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Epidermal Growth Factor Receptor-dependent and -independent Cell-signaling Pathways Originating from the Urokinase Receptor

2003; Elsevier BV; Volume: 278; Issue: 3 Linguagem: Inglês

10.1074/jbc.m210877200

ISSN

1083-351X

Autores

Minji Jo, Keena S. Thomas, Denise M. O'Donnell, Steven L. Gonias,

Tópico(s)

Cell Adhesion Molecules Research

Resumo

Urokinase-type plasminogen activator (uPA) and vitronectin activate cell-signaling pathways by binding to the uPA receptor (uPAR). Because uPAR is glycosylphosphatidylinositol-anchored, the signaling receptor is most likely a uPAR-containing multiprotein complex. This complex may be heterogeneous within a single cell and among different cell types. The goal of this study was to elucidate the role of the EGF receptor (EGFR) as a component of the uPAR-signaling machinery. uPA activated extracellular signal-regulated kinase (ERK) in COS-7 cells and in COS-7 cells that overexpress uPAR, and this response was blocked by the EGFR inhibitor, tyrphostin AG1478, implicating the EGFR in the pathway that links uPAR to ERK. By contrast, Rac1 activation, which occurred as a result of uPAR overexpression, was EGFR-independent. COS-7 cell migration was stimulated, in an additive manner, by uPAR-dependent pathways leading to ERK and Rac1. AG1478 inhibited only the ERK-dependent component of the response. CHO-K1 cells do not express EGFR; however, these cells demonstrated ERK activation in response to uPA, indicating the presence of an EGFR-independent alternative pathway. As anticipated, this response was insensitive to AG1478. When CHO-K1 cells were transfected to express EGFR or a kinase-inactive mutant of EGFR, ERK activation in response to uPA was unchanged; however, the EGFR-expressing cells acquired sensitivity to AG1478. We conclude that the EGFR may function as a transducer of the signal from uPAR to ERK, but not Rac1. In the absence of EGFR, an alternative pathway links uPAR to ERK; however, this pathway is apparently silenced by EGFR expression. Urokinase-type plasminogen activator (uPA) and vitronectin activate cell-signaling pathways by binding to the uPA receptor (uPAR). Because uPAR is glycosylphosphatidylinositol-anchored, the signaling receptor is most likely a uPAR-containing multiprotein complex. This complex may be heterogeneous within a single cell and among different cell types. The goal of this study was to elucidate the role of the EGF receptor (EGFR) as a component of the uPAR-signaling machinery. uPA activated extracellular signal-regulated kinase (ERK) in COS-7 cells and in COS-7 cells that overexpress uPAR, and this response was blocked by the EGFR inhibitor, tyrphostin AG1478, implicating the EGFR in the pathway that links uPAR to ERK. By contrast, Rac1 activation, which occurred as a result of uPAR overexpression, was EGFR-independent. COS-7 cell migration was stimulated, in an additive manner, by uPAR-dependent pathways leading to ERK and Rac1. AG1478 inhibited only the ERK-dependent component of the response. CHO-K1 cells do not express EGFR; however, these cells demonstrated ERK activation in response to uPA, indicating the presence of an EGFR-independent alternative pathway. As anticipated, this response was insensitive to AG1478. When CHO-K1 cells were transfected to express EGFR or a kinase-inactive mutant of EGFR, ERK activation in response to uPA was unchanged; however, the EGFR-expressing cells acquired sensitivity to AG1478. We conclude that the EGFR may function as a transducer of the signal from uPAR to ERK, but not Rac1. In the absence of EGFR, an alternative pathway links uPAR to ERK; however, this pathway is apparently silenced by EGFR expression. urokinase-type plasminogen activator urokinase-type plasminogen activator receptor extracellular signal-regulated kinase epidermal growth factor receptor epidermal growth factor diisopropyl phospho green fluorescent protein fetal bovine serum uPAR-overexpressing COS-7 cells kinase-inactive bovine serum albumin p21-activated kinase Binding of the serine proteinase, urokinase-type plasminogen activator (uPA),1 to its cell surface receptor, uPAR, promotes plasminogen activation at the cell surface and may lead to the activation of metalloproteinases (1Andreasen P.A. Kjoller L. Christensen L. Duffy M.J. Int. J. Cancer. 