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• W2023007685 abstract "The multiple β-actin rich pseudopodial protrusions of the invasive variant of Moloney sarcoma virus (MSV)-transformed epithelial MDCK cells (MSV-MDCK-INV) are strongly labeled for phosphotyrosine. Increased tyrosine phosphorylation among a number of proteins was detected in MSV-MDCK-INV cells relative to untransformed and MSV-transformed MDCK cells, especially for the hepatocyte growth factor receptor (HGF-R), otherwise known as c-met proto-oncogene. Cell surface expression of HGF-R was similar in the three cell lines, indicating that HGF-R is constitutively phosphorylated in MSV-MDCK-INV cells. Both the tyrosine kinase inhibitor herbimycin A and the HGFα antibody abolished HGF-R phosphorylation, induced retraction of pseudopodial protrusions, and promoted the establishment of cell-cell contacts as well as the apparition of numerous stabilizing stress fibers in MSV-MDCK-INV cells. Furthermore, anti-HGFα antibody abolished cell motility among MSV-MDCK-INV cells. Conditioned medium from MSV-MDCK-INV cells induced MDCK cell scattering, indicating that HGF is secreted by MSV-MDCK-INV cells. HGF titration followed by a subsequent washout of the antibodies led to renewed pseudopodial protrusion and cellular movement. We therefore show that activation of the tyrosine kinase activity of HGF-R/Met via an autocrine HGF loop is directly responsible for pseudopodial protrusion, thereby explaining the motile and invasive potential of this model epithelium-derived tumor cell line. The multiple β-actin rich pseudopodial protrusions of the invasive variant of Moloney sarcoma virus (MSV)-transformed epithelial MDCK cells (MSV-MDCK-INV) are strongly labeled for phosphotyrosine. Increased tyrosine phosphorylation among a number of proteins was detected in MSV-MDCK-INV cells relative to untransformed and MSV-transformed MDCK cells, especially for the hepatocyte growth factor receptor (HGF-R), otherwise known as c-met proto-oncogene. Cell surface expression of HGF-R was similar in the three cell lines, indicating that HGF-R is constitutively phosphorylated in MSV-MDCK-INV cells. Both the tyrosine kinase inhibitor herbimycin A and the HGFα antibody abolished HGF-R phosphorylation, induced retraction of pseudopodial protrusions, and promoted the establishment of cell-cell contacts as well as the apparition of numerous stabilizing stress fibers in MSV-MDCK-INV cells. Furthermore, anti-HGFα antibody abolished cell motility among MSV-MDCK-INV cells. Conditioned medium from MSV-MDCK-INV cells induced MDCK cell scattering, indicating that HGF is secreted by MSV-MDCK-INV cells. HGF titration followed by a subsequent washout of the antibodies led to renewed pseudopodial protrusion and cellular movement. We therefore show that activation of the tyrosine kinase activity of HGF-R/Met via an autocrine HGF loop is directly responsible for pseudopodial protrusion, thereby explaining the motile and invasive potential of this model epithelium-derived tumor cell line. Cell motility is required for physiological processes of wound repair and organogenesis as well as for the pathologic process of tumor invasion; a critical element of cell motility and invasion is thede novo polarized protrusion of pseudopodia via localized actin polymerization (1Clark P. J. Cell Sci. 1994; 107: 1265-1275Google Scholar, 2Stossel T.P. Science. 