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• W2044857500 abstract "Binding of the urokinase-type plasminogen activator (uPA) to its receptor activates diverse cell signaling pathways. How these signals are integrated so that cell physiology is altered remains unclear. In this study, we demonstrated that migration of MCF-7 breast cancer cells and HT-1080 fibrosarcoma cells on serum-coated surfaces is stimulated by agents that activate ERK, including uPA, epidermal growth factor, and constitutively active MEK1. The promigratory activity of these agents was entirely blocked not only by the MEK-specific antagonist PD098059, but also by antagonists of the Rho-Rho kinase pathway, including Y-27632 and dominant-negative RhoA (RhoA-N19). uPA did not significantly increase the level of GTP-bound RhoA, suggesting that the constitutive activity of the Rho-Rho kinase pathway may be sufficient to support ERK-stimulated cell migration. Paradoxically, Y-27632 and RhoA-N19 increased ERK phosphorylation in MCF-7 cells, providing further evidence that ERK activation alone does not promote cell migration when Rho kinase is antagonized. When MCF-7 cell migration was stimulated by ERK-independent processes such as expression of the β3 integrin subunit or changing the substratum to type I collagen, Y-27632 and RhoA-N19 failed to inhibit the response. This study supports a model in which the Ras-ERK and Rho-Rho kinase pathways cooperate to promote cell migration. Neutralizing either pathway is sufficient to block the response to agents that stimulate cell migration by activating ERK. Binding of the urokinase-type plasminogen activator (uPA) to its receptor activates diverse cell signaling pathways. How these signals are integrated so that cell physiology is altered remains unclear. In this study, we demonstrated that migration of MCF-7 breast cancer cells and HT-1080 fibrosarcoma cells on serum-coated surfaces is stimulated by agents that activate ERK, including uPA, epidermal growth factor, and constitutively active MEK1. The promigratory activity of these agents was entirely blocked not only by the MEK-specific antagonist PD098059, but also by antagonists of the Rho-Rho kinase pathway, including Y-27632 and dominant-negative RhoA (RhoA-N19). uPA did not significantly increase the level of GTP-bound RhoA, suggesting that the constitutive activity of the Rho-Rho kinase pathway may be sufficient to support ERK-stimulated cell migration. Paradoxically, Y-27632 and RhoA-N19 increased ERK phosphorylation in MCF-7 cells, providing further evidence that ERK activation alone does not promote cell migration when Rho kinase is antagonized. When MCF-7 cell migration was stimulated by ERK-independent processes such as expression of the β3 integrin subunit or changing the substratum to type I collagen, Y-27632 and RhoA-N19 failed to inhibit the response. This study supports a model in which the Ras-ERK and Rho-Rho kinase pathways cooperate to promote cell migration. Neutralizing either pathway is sufficient to block the response to agents that stimulate cell migration by activating ERK. The urokinase-type plasminogen activator (uPA) 1The abbreviations used are: uPAurokinase-type plasminogen activatoruPARurokinase-type plasminogen activator receptorERKextracellular signal-regulated kinaseMEKmitogen-activated protein kinase/extracellular signal-regulated kinase kinaseEGFepidermal growth factorDFPdiisopropyl fluorophosphateHAhemagglutininGFPgreen fluorescent proteinGST-TRBDglutathione S-transferase-Rhotekin Rho-binding domainLPAlysophosphatidic acid is a serine proteinase that activates diverse cell signaling pathways by binding to its cell-surface receptor (uPAR) (1.Ossowski L. Aguirre-Ghiso J.A. Curr. Opin. Cell Biol. 2000; 12: 613-620Crossref PubMed Scopus (359) Google Scholar, 2.Blasi F. Thromb. Haemostasis. 