1997; 72: 1-22Crossref PubMed Scopus (1449) Google Scholar). 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Adamski J. Zelent A. Ossowski L. EMBO J. 1994; 13: 3983-3991Crossref PubMed Scopus (174) Google Scholar, 11Shapiro R.L. Duquette J.G. Roses D.F. Nunes I. Harris M.N. Kamino H. Wilson E.L. Rifkin D.B. Cancer Res. 1996; 56: 3597-3604PubMed Google Scholar). In addition to uPA, uPAR binds directly to the extracellular matrix protein, vitronectin (12Wei Y. Waltz D.A. Rao N. Drummond R.J. Rosenberg S. Chapman H.A. J. Biol. Chem. 1994; 269: 32380-32388Abstract Full Text PDF PubMed Google Scholar, 13Kanse S.M. Kost C. Wilhelm O.G. Andreasen P.A. Preissner K.T. Exp. Cell Res. 1996; 224: 344-353Crossref PubMed Scopus (232) Google Scholar), and may thereby function as an adhesion receptor. The binding sites for uPA and vitronectin, in the structure of uPAR, are distinct but interactive because uPA-binding increases the affinity of uPAR for vitronectin (14Waltz D.A. Chapman H.A. J. Biol. Chem. 1994; 269: 14746-14750Abstract Full Text PDF PubMed Google Scholar, 15Wei Y. Lukashev M. Simon D.I. Bodary S.C. Rosenberg S. Doyle M.V. Chapman H.A. Science. 1996; 273: 1551-1555Crossref PubMed Scopus (698) Google Scholar). When uPA or vitronectin bind to uPAR, cell-signaling responses are elicited. Vitronectin-binding activates a pathway that includes the small GTPase, Rac1, and may drive new actin polymerization in the migrating cell (16Kjoller L. Hall A. J. Cell Biol. 2001; 152: 1145-1157Crossref PubMed Scopus (174) Google Scholar). uPA-binding activates diverse signaling pathways, and the nature of the response may be cell type-specific (17Ossowski L. Aguirre-Ghiso J.A. Curr. Opin. Cell Biol. 2000; 12: 613-620Crossref PubMed Scopus (359) Google Scholar, 18Kjoller L. Biol. Chem. 2002; 383: 5-19Crossref PubMed Scopus (102) Google Scholar). In multiple cells, binding of uPA to uPAR activates the Ras-extracellular signal-regulated kinase (ERK) pathway (19Nguyen 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, 20Nguyen 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, 21Tang 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, 22Aguirre Ghiso J.A. Kovalski K. Ossowski L. J. Cell Biol. 1999; 147: 89-104Crossref PubMed Scopus (467) Google Scholar). Because myosin light chain kinase is activated downstream of ERK, this signaling pathway also may stimulate cell migration (20Nguyen 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). uPAR is linked to the plasma membrane only by a glycosylphosphatidylinositol anchor and thus lacks transmembrane and intracytoplasmic domains (23Ploug 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). For this reason, it is generally assumed that the complete uPAR-signaling receptor is a multiprotein complex. In support of this hypothesis, soluble uPAR binds to the cell surface and elicits many of the same responses as uPA (24Fazioli F. Resnati M. Sidenius N. Higashimoto Y. Appella E. Blasi F. EMBO J. 1997; 16: 7279-7286Crossref PubMed Scopus (230) Google Scholar, 25Degryse B. Resnati M. Rabbani S.A. Villa A. Fazioli F. Blasi F. Blood. 1999; 94: 649-662Crossref PubMed Google Scholar, 26Nguyen 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 true function of uPA may be to alter the conformation of uPAR, promoting interactions with other proteins in the plasma membrane that are required for cell-signaling (27Blasi F. Thromb. Haemost. 