1993; 260: 1086-1094Google Scholar, 3Nabi I.R. J. Cell Sci. 1999; 112: 1803-1811Google Scholar, 4Mitchison T.J. Cramer L.P. Cell. 1996; 84: 371-379Google Scholar, 5Lauffenburger D.A. Horwitz A.F. Cell. 1996; 84: 359-369Google Scholar). Pseudopodia formation is associated with cell motility in vitro and tumor cell motility in vivo, and pseudopodial protrusion of surrounding extracellular matrix has been well characterized as an essential element of tumor cell invasion (6Muller-Glauser W. Haemmerli G. Strauli P. Cell Biol. Int. Rep. 1985; 9: 447-461Google Scholar, 7Arencibia I. Suarez N.C. Wolf-Watz H. Sundqvist K.G. J. Immunol. 1997; 159: 1853-1859Google Scholar, 8Mohler J.L. Partin A.W. Isaacs W.B. Coffey D.S. J. Urol. 1987; 137: 544-547Google Scholar, 9Guirguis R. Margulies I. Taraboletti G. Schiffmann E. Liotta L. Nature. 1987; 329: 261-263Google Scholar, 10Yamamura S. Sadahira Y. Ruan F. Hakomori S. Igarashi Y. FEBS Lett. 1996; 382: 193-197Google Scholar, 11Russo R.G. Liotta L.A. Thorgeirsson U. Brundage R. Schiffmann E. J. Cell Biol. 1981; 91: 459-467Google Scholar, 12Otsubo Y. Kameyama Y. J. Oral Pathol. 1982; 11: 159-173Google Scholar, 13Mueller S.C. Chen W.T. J. Cell Sci. 1991; 99: 213-225Google Scholar, 14Kramer R.H. Bensch K.G. Wong J. Cancer Res. 1986; 46: 1980-1989Google Scholar, 15Ausprunk D.H. Folkman J. Microvasc. Res. 1977; 14: 53-65Google Scholar). Hepatocyte growth factor (HGF) 1The abbreviations used are: HGF, hepatocyte growth factor; HGF-R, HGF receptor; MSV, Moloney sarcoma virus; MDCK, Madin Darby canine kidney; MSV-MDCK, Moloney sarcoma virus transformed epithelial MDCK; MSV-MDCK-INV, invasive variant of Moloney sarcoma virus transformed epithelial MDCK cells; CM-INV, conditioned medium from MSV-MDCK-INV cells; FBS, fetal bovine serum; DMEM, Dulbecco's minimal essential medium; HRP, horseradish peroxidase. /scatter factor, secreted by cells of mesodermal and mesenchymal origin, was originally identified for its ability to disrupt epithelial cell-cell interactions and to trigger invasive growth (16Balkovetz D.F. Microsc. Res. Tech. 1998; 43: 456-463Google Scholar, 17Balkovetz D.F. Lipschutz J.H. Int. Rev. Cytol. 1999; 186: 225-261Google Scholar, 18Bardelli A. Pugliese L. Comoglio P.M. Biochim. Biophys. Acta. 1997; 1333: M41-M51Google Scholar, 19Birchmeier C. Birchmeier W. Annu. Rev. Cell Biol. 1993; 9: 511-540Google Scholar, 20Birchmeier W. Brinkmann V. Niemann C. Meiners S. DiCesare S. Naundorf H. Sachs M. CIBA Found. Symp. 1997; 212: 230-240Google Scholar, 21Stoker M. Gherardi E. Perryman M. Grey J. Nature. 1987; 327: 239-242Google Scholar, 22Takayama H. LaRochelle W.J. Sharp R. Otsuka T. Kriebel P. Anver M. Aaronson S.A. Merlino G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 701-706Google Scholar, 23Vande Woude G. Jeffers M. Cortner J. Alvord G. Tsarfaty I. Resau J. CIBA Found. Symp. 1997; 212: 119-130Google Scholar). HGF exhibits powerful mitogenic, motogenic, and morphogenic activities on epithelial and endothelial cells expressing the HGF receptor (HGF-R), also known as the met proto-oncogene (17Balkovetz D.F. Lipschutz J.H. Int. Rev. Cytol. 1999; 186: 225-261Google Scholar, 24Bottaro D.P. Rubin J.S. Faletto D.L. Chan A.M.-L. Kmiecik T.E. Vande Woude G.F. Aaronson S.A. Science. 1991; 251: 802-804Google Scholar), whose two end results are the modification of the actin cytoskeleton and the disruption of epithelial cell-cell adhesions (16Balkovetz D.F. Microsc. Res. Tech. 1998; 43: 456-463Google Scholar). HGF-R activation promotes receptor auto-phosphorylation on tyrosine residues and activation of downstream signaling events including the ras (25Ridley A.J. Comoglio P.M. Hall A. Mol. Cell. Biol. 1995; 15: 1110-1122Google Scholar), phosphatidylinositol 3-kinase (26Graziani A. Gramaglia D. Cantley L.C. Comoglio P.M. J. Biol. Chem. 1991; 266: 22087-22090Google Scholar, 27Rahimi N. Tremblay E. McAdam L. Park M. Schwall R. Elliott B. Cell Growth Differ. 