1999; 82: 298-304Crossref PubMed Scopus (171) Google Scholar, 3.Chapman H.A. Wei Y. Simon D.I. Waltz D.A. Thromb. Haemostasis. 1999; 82: 291-297Crossref PubMed Scopus (133) Google Scholar). Because uPAR is glycosylphosphatidylinositol-anchored and thus lacks transmembrane and intracytoplasmic domains (4.Ploug M. Ronne E. Behrendt N. Jensen A.L. Blasi F. Dano K. J. Biol. Chem. 1991; 266: 1926-1933Abstract Full Text PDF PubMed Google Scholar), it is assumed that the complete signaling receptor is a uPAR-containing protein complex. The function of uPA may be to induce uPAR conformational change so that the required interactions with other plasma membrane proteins are established and signaling is initiated (2.Blasi F. Thromb. Haemostasis. 1999; 82: 298-304Crossref PubMed Scopus (171) Google Scholar). In support of this hypothesis, soluble uPAR mimics many of the activities of uPA, probably because soluble uPAR adopts the same conformation as plasma membrane-associated uPAR after complex formation with uPA (5.Fazioli F. Resnati M. Sidenius N. Higashimoto Y. Appella E. Blasi F. EMBO J. 1997; 16: 7279-7286Crossref PubMed Scopus (230) Google Scholar, 6.Degryse B. Resnati M. Rabbani S.A. Villa A. Fazioli F. Blasi F. Blood. 1999; 94: 649-662Crossref PubMed Google Scholar, 7.Nguyen D.H. Webb D.J. Catling A.D. Song Q. Dhakephalkar A. Weber M.J. Ravichandran K.S. Gonias S.L. J. Biol. Chem. 2000; 275: 19382-19388Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). uPAR may also regulate cell signaling by uPA-independent pathways (8.Kjoller L. Hall A. J. Cell Biol. 2001; 152: 1145-1157Crossref PubMed Scopus (174) Google Scholar, 9.Nguyen D.H. Catling A.D. Webb D.J. Sankovic M. Walker L.A. Somlyo A.V. Weber M.J. Gonias S.L. J. Cell Biol. 1999; 146: 149-164Crossref PubMed Scopus (299) Google Scholar). How the cell integrates uPA-dependent and uPA-independent uPAR signals remains to be determined. urokinase-type plasminogen activator urokinase-type plasminogen activator receptor extracellular signal-regulated kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase epidermal growth factor diisopropyl fluorophosphate hemagglutinin green fluorescent protein glutathione S-transferase-Rhotekin Rho-binding domain lysophosphatidic acid In many cell types, binding of uPA to uPAR results in activation of the mitogen-activated protein kinase ERK (7.Nguyen D.H. Webb D.J. Catling A.D. Song Q. Dhakephalkar A. Weber M.J. Ravichandran K.S. Gonias S.L. J. Biol. Chem. 2000; 275: 19382-19388Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 10.Nguyen D.H. Hussaini I.M. Gonias S.L. J. Biol. Chem. 1998; 273: 8502-8507Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 11.Kanse S.M. Benzakour O. Kanthou C. Kost C. Lijnen H.R. Preissner K.T. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 2848-2854Crossref PubMed Scopus (77) Google Scholar, 12.Konakova M. Hucho F. Schleuning W.D. Eur. J. Biochem. 1998; 253: 421-429Crossref PubMed Scopus (94) Google Scholar, 13.Tang H. Kerins D.M. Hao Q. Inagami T. Vaughan D.E. J. Biol. Chem. 1998; 273: 18268-18272Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). This signaling response may be of central importance because the level of activated ERK controls many physiologic processes, including cell growth, differentiation, apoptosis, and migration, and in cancer, invasion and metastasis (1.Ossowski L. Aguirre-Ghiso J.A. Curr. Opin. Cell Biol. 2000; 12: 613-620Crossref PubMed Scopus (359) Google Scholar). uPA-induced ERK activation in MCF-7 breast cancer cells is entirely dependent on the small