1999; 82: 298-304Crossref PubMed Scopus (171) Google Scholar). A question of considerable importance concerns the nature of the uPAR-containing multiprotein complex involved in cell-signaling. uPAR associates with β1, β2, β3, and β5 integrins (15Wei Y. Lukashev M. Simon D.I. Bodary S.C. Rosenberg S. Doyle M.V. Chapman H.A. Science. 1996; 273: 1551-1555Crossref PubMed Scopus (698) Google Scholar, 28Wei Y. Eble J.A. Wang Z. Kreidberg J.A. Chapman H.A. Mol. Biol. Cell. 2001; 12: 2975-2986Crossref PubMed Scopus (223) Google Scholar, 29Xue W. Mizukami I. Todd 3rd, R.F. Petty H.R. Cancer Res. 1997; 57: 1682-1689PubMed Google Scholar, 30Kindzelskii A.L. Eszes M.M. Todd 3rd, R.F. Petty H.R. Biophys. 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Liu Q. Wilkins J.A. Chapman H.A. J. Cell Biol. 1999; 144: 1285-1294Crossref PubMed Scopus (369) Google Scholar). The transmembrane protein, gp130, associates with uPAR and serves as a critical adaptor protein leading to activation of the JAK/STAT pathway (34Koshelnick Y. Ehart M. Hufnagl P. Heinrich P.C. Binder B.R. J. Biol. Chem. 1997; 272: 28563-28567Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). The G protein-coupled receptor, FPR-like receptor-1/lipoxin A4 receptor (FPRL1/LXA4R), binds soluble uPAR, mediates uPAR-initiated chemotaxis in monocytes, and is necessary for signaling to the tyrosine kinase, Hck (35Resnati M. Pallavicini I. Wang J.M. Oppenheim J. Serhan C.N. Romano M. Blasi F. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1359-1364Crossref PubMed Scopus (324) Google Scholar). Finally, Liu et al. (36Liu D. Aguirre Ghiso J. Estrada Y. Ossowski L. Cancer Cell. 2002; 1: 445-457Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar) identified a complex that includes uPAR, α5β1, and the epidermal growth factor receptor (EGFR), mainly in cells that express high levels of uPAR. After uPA stimulation, focal adhesion kinase operated downstream of α5β1 to activate the EGFR, and this was necessary for signaling to ERK. Specific antagonists of EGFR completely blocked ERK phosphorylation. Thus, it was proposed that EGFR serves as a critical adaptor protein in the pathway that links uPAR to ERK. The EGFR also may be transactivated by G-protein coupled receptors and receptor tyrosine kinases such as the platelet-derived growth factor β receptor (37Prenzel N. Zwick E. Daub H. Leserer M. Abraham R. Wallasch C. Ullrich A. Nature. 1999; 402: 884-888Crossref PubMed Scopus (1501) Google Scholar, 38Prenzel N. Zwick E. Leserer M. Ullrich A. Breast Cancer Res. 2000; 2: 184-190Crossref PubMed Scopus (127) Google Scholar). The diversity of adaptor proteins, which associate with uPAR and promote uPAR signaling, suggests that the uPAR-multiprotein signaling complex may be large and heterogeneous. Components of the uPAR signaling complex may be affected by multiple properties of the cell, including membrane protein expression. We are particularly interested in signaling pathways leading to activation of Rac1 and ERK, because these pathways play pivotal roles in cell migration. In this study, we demonstrate that the EGFR is indeed essential in the pathway that links uPAR to ERK in cells that express EGFR. However, in cells that lack EGFR, alternative EGFR-independent pathways are operational, and uPA is still able to activate ERK. uPAR-dependent Rac1 activation is EGR-independent. Two-chain uPA was kindly provided by Drs. Jack Henkin and Andrew Mazar, previously of Abbott Laboratories (Abbott Park, IL). uPA was treated with diisopropyl fluorophosphate to generate DIP-uPA, as previously described (19Nguyen 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). DIP-uPA binds to uPAR with unchanged affinity but lacks proteinase activity. Recombinant human EGF was purchased from R&D Systems, Inc. (Minneapolis, MN). The mitogen-activated protein kinase kinase inhibitor, PD098059, was from Calbiochem (San