (8.Tanaka H. Yamashita T. Asada M. Mizutani S. Yoshikawa H. Tohyama M. J. Cell Biol. 2002; 158: 321-329Crossref PubMed Scopus (133) Google Scholar). To determine whether endogenous p21 physically interacts with ROCK in vivo, we immunoprecipitated ROCK from Ras-transformed NIH3T3 cells in which p21 is highly expressed. We found that endogenous p21 was coprecipitated with an anti-ROCK-I antibody (Fig. 4A), demonstrating that endogenous p21 forms a complex with ROCK-I in vivo. Next, we sought to determine whether ectopic expression of cytoplasmic p21 in NIH3T3 cells could directly interfere with ROCK/LIMK/cofilin pathway. For this purpose, we measured the level of phosphorylation of exogenously expressed cofilin after cotransfecting EGFP-tagged cofilin in combination with ΔNLS-p21 or controls. The phosphorylation of exogenous EGFP-cofilin was significantly reduced by ΔNLS-p21 protein but not by the wild-type p21 (Fig. 4B). In addition, treatment of a specific ROCK inhibitor, Y27632 (10 μm), effectively blocked the phosphorylation of EGFP-cofilin, an observation consistent with the notion that endogenous ROCK is an upstream effector mediating LIM kinase-dependent phosphorylation of exogenous EGFP-cofilin as well (Fig. 4B). Taken together, these results suggest that association of p21 with ROCK in the cytoplasm is the mechanism by which ROCK/LIMK/cofili" @default.
• W2052022100 created "2016-06-24" @default.
• W2052022100 creator A5039994535 @default.
• W2052022100 creator A5065126365 @default.
• W2052022100 date "2004-01-01" @default.
• W2052022100 modified "2023-10-09" @default.
• W2052022100 title "Cytoplasmic p21Cip1 Is Involved in Ras-induced Inhibition of the ROCK/LIMK/Cofilin Pathway" @default.
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