GTPase Ras (7.Nguyen D.H. Webb D.J. Catling A.D. Song Q. Dhakephalkar A. Weber M.J. Ravichandran K.S. Gonias S.L. J. Biol. Chem. 2000; 275: 19382-19388Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), which presumably functions through the downstream effectors Raf and MEK (14.McCormick F. Curr. Opin. Genet. Dev. 1994; 4: 71-76Crossref PubMed Scopus (210) Google Scholar). In aggressive cancer cells, endogenously produced uPA and uPAR may form a self-contained autocrine pathway, which serves as a major determinant of the basal level of ERK activation, in the absence of exogenous stimulants (15.Aguirre-Ghiso J.A. Kovalski K. Ossowski L. J. Cell Biol. 1999; 147: 89-104Crossref PubMed Scopus (467) Google Scholar, 16.Aguirre-Ghiso J.A. Liu D. Mignatti A. Kovalski K. Ossowski L. Mol. Biol. Cell. 2001; 12: 863-879Crossref PubMed Scopus (383) Google Scholar, 17.Ma Z. Webb D.J. Jo M. Gonias S.L. J. Cell Sci. 2001; 114: 3387-3396Crossref PubMed Google Scholar). uPA promotes migration of MCF-7 and HT-1080 cells on serum- or vitronectin-coated surfaces by a mechanism that requires ERK (9.Nguyen D.H. Catling A.D. Webb D.J. Sankovic M. Walker L.A. Somlyo A.V. Weber M.J. Gonias S.L. J. Cell Biol. 1999; 146: 149-164Crossref PubMed Scopus (299) Google Scholar, 10.Nguyen D.H. Hussaini I.M. Gonias S.L. J. Biol. Chem. 1998; 273: 8502-8507Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). In this pathway, ERK activates myosin light chain kinase, which induces serine phosphorylation of the myosin II regulatory light chain and thereby promotes contraction of the actomyosin cytoskeleton (9.Nguyen D.H. Catling A.D. Webb D.J. Sankovic M. Walker L.A. Somlyo A.V. Weber M.J. Gonias S.L. J. Cell Biol. 1999; 146: 149-164Crossref PubMed Scopus (299) Google Scholar, 18.Klemke R.L. Cai S. Giannini A.L. Gallagher P.J. de Lanerolle P. Cheresh D.A. J. Cell Biol. 1997; 137: 481-492Crossref PubMed Scopus (1103) Google Scholar). Why increased cytoskeletal contractility is important for the migrating cell remains incompletely understood; however, it is thought that this activity facilitates retraction of the cell tail. uPAR-initiated signaling is also important in the mechanism by which uPA promotes smooth muscle cell migration; however, in smooth muscle cells, the response is blocked by inhibitors of a distinct signaling pathway that includes the Janus kinase Tyk2 and phosphatidylinositol 3-kinase (19.Kusch A. Tkachuk S. Haller H. Dietz R. Gulba D.C. Lipp M. Dumler I. J. Biol. Chem. 2000; 275: 39466-39473Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The activity of the Ras-ERK pathway in smooth muscle cell migration has not been explored. Nevertheless, the function of Tyk2 and phosphatidylinositol 3-kinase in smooth muscle cell migration (19.Kusch A. Tkachuk S. Haller H. Dietz R. Gulba D.C. Lipp M. Dumler I. J. Biol. Chem. 2000; 275: 39466-39473Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) raises the possibility that distinct signaling pathways may be preeminent in determining physiologic responses to uPA in different cell types. The focus of this study is the function of the small GTPase Rho and the major Rho effector, Rho kinase (also known as p160ROCK), in uPA-promoted MCF-7 and HT-1080 cell migration. RhoA and homologous proteins in the Rho family function primarily in processes that require remodeling of the actin cytoskeleton (20.Kaibuchi K. Kuroda S. Amano M. Annu. Rev. Biochem. 1999; 68: 459-486Crossref PubMed Scopus (891) Google Scholar). In stationary cells, RhoA promotes the formation of actin stress fibers and clustering of proteins in focal adhesions (20.Kaibuchi K. Kuroda S. Amano M. Annu. Rev. Biochem. 