Diego, CA). Expression constructs encoding human uPAR and green fluorescent protein (GFP) were previously described (20Nguyen 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). The expression construct, which encodes dominant-negative Rac1 (N17Rac1), was kindly provided by Dr. Robert Nakamoto (University of Virginia). Constructs encoding wild-type full-length EGFR and kinase-inactive EGFR (KI-EGFR) were kindly provided by Dr. Sarah Parsons (University of Virginia). In KI-EGFR, mutation at residue 721 (Lys→Ala) abolishes kinase activity (39Chen W.S. Lazar C.S. Poenie M. Tsien R.Y. Gill G.N. Rosenfeld M.G. Nature. 1987; 328: 820-823Crossref PubMed Scopus (413) Google Scholar, 40Honegger A.M. Dull T.J. Felder S. Van Obberghen E. Bellot F. Szapary D. Schmidt A. Ullrich A. Schlessinger J. Cell. 1987; 51: 199-209Abstract Full Text PDF PubMed Scopus (312) Google Scholar). Rac/Cdc42 assay reagent (PAK-PBD1), which includes residues 67–150 of p21-activated kinase (PAK-1) fused to glutathione-S-transferase and coupled to glutathione-agarose was from Upstate Biotechnology (Lake Placid, NY). Antibody that specifically detects phosphorylated ERK1 and ERK2 was from Cell Signaling Technology (Beverly, MA). Polyclonal antibody that recognizes total ERK1 and ERK2 was from Zymed Laboratories Inc.(San Francisco, CA). Rac1-specific monoclonal antibody was from BD Biosciences. Polyclonal anti-human uPAR antibody was from American Diagnostica. Horseradish peroxidase-conjugated antibodies specific for mouse IgG and rabbit IgG were from Amersham Biosciences. Tyrphostin AG1478, protease inhibitor mixture, sodium orthovanadate, dithiothreitol, G418, and bovine serum albumin were from Sigma. COS-7 cells (ATCC) were maintained in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Hyclone), penicillin (100 units/ml), and streptomycin (100 μg/ml). Chinese hamster ovary cells (CHO-K1) (ATCC) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% FBS, penicillin (100 units/ml), streptomycin (100 μg/ml), and nonessential amino acids (0.1 mm). Cells were passaged using Trypsin-EDTA (Invitrogen) and maintained in culture for 48 h before performing experiments. COS-7 cells were transfected to overexpress human uPAR by incubation for 48 h with 2 μg of the uPAR expression construct in the presence of EffecteneTM (Qiagen). uPAR expression was demonstrated by immunoblot analysis. An equivalent protocol was used to transfect CHO-K1 cells to express wild-type EGFR or KI-EGFR. Cultures were selected in G418 (1 mg/ml) for 30 days and then maintained in DMEM supplemented with 10% FBS and 0.5 mg/ml G418. Migration of COS-7 and uPAR-overexpressing COS-7 cells (COS-7/uPAR) was studied using 6.5-mm Transwell chambers with 8-μm pores (Costar), as previously described (19Nguyen 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 precoated with 20% FBS on the underside only. Both membrane surfaces were then blocked with 5 mg/ml bovine serum albumin for 2 h at 37 °C. Cells (105in 100 μl) in serum-free medium were pretreated with tyrphostin AG1478 (50 nm) or PD098059 (50 μm) for 15 min in suspension and then with DIP-uPA or EGF for an additional 15 min. The cells were then added to the upper chamber of each Transwell unit in the presence of the same agents. The lower chamber was supplemented with DIP-uPA or EGF when these agents were added to the top chamber. Migration was allowed to occur for 6 h at 37 °C. Cell migration was determined by crystal violet staining, as previously described (20Nguyen 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, COS-7 and COS-7/uPAR cells were transiently co-transfected with the constructs encoding N17Rac1 (2 μg) and with pEGFP (0.5 μg), which encodes GFP, by incubation with EffecteneTM (Qiagen) for 24 h. Co-transfection efficiencies were essentially 100%, when determined as previously described (20Nguyen 