1996; 7: 263-670Google Scholar) phospholipase C-γ (28Gual P. Giordano S. Williams T.A. Rocchi S. Van Obberghen E. Comoglio P.M. Oncogene. 2000; 19: 1509-1518Google Scholar), and mitogen-activated protein kinase (29Adachi T. Nakashima S. Saji S. Nakamura T. Nozawa Y. Hepatology. 1996; 23: 1244-1253Google Scholar) related pathways. Deregulated control of the invasive-growth phenotype by oncogenically activated Met confers invasive and metastatic properties to cancer cells (18Bardelli A. Pugliese L. Comoglio P.M. Biochim. Biophys. Acta. 1997; 1333: M41-M51Google Scholar, 22Takayama H. LaRochelle W.J. Sharp R. Otsuka T. Kriebel P. Anver M. Aaronson S.A. Merlino G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 701-706Google Scholar). Breast (30Tuck A.B.M.P. Sterns E.E. Boag A. Elliott B.E. Am. J. Pathol. 1996; 148: 225-232Google Scholar), ovarian (31Auersperg N. Maines-Bandiera S.L. Dyck H.G. J. Cell. Physiol. 1997; 173: 261-265Google Scholar), prostate (32Humphrey P.A. Zhu X. Zarnegar R. Swanson P.E. Ratliff T.L. Vollmer R.T. Day M.L. Am. J. Pathol. 1995; 147: 386-396Google Scholar), gastric (33Nakajima M. Sawada H. Yamada Y. Watanabe A. Tatsumi M. Yamashita J. Matsuda M. Sakaguchi T. Hirao T. Nakano H. Cancer. 1999; 85: 1894-1902Google Scholar), thyroid (34Scarpino S. Stoppacciaro A. Colarossi C. Cancellario F. Marzullo A. Marchesi M. Biffoni M. Comoglio P.M. Prat M. Ruco L.P. J. Pathol. 1999; 189: 570-575Google Scholar), hepatocellular (35Ueki T. Fujimoto J. Suzuki T. Yamamoto H. Okamoto E. Hepatology. 1997; 25: 619-623Google Scholar), and renal (36Schmidt L. Junker K. Weirich G. Glenn G. Choyke P. Lubensky I. Zhuang Z. Jeffers M. Vande Woude G. Neumann H. Walther M. Linehan W.M. Zbar B. Cancer Res. 1998; 58: 1719-1722Google Scholar) carcinomas as well as osteosarcoma (37Ferracini R. Di Renzo M.F. Scotlandi K. Baldini N. Oliviero M. Lollini P.-L. Cremona O. Campanacci M. Comoglio P.M. Oncogene. 1995; 10: 739-749Google Scholar, 38Scotlandi K. Baldini N. Oliviero M. Flavia Di Renzo M. Martano M. Serra M. Manara M.C. Comoglio P.M. Ferracini R. Am. J. Pathol. 1996; 149: 1209-1219Google Scholar) and myeloma (39Borset M. Lien E. Espevik T. Helseth E. Waage A. Sundan A. J. Biol. Chem. 1996; 271: 24655-24661Google Scholar) are indeed associated with HGF-R overexpression or increased HGF-R tyrosine phosphorylation. Notably, protein overexpression was found to be associated with amplification of the met gene in only a few primary carcinomas, but in a significant proportion of the metastases examined (18Bardelli A. Pugliese L. Comoglio P.M. Biochim. Biophys. Acta. 1997; 1333: M41-M51Google Scholar, 33Nakajima M. Sawada H. Yamada Y. Watanabe A. Tatsumi M. Yamashita J. Matsuda M. Sakaguchi T. Hirao T. Nakano H. Cancer. 1999; 85: 1894-1902Google Scholar, 38Scotlandi K. Baldini N. Oliviero M. Flavia Di Renzo M. Martano M. Serra M. Manara M.C. Comoglio P.M. Ferracini R. Am. J. Pathol. 1996; 149: 1209-1219Google Scholar). Targeting of a constitutively active Tpr-Met to the plasma membrane via a c-Src myristoylation signal induces enhanced cellular transformation and the formation of cellular protrusions in MDCK cells (40Kamikura D.M. Khoury H. Maroun C. Naujokas M.A. Park M. Mol. Cell. Biol. 2000; 20: 3482-3496Google Scholar). However, whether the acquisition of a motile and metastatic phenotype by tumor cells due to HGF-R activation is indirectly due to disruption of epithelial cell-cell contacts and induction of an epithelial-mesenchymal transformation or whether autocrine HGF-R activation specifically induces cell motility and, more particularly, pseudopodial protrusion, has yet to be directly demonstrated. To determine the molecular basis for the acquisition of motile and invasive properties following transformation of polarized epithelial cells, we have established a model system based on Moloney sarcoma virus (MSV) transformants of the polarized epithelial MDCK cell line that exhibit decreased expression of E-cadherin (41Behrens J. Mareel M.M. Van Roy R.M. Birchmeier W. J. Cell Biol. 1989; 108: 2435-2447Google Scholar, 42Simard D. Nabi I.R. Biochem. Biophys. Res. Commun. 