1999; 68: 459-486Crossref PubMed Scopus (891) Google Scholar, 21.Ridley A.J. Comoglio P.M. Hall A. Mol. Cell. Biol. 1995; 15: 1110-1122Crossref PubMed Google Scholar, 22.Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5230) Google Scholar, 23.Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3747) Google Scholar). However, recent studies suggest that RhoA, RhoC, and Rho kinase may also be important in cell migration and cancer invasion (24.Takaishi K. Sasaki T. Kato M. Yamochi W. Kuroda S. Nakamura T. Takeichi M. Takai Y. Oncogene. 1994; 9: 273-279PubMed Google Scholar, 25.Yoshioka K. Matsumura F. Akedo H. Itoh K. J. Biol. Chem. 1998; 273: 5146-5154Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 26.Itoh K. Yoshioka K. Akedo H. Uehata M. Ishizaki T. Narumiya S. Nat. Med. 1999; 5: 221-225Crossref PubMed Scopus (560) Google Scholar, 27.Clark E.A. Golub T.R. Lander E.S. Hynes R.O. Nature. 2000; 406: 532-535Crossref PubMed Scopus (1309) Google Scholar, 28.Somlyo A.V. Bradshaw D. Ramos S. Murphy C. Myers C.E. Somlyo A.P. Biochem. Biophys. Res. Commun. 2000; 269: 652-659Crossref PubMed Scopus (213) Google Scholar, 29.Worthylake R.A. Lemoine S. Watson J.M. Burridge K. J. Cell Biol. 2001; 154: 147-160Crossref PubMed Scopus (412) Google Scholar). How these proteins promote cell migration is incompletely understood. By inactivating myosin light chain phosphatase, Rho kinase may complement the function of myosin light chain kinase in promoting myosin regulatory light chain phosphorylation and actomyosin contractility (28.Somlyo A.V. Bradshaw D. Ramos S. Murphy C. Myers C.E. Somlyo A.P. Biochem. Biophys. Res. Commun. 2000; 269: 652-659Crossref PubMed Scopus (213) Google Scholar, 30.Kimura K. 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 (2444) Google Scholar). Rho kinase also activates a downstream pathway that culminates in the inactivation of cofilin, an actin-depolymerizing agent (31.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 (1296) Google Scholar). Furthermore, Rho kinase may regulate the avidity of cellular integrins for the extracellular matrix, allowing the migrating cell to retract its tail (28.Somlyo A.V. Bradshaw D. Ramos S. Murphy C. Myers C.E. Somlyo A.P. Biochem. Biophys. Res. Commun. 2000; 269: 652-659Crossref PubMed Scopus (213) Google Scholar, 29.Worthylake R.A. Lemoine S. Watson J.M. Burridge K. J. Cell Biol. 2001; 154: 147-160Crossref PubMed Scopus (412) Google Scholar). We undertook these studies because of the apparent overlap in the downstream targets of the Rho-Rho kinase and Ras-ERK pathways and because of the accumulating evidence that antagonists of Rho kinase inhibit cancer progression. Although we began our studies by examining the activity of the Rho-Rho kinase pathway in uPA-treated cells, we also examined cells treated with epidermal growth factor (EGF) or transfected to express constitutively active MEK1. Our results indicate that the Rho-Rho kinase and Ras-ERK pathways function independently but cooperatively to stimulate MCF-7 and HT-1080 cell migration on serum-coated surfaces. Antagonists of the Rho-Rho kinase pathway entirely blocked uPA-, EGF-, and MEK1-promoted cell migration even though these antagonists independently activated ERK. When MCF-7 cell migration was stimulated by mechanisms that do not require ERK, Rho kinase also was not required. These results support a model in which activation and/or the constitutive activity of multiple independent signaling pathways may be essential to stimulate cell migration in response to uPA. Antagonizing any one of the pathways may be sufficient to eliminate the response completely. Two-chain uPA was kindly provided by Drs. Jack Henkin and Andrew Mazar (Abbott). uPA was treated with diisopropyl