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). Migration experiments were performed using Biocoat cell culture inserts (BD Biosciences) instead of Transwell chambers. The membrane-coating method and protocol for pretreating cells was unchanged. Cell migration was determined by counting green-fluorescing cells. COS-7 cells, COS-7/uPAR cells, CHO-K1 cells, and EGFR-overexpressing CHO-K1 cells were cultured on 60-mm plates until 80–90% confluent and then were serum-starved for 24 h. When indicated, the cells were preincubated with tyrphostin AG1478 (50 nm) for 2 h. DIP-uPA (10 nm) was then added for 10 min. Cell extracts were prepared in 10 mm HEPES, 150 mm NaCl, 2 mm EDTA, 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 bicinchoninic acid assay. Equal amounts of each extract were subjected to SDS-PAGE on 10% slabs, electrotransferred to polyvinylidene diflouride membranes, and probed with specific antibodies for phosphorylated and total ERK. Affinity precipitation of active Rac1 was performed using the fusion protein, PAK1-PBD, which specifically recognizes the active GTP-bound forms of Rac1 and Cdc42, as previously described (16Kjoller L. Hall A. J. Cell Biol. 2001; 152: 1145-1157Crossref PubMed Scopus (174) Google Scholar, 41Benard V. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1999; 274: 13198-13204Abstract Full Text Full Text PDF PubMed Scopus (672) Google Scholar). COS-7 and COS-7/uPAR cells (2 × 105) were cultured in 10-cm plates for 18 h. Some cultures were pretreated with tyrphostin AG1478 (50 nm) for 2 h. Cultures then were 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, 0.5m NaCl, 10 mm MgCl2, pH 7.2, supplemented with protease inhibitor mixture and 1 mmsodium orthovanadate. The extracts were incubated with 15 μg of PAK1-PBD 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 the PAK1-PBD and associated proteins. Immunoblot analysis was performed to detect active Rac1. Samples of each cell extract were also subjected to immunoblot analysis before incubation with PAK1-PBD to determine total Rac1. In this study, COS-7 cells, which express EGFR (42Daub H. Wallasch C. Lankenau A. Herrlich A. Ullrich A. EMBO J. 1997; 16: 7032-7044Crossref PubMed Scopus (588) Google Scholar), were transfected to overexpress uPAR. As shown in Fig. 1 A, low levels of uPAR were detected in the parental cells; however, the transfected cells (COS-7/uPAR) expressed high levels of uPAR. When treated with EGF (10 ng/ml), COS-7 and COS-7/uPAR cells demonstrated significant ERK activation, as determined by immunoblot analysis (Fig. 1 B), confirming that these cells express EGFR. Both cell types also responded to DIP-uPA (10 nm) and demonstrated ERK activation within 10 min. The major difference observed between the COS-7 and COS-7/uPAR cells was an increase in the basal level of activated ERK (in the absence of exogenously added agents) in the COS-7/uPAR cells. This may reflect autocrine uPAR activation by endogenously produced uPA, as has been previously described (43Ma Z. Webb D.J., Jo, M. Gonias S.L. J. Cell Sci. 2001; 114: 3387-3396Crossref PubMed Google Scholar). The ability of uPA to activate ERK in parental COS-7 cells was not surprising, despite the low level of uPAR, because uPA activates ERK in MCF-7 cells, which express only 3,000–4,000 copies of cell-surface uPAR/cell (19Nguyen 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). To determine the role of the EGFR in uPA-stimulated ERK activation, we pretreated COS-7 and COS-7/uPAR cells with the EGFR-specific inhibitor, tyrphostin AG1478 (50 nm). This drug completely blocked ERK activation in response to DIP-uPA in both cell types (Fig. 1 C). A slight decrease in the basal level of activated ERK was also observed. These results suggest that the EGFR plays an essential role in the pathway by which uPA activates ERK in COS-7 cells and that the level of uPAR expression is not critical in determining