fluorophosphate (DFP) to generate DFP-uPA as previously described (10.Nguyen D.H. Hussaini I.M. Gonias S.L. J. Biol. Chem. 1998; 273: 8502-8507Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). DFP-uPA binds to uPAR with unchanged affinity, but lacks proteinase activity. Recombinant human EGF was purchased from R&D Systems, Inc. (Minneapolis, MN). The MEK inhibitor PD098059 was from Calbiochem. The Rho kinase inhibitor Y-27632 was from Mitsubishi Pharma Corp. (Tokyo, Japan). Expression constructs encoding constitutively active MEK1, hemagglutinin (HA)-tagged ERK1, and green fluorescent protein (GFP) were previously described (9.Nguyen D.H. Catling A.D. Webb D.J. Sankovic M. Walker L.A. Somlyo A.V. Weber M.J. Gonias S.L. J. Cell Biol. 1999; 146: 149-164Crossref PubMed Scopus (299) Google Scholar). An expression construct that encodes dominant-negative RhoA (RhoA-N19) was kindly provided by Dr. Robert Nakamoto (University of Virginia). A construct that encodes the Rho-binding domain of Rhotekin as a glutathione S-transferase fusion protein (GST-TRBD) in pGEX was provided by Dr. Martin Schwartz (Scripps Research Institute, La Jolla, CA) and expressed in BL21 cells. Purified GST-TRBD was also purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). The full-length cDNA encoding the human β3 integrin subunit was kindly provided by Dr. David Cheresh (Scripps Research Institute) and subcloned into pBK-CMV. Antibody that specifically detects phosphorylated ERK1 and ERK2 was from Calbiochem. Polyclonal antibody that recognizes total ERK1 and ERK2 was from Zymed Laboratories Inc. (South San Francisco, CA). RhoA-specific monoclonal antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-HA monoclonal antibody 12CA5 was from Babco. Horseradish peroxidase-conjugated antibodies against mouse IgG and rabbit IgG were from Amersham Biosciences. Lysophosphatidic acid (LPA), protease inhibitor mixture, sodium orthovanadate, dithiothreitol, and bovine serum albumin were from Sigma. Low-passage MCF-7 cells (passage 8) were kindly provided by Dr. Sally Parsons (University of Virginia) and cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone Laboratories), 100 units/ml penicillin, and 100 μg/ml streptomycin. HT-1080 cells (American Type Culture Collection, Manassas, VA) were cultured in minimum essential medium with 0.1 mm nonessential amino acids. Cells were passaged using enzyme-free dissociation buffer (Invitrogen) and maintained in culture for 48 h before experiments were performed. Migration of MCF-7 and HT-1080 cells was studied using 6.5-mm Transwell chambers with 8-μm pores (Costar Corp.) as previously described (10.Nguyen D.H. Hussaini I.M. Gonias S.L. J. Biol. Chem. 1998; 273: 8502-8507Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). The Transwell membranes were coated with 20% fetal bovine serum or type I collagen (25 μg/ml) (BD PharMingen) on the underside only. Both membrane surfaces were then blocked with 5 mg/ml bovine serum albumin for 2 h at 37 °C. Cells (105 cells/100 μl) in serum-free medium were pretreated first with PD098059 (50 μm) or Y-27632 (25 μm) for 15 min in suspension and then with DFP-uPA or EGF for an additional 15 min. The cells then were added to the upper chamber of each Transwell unit in the presence of the same agents. The lower chamber was supplemented with DFP-uPA or EGF when these agents were added to the top chamber. Fetal bovine serum (10%) was also added to the lower chamber in MCF-7 cell migration experiments. Migration was allowed to occur for 6 h at 37 °C. MCF-7 cell migration to the underside of the membrane was determined by counting cells stained with Diff-Quik C (Dade Diagnostics) (10.Nguyen D.H. Hussaini I.M. Gonias S.L. J. Biol. Chem. 1998; 273: 8502-8507Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). HT-1080 cell migration was determined by crystal violet staining as previously described (9.Nguyen D.H. Catling A.D. Webb D.J. Sankovic M. Walker L.A. Somlyo A.V. Weber M.J. Gonias S.L. J. Cell Biol. 1999; 146: 149-164Crossref PubMed Scopus (299) Google Scholar). In some experiments, MCF-7 cells were transiently transfected to express constitutively active MEK1, RhoA-N19, or the β3integrin subunit (2 μg of each construct) by incubation with Effectene (QIAGEN Inc.) for 24 h. The same cells were cotransfected to express GFP (0.5 μg). Transfection and cotransfection efficiencies were determined as previously described (9.Nguyen D.H. Catling A.D. Webb D.J. Sankovic M. Walker L.A. Somlyo A.V. Weber M.J. Gonias S.L. J. Cell Biol. 1999; 146: 149-164Crossref PubMed Scopus (299) Google Scholar). Importantly, cotransfection efficiencies were always ∼100%. Migration experiments were performed as described above, except that Biocoat cell culture inserts (BD PharMingen) were used instead of Transwell chambers. Cell migration was determined by counting green fluorescing cells. MCF-7 cells were cultured on six-well plates until 80–90% confluent and then serum-starved for 24 h. Where indicated, the cells were preincubated with PD098059 (50 μm) or Y-27632 (25 μm) for 15 min. DFP-uPA (10 nm) was then added for 2 min. Cell extracts were prepared in 10 mm HEPES, 150 mm NaCl, 2 mm EDTA, and 1% (v/v) Nonidet P-40 (pH 7.5) containing protease inhibitor mixture and sodium orthovanadate (1 mm). The protein concentration in each extract was determined by fluoraldehyde assay. Equal amounts of each extract were then subjected SDS-PAGE on 10% slabs, electrotransferred to polyvinylidene difluoride membranes, and probed for phosphorylated and total ERKs by immunoblot analysis. To study ERK phosphorylation in MCF-7 cells that were transiently transfected with constructs encoding constitutively active MEK1 (2 μg) and/or RhoA-N19 (2 μg), cells were cotransfected to express HA-tagged ERK1 (0.5 μg) using Effectene. Cells were maintained in serum-containing medium for 24 h to optimize transfection and expression and then serum-starved for 24 h. Treatments with PD098059 or Y-27632 were executed for 15 min. The cells were then extracted with ice-cold radioimmune precipitation assay buffer (20 mm sodium phosphate, 150 mm NaCl (pH 7.4), 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, and 1% (v/v) Nonidet P-40) containing protease inhibitor mixture and sodium orthovanadate (1 mm). The extracts were precleared with protein G-Sepharose and then incubated with antibody 12CA5 for 12 h at 4 °C, followed by protein G-Sepharose for 1 h. The pellets were washed four times and analyzed by SDS-PAGE and immunoblotting as described above. Affinity precipitation of active Rho was performed using the fusion protein GST-TRBD, which specifically recognizes the active GTP-bound form of Rho (GTP-Rho), as previously described (32.Ren X.D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1369) Google Scholar). MCF-7 cells were cultured in 15-cm2 plates until 80–90% confluent and then serum-starved for 24 h. Some cells were pretreated with PD098059 (50 μm) for 15 min. Cultures were then exposed to DFP-uPA (10 nm) or LPA (10 μm) for the indicated times; washed with ice-cold phosphate-buffered saline; and extracted in 1% (v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 50 mm Tris-HCl, 0.5 m NaCl, and 10 mm MgCl2 (pH 7.2) supplemented with protease inhibitor mixture and 1 mm sodium orthovanadate. The extracts were incubated with 30 μg of GST-TRBD coupled to glutathione-Sepharose for 45 min at 