whether the EGFR participates in this signaling event. Kjoller et al. (16Kjoller L. Hall A. J. Cell Biol. 2001; 152: 1145-1157Crossref PubMed Scopus (174) Google Scholar) demonstrated that uPAR overexpression is associated with an increase in Rac1 activation and increased cell migration. This response requires vitronectin binding to uPAR and not uPA. Because of the role of the EGFR in uPA-induced ERK activation in COS-7 cells, we performed experiments to determine whether the EGFR is necessary in the pathway that links uPAR to Rac1. As shown in Fig. 2, GTP-bound Rac1 was increased in the uPAR-overexpressing COS-7 cells (p < 0.05, n = 3), confirming the results of Kjolleret al. (16Kjoller L. Hall A. J. Cell Biol. 2001; 152: 1145-1157Crossref PubMed Scopus (174) Google Scholar). Tyrphostin AG1478 had no effect on the level of GTP-Rac1 in COS-7 or COS-7/uPAR cells. These results suggest that the adaptor protein interactions, which facilitate uPAR signaling either to ERK or Rac1, are distinct. uPAR overexpression was associated with a modest but significant increase (p < 0.01, n = 9) in COS-7 cell migration through serum-coated Transwell membranes (Fig. 3 A). In the absence of uPA, tyrphostin AG1478 had no effect on the migration rate of COS-7/uPAR cells. uPA further stimulated the migration of COS-7/uPAR cells (1.9-fold; p < 0.01, n = 5), and this response was blocked by tyrphostin AG1478. Tyrphostin AG1478 also blocked the effects of EGF on COS-7/uPAR cell migration, as anticipated. We hypothesized that uPAR overexpression promotes cell migration by its effects on Rac1 activation, which is independent of EGFR, and that uPA and EGF stimulate cell migration by their effects on ERK activation, which is EGFR-dependent. To test this hypothesis, we examined COS-7/uPAR cell migration after treatment with the mitogen-activated protein kinase kinase inhibitor, PD098053 (50 μm). As shown in Fig. 3 B, PD098059 blocked the effects of uPA on COS-7/uPAR cell migration without affecting the basal level of migration of these cells. To examine the role of Rac1, we transfected COS-7/uPAR cells to express dominant-negative Rac1 (N17Rac1). The cells were co-transfected with pEGFP, so that migration of N17Rac1-expressing cells could be determined by counting fluorescent cells. As shown in Fig. 3 C, N17Rac1 neutralized the increase in cell migration that was associated with uPAR overexpression in COS-7/uPAR cells, consistent with our model. uPA failed to stimulate migration of N17Rac1-expressing cells, suggesting that ERK activation may not promote cell migration when the constitutive activity of Rac1 is neutralized. A similar relationship was recently defined, regarding ERK and RhoA in uPA-stimulated MCF-7 cell migration (44Jo M. Thomas K.S. Somlyo A.V. Somlyo A.P. Gonias S.L. J. Biol. Chem. 2002; 277: 12479-12485Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). CHO-K1 cells express cell-surface uPAR but not EGFR (45Bringman T.S. Lindquist P.B. Derynck R. Cell. 1987; 48: 429-440Abstract Full Text PDF PubMed Scopus (179) Google Scholar). Thus, we chose this cell line to further probe the dependence on EGFR for uPAR-signaling to ERK. CHO-K1 cells were transfected to express wild-type EGFR or a kinase-inactive mutant of the EGFR. Expression was confirmed by immunoblot analysis (results not shown). As shown in Fig. 4 A, EGF did not stimulate ERK activation in the parental cells, as anticipated because of the lack of EGFR. Cells that were transfected to express KI-EGFR also failed to respond to EGF. By contrast, CHO-K1 cells that expressed wild-type EGFR demonstrated significant ERK phosphorylation in response to EGF. DIP-uPA stimulated ERK activation in the parental CHO-K1 cells, despite the lack of EGFR (Fig. 4 B). Thus, we hypothesized that these cells use an EGFR-independent pathway to couple uPAR to ERK. To rule out the possibility that trace