4 °C. The glutathione-Sepharose was washed four times and then treated with SDS sample buffer to dissociate GST-TRBD and GTP-Rho. Immunoblot analysis was performed to detect active RhoA. Samples of each cell extract were also subjected to immunoblot analysis prior to incubation with GST-TRBD to quantitate total RhoA. We previously demonstrated an essential role for the Ras-ERK pathway in uPA-stimulated MCF-7 breast cancer cell and HT-1080 fibrosarcoma cell migration (10.Nguyen D.H. Hussaini I.M. Gonias S.L. J. Biol. Chem. 1998; 273: 8502-8507Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Synthetic inhibitors and dominant-negative mutants of MEK1 and Ha-Ras, which prevent ERK activation, block cell migration in response to uPA (9.Nguyen D.H. Catling A.D. Webb D.J. Sankovic M. Walker L.A. Somlyo A.V. Weber M.J. Gonias S.L. J. Cell Biol. 1999; 146: 149-164Crossref PubMed Scopus (299) Google Scholar). Furthermore, dominant-negative mutants of signaling proteins that frequently couple signaling receptors to Ha-Ras, including focal adhesion kinase, c-Src, and Shc, also block cell migration in response to uPA (7.Nguyen D.H. Webb D.J. Catling A.D. Song Q. Dhakephalkar A. Weber M.J. Ravichandran K.S. Gonias S.L. J. Biol. Chem. 2000; 275: 19382-19388Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). The same agents have no effect on cell migration in the absence of uPA. Taken together, these results could be interpreted to indicate an exclusive role for the Ras-ERK pathway in uPA-stimulated cell migration. In cell migration, the Rho-Rho kinase and Ras-ERK pathways may affect similar processes such as tail retraction (18.Klemke R.L. Cai S. Giannini A.L. Gallagher P.J. de Lanerolle P. Cheresh D.A. J. Cell Biol. 1997; 137: 481-492Crossref PubMed Scopus (1103) Google Scholar, 28.Somlyo A.V. Bradshaw D. Ramos S. Murphy C. Myers C.E. Somlyo A.P. Biochem. Biophys. Res. Commun. 2000; 269: 652-659Crossref PubMed Scopus (213) Google Scholar, 29.Worthylake R.A. Lemoine S. Watson J.M. Burridge K. J. Cell Biol. 2001; 154: 147-160Crossref PubMed Scopus (412) Google Scholar). To assess the function of the Rho-Rho kinase pathway in uPA-stimulated cell migration, we studied MCF-7 and HT-1080 cells in Transwell membranes that were precoated with serum for 2 h. Under these conditions, the membranes become coated primarily with vitronectin, which serves as the major cell attachment and spreading factor (33.Hayman E.G. Pierschbacher M.D. Ohgren Y. Ruoslahti E. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4003-4007Crossref PubMed Scopus (326) Google Scholar). Our previous work showed that identical results are obtained when membranes are coated with serum or purified vitronectin (7.Nguyen D.H. Webb D.J. Catling A.D. Song Q. Dhakephalkar A. Weber M.J. Ravichandran K.S. Gonias S.L. J. Biol. Chem. 2000; 275: 19382-19388Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 9.Nguyen D.H. Catling A.D. Webb D.J. Sankovic M. Walker L.A. Somlyo A.V. Weber M.J. Gonias S.L. J. Cell Biol. 1999; 146: 149-164Crossref PubMed Scopus (299) Google Scholar, 10.Nguyen D.H. Hussaini I.M. Gonias S.L. J. Biol. Chem. 1998; 273: 8502-8507Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Cells were added to the upper chamber and allowed to migrate for 6 h. Fig. 1A shows that DFP-uPA promoted MCF-7 cell migration, as previously reported (10.Nguyen D.H. Hussaini I.M. Gonias S.L. J. Biol. Chem. 1998; 273: 8502-8507Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). When cells were pretreated with the MEK inhibitor PD098059 (50 μm), the response to uPA was entirely blocked. PD098059 had no effect on cell migration in the absence of uPA, as anticipated because, in unstimulated MCF-7 cells, the level of activated ERK is extremely low (10.Nguyen D.H. Hussaini I.M. Gonias S.L. J. Biol. Chem. 1998; 273: 8502-8507Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). We next examined the Rho kinase inhibitor Y-27632. As shown in Fig. 1A, Y-27632 (25 μm), like PD098059, completely blocked uPA-stimulated MCF-7 cell migration and had no effect on cell migration in the absence of uPA. Inhibition of uPA-stimulated MCF-7 cell migration was Y-27632 concentration-dependent when the inhibitor was added at final concentrations of 2–25 μm(data not shown). When uPA-stimulated cell migration was maximally inhibited by Y-27632, simultaneous addition of PD098059 had no further effect. In control experiments, performed as previously described (10.Nguyen D.H. Hussaini I.M. Gonias S.L. J. Biol. Chem. 1998; 273: 8502-8507Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar), neither Y-27632 nor PD098059 inhibited MCF-7 cell adhesion to vitronectin (data not shown). Fig. 1B shows the results of cell migration experiments performed with HT-1080 cells. uPA stimulated HT-1080 cell migration, and PD098059 (50 μm) completely blocked this response, as anticipated (9.Nguyen D.H. Catling A.D. Webb D.J. Sankovic M. Walker L.A. Somlyo A.V. Weber M.J. Gonias S.L. J. Cell Biol. 1999; 146: 149-164Crossref PubMed Scopus (299) Google Scholar). PD098059 had no effect on HT-1080 cell migration in the absence of uPA. Similarly, Y-27632 (25 μm) blocked the effects of uPA on HT-1080 cell migration and had no effect on cell migration in the absence of uPA. When HT-1080 cells were treated simultaneously with PD098059 and Y-27632 at concentrations that were sufficient to independently block uPA-stimulated cell migration, the combination of drugs was no more effective than either agent added independently. We next sought to determine whether Rho kinase is important when MCF-7 cell migration is stimulated by agents other than uPA. EGF activates multiple signaling pathways, including the Ras-ERK pathway, by binding to its receptor tyrosine kinase (34.van der Geer P. Hunter T. Lindberg R.A. Annu. Rev. Cell Biol. 1994; 10: 251-337Crossref PubMed Scopus (1252) Google Scholar). As shown in Fig. 2A, EGF increased MCF-7 cell migration on serum-coated membranes by 3.1-fold. The response to EGF was completely blocked by PD098059 (50 μm), indicating an essential role for MEK and its downstream effector ERK. Y-27632 also completely blocked EGF-stimulated cell migration. The combination of PD098059 and Y-27632 was no more effective than either agent added separately. EGF promotes cell migration by activating interrelated downstream signaling pathways (34.van der Geer P. Hunter T. Lindberg R.A. Annu. Rev. Cell Biol. 1994; 10: 251-337Crossref PubMed Scopus (1252) Google Scholar, 35.Hughes P.E. Renshaw M.W. Pfaff M. Forsyth J. Keivens V.M. Schwartz M.A. Ginsberg M.H. Cell. 1997; 88: 521-530Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). To activate ERK specifically without simultaneously activating other signaling pathways, we transfected MCF-7 cells to express constitutively active MEK1. The cells were cotransfected to express GFP. Cotransfection efficiencies were essentially 100%, allowing us to identify active MEK1-expressing cells, after migration through membranes, by fluorescence microscopy (7.Nguyen D.H. Webb D.J. Catling A.D. Song Q. Dhakephalkar A. Weber M.J. Ravichandran K.S. Gonias S.L. J. Biol. Chem" @default.
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• W2044857500 title "Cooperativity between the Ras-ERK and Rho-Rho Kinase Pathways in Urokinase-type Plasminogen Activator-stimulated Cell Migration" @default.
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