levels of EGFR are responsible for the activation of ERK in uPA-treated CHO-K1 cells, we pretreated these cells with tyrphostin AG1478; however, the EGFR kinase antagonist was without effect. DIP-uPA promoted ERK activation equally well in cells that were transfected to express wild-type EGFR or KI-EGFR (Fig. 4 C). Tyrphostin AG1478 inhibited ERK activation in response to uPA only in the cells that express wild-type EGFR. Thus, whereas CHO-K1 cells apparently have an alternative EGFR-independent pathway to couple uPAR to ERK, this pathway is not operational when EGFR is expressed. uPA-binding to uPAR activates multiple cell-signaling proteins and systems, including focal adhesion kinase, ERK, protein kinase Cε, the Src-family tyronine kinase Hck, and the JAK/STAT pathway (17Ossowski L. Aguirre-Ghiso J.A. Curr. Opin. Cell Biol. 2000; 12: 613-620Crossref PubMed Scopus (359) Google Scholar, 18Kjoller L. Biol. Chem. 2002; 383: 5-19Crossref PubMed Scopus (102) Google Scholar). Vitronectin binding to uPAR activates Rac1 (16Kjoller L. Hall A. J. Cell Biol. 2001; 152: 1145-1157Crossref PubMed Scopus (174) Google Scholar). Various adaptor proteins that associate with uPAR in the plasma membrane have been implicated as transducers of uPAR-signaling across the plasma membrane. FPRL1/LXA4R has been implicated in signaling to Hck (35Resnati M. Pallavicini I. Wang J.M. Oppenheim J. Serhan C.N. Romano M. Blasi F. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1359-1364Crossref PubMed Scopus (324) Google Scholar), gp130 in signaling to JAK1 (34Koshelnick Y. Ehart M. Hufnagl P. Heinrich P.C. Binder B.R. J. Biol. Chem. 1997; 272: 28563-28567Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar), and EGFR in signaling to ERK (36Liu D. Aguirre Ghiso J. Estrada Y. Ossowski L. Cancer Cell. 2002; 1: 445-457Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar). An important question that arises is whether these adaptor proteins are truly compartmentalized in function, transmitting uPAR signals to some signaling pathways but not to others. In this case, how a cell responds to uPA may be determined by the composition of the uPAR-signaling receptor complex. Furthermore, the composition of the uPAR-signaling receptor complex may be dynamic and thus altered by changes in membrane protein expression or catabolism. In this study, our goal was to elucidate the function of the EGFR in uPAR-initiated cell signaling. Our results provide evidence for compartmentalized function of uPAR-associated adaptor proteins in cell signaling. In a single cell type (COS-7), the EGFR was essential for transmitting uPAR signals to ERK but not Rac1. The branch point remains to be determined. In epidermoid carcinoma cells, EGFR was activated downstream of uPAR-integrin complex and focal adhesion kinase; however, EGFR was recovered in complex with uPAR and α5β1, so these reactions probably occurred within the context of a single multicomponent complex (36Liu D. Aguirre Ghiso J. Estrada Y. Ossowski L. Cancer Cell. 2002; 1: 445-457Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar). Rac1 activation may occur downstream of tyrosine kinases and G protein-coupled receptors and require the activity of phosphatidylinositol 3-kinase or p130Cas (46Bollag G. McCormick F. Annu. Rev. Cell Biol. 1991; 7: 601-632Crossref PubMed Scopus (289) Google Scholar, 47Timokhina I. Kissel H. Stella G. Besmer P. EMBO J. 1998; 17: 6250-6262Crossref PubMed Scopus (234) Google Scholar, 48Ma A.D. Metjian A. Bagrodia S. Taylor S. Abrams C.S. Mol. Cell. Biol. 1998; 18: 4744-4751Crossref PubMed Google Scholar, 49Cho S.Y. Klemke R.L. J. Cell Biol. 2002; 156: 725-736Crossref PubMed Scopus (151) Google Scholar). Rac1 is also activated downstream of integrins (50Bialkowska K. Kulkarni S., Du, X. Goll D.E. Saido T.C. Fox J.E. J. Cell Biol. 2000; 151: 685-696Crossref PubMed Scopus (91) Google Scholar). Thus, the integrin-uPAR complex may signal directly to Rac1. In CHO-K1 cells, EGFR is not expressed (45Bringman T.S. Lindquist P.B. Derynck R. Cell. 1987; 48: 429-440Abstract Full Text PDF PubMed Scopus (179) Google Scholar). We demonstrated that EGF does not activate ERK in CHO-K1 cells, as anticipated because of the lack of receptor; however, uPA did activate ERK. Thus, an EGFR-independent pathway exists for transmitting signals from uPAR to ERK in CHO-K1 cells and possibly in other cells as well. In MCF-7 cells, focal adhesion kinase, c-Src and Shc function upstream of Ras in the pathway that couples uPAR to ERK (26Nguyen 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). These same factors may directly couple integrins to ERK, without the EGFR as a necessary intermediate (51Howe A.K. Aplin A.E. Juliano R.L. Curr. Opin. Genet. Dev. 2002; 12: 30-35Crossref PubMed Scopus (249) Google Scholar, 52Giancotti F.G. Ruoslahti E. Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3829) Google Scholar). Thus, uPAR and integrins may form an adequate signaling complex to activate the Ras-ERK pathway in the absence of EGFR. It is also possible that when EGFR is not expressed, an alternative adaptor protein associates with the uPAR multiprotein signaling-receptor complex. This alternative adaptor protein could be another member of the EGF receptor family or an unrelated membrane protein. In this regard, tyrphostin AG1478 is specific for EGFR (53Levitzki A. Gazit A. Science. 1995; 267: 1782-1788Crossref PubMed Scopus (1628) Google Scholar). An interesting and potentially important observation concerns the effects of EGFR expression on the response of CHO-K1 cells to uPA. An obvious change in the magnitude or kinetics of ERK activation in response to DIP-uPA was not observed; however, the response became sensitive to tyrphostin AG1478. This result suggests that when EGFR is expressed, it associates with uPAR in the plasma membrane and assumes a dominant role in the uPA-dependent signaling complex that activates ERK. The inability of uPA to activate ERK, in EGFR-expressing CHO-K1 cells, when these cells are pretreated with tyrphostin AG1478, suggests that EGFR silences the alternative pathway. One explanation for these data is that EGFR displaces an alternative adaptor protein from the uPAR signaling receptor complex; however, if this model is correct, we would expect KI-EGFR to inhibit uPAR-signaling to ERK, which was not observed. The inability of KI-EGFR to silence the alternative pathway may reflect conformational variation in the mutant receptor so that it does not associate with uPAR, or possibly insufficient expression in our experiments. From these studies, we propose that cell signaling initiated from the uPAR-containing multiprotein complex is compartmentalized. By this we mean that different proteins that are associated with uPAR may be responsible for triggering different signaling pathways. Furthermore, we hypothesize that the uPAR signaling-receptor complex is dynamic. When a protein like EGFR is expressed in cells, it may enter the complex and alter its properties. This model provides an explanation for the diversity of adaptor proteins shown by others to facilitate uPAR signaling. This model also provides a possible explanation for differences in signaling responses observed in different cell types. In processes such as breast cancer, the EGFR and uPAR are both important determinants of disease progression. Biochemical and functional interactions between these two receptors, described here and elsewhere (36Liu D. Aguirre Ghiso J. Estrada Y. Ossowski L. Cancer Cell. 2002; 1: 445-457Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar), raise the possibility that extensive cross-talk may occur in cancer cells. Understanding this cross-talk is an important goal for the future.

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