1996; 219: 122-127Google Scholar). An invasive MSV-MDCK cell variant (MSV-MDCK-INV), selected for its capacity to pass through a Matrigel® coated filter unit, exhibits increased expression of β-actin, the loss of actin stress fibers, and the expression of multiple β-actin-rich pseudopodia (43Le P.U. Nguyen T.N. Drolet-Savoie P. Leclerc N. Nabi I.R. Cancer Res. 1998; 58: 1631-1635Google Scholar). Subsequent studies have identified necessary roles for the Na-H exchanger NHE1 (44Lagana A. Vadnais J. Le P.U. Nguyen T.N. Laprade R. Nabi I.R. Noël J. J. Cell Sci. 2000; 113: 3649-3662Google Scholar) and for glycolysis in the formation of the pseudopodial protrusions of MSV-MDCK-INV cells (45Nguyen T.N. Wang H.J. Zalzal S. Nanci A. Nabi I.R. Exp. Cell Res. 2000; 258: 171-183Google Scholar). The β-actin rich pseudopodia of MSV-MDCK-INV cells are presented here as expressing a high degree of tyrosine-phosphorylated proteins, namely a 160-kDa protein identified as HGF-R or Met. Furthermore, constitutive phosphorylation of HGF-R caused by autocrine secretion of HGF is shown to regulate the expression of multiple pseudopodial protrusions and the acquisition of an invasive phenotype by MSV-MDCK-INV cells. Autocrine activation of the HGF-R tyrosine kinase is therefore associated not with epithelial transformation but more particularly with acquisition of tumor cell motility during tumor progression. Texas Red-conjugated phalloidin was purchased from Molecular Probes (Eugene, OR). Monoclonal antibody to phosphotyrosine (p-Tyr) (PY99) and polyclonal antibody to HGFα (H-145) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibody to c-Met (DO-24) was obtained from Upstate Biotechnologies (Lake Placid, NY) and phospho-specific polyclonal c-Met antibodies were purchased from BioSource International (MediCorp, Montréal, Canada). Antibodies to β-actin and ezrin as well as herbimycin were purchased from Sigma. Recombinant human HGFα (rHGF) was obtained from R & D System and from Sigma.N-hydroxysulfosuccinimide-long chain-biotin was obtained from Pierce (Rockford, IL). Streptavidin-HRP, polyvinylidene difluoride, and Hybond C extra membranes were from AmershamBiosciences, and ECL reagent was purchased from Mandel (St.-Laurent, Quebec). Fetal bovine serum (FBS), glutamine, essential amino acids, vitamins, media, penicillin, and streptomycin were purchased from Invitrogen (Burlington, ON). Centricon Plus-80 filter units were from Millipore (Toronto, Ontario). MDCK strain I, MDCK strain II, MSV-MDCK (42Simard D. Nabi I.R. Biochem. Biophys. Res. Commun. 1996; 219: 122-127Google Scholar), and MSV-MDCK-INV cells (43Le P.U. Nguyen T.N. Drolet-Savoie P. Leclerc N. Nabi I.R. Cancer Res. 1998; 58: 1631-1635Google Scholar) were cultured in Dulbecco's minimum essential medium containing 25 mm NaHCO3(DMEM), 10% FBS, glutamine, 1% essential amino acids, vitamins, penicillin and streptomycin under 5% CO2 atmosphere at 37 °C. For video microscopy recording, cells were incubated in DMEM medium containing 10 mm HEPES (DMEM-HEPES), 5% FBS, and supplemented with glutamine, vitamins, penicillin and streptomycin. To collect conditioned medium from MSV-MDCK-INV cells (CM-INV), cells were plated in DMEM containing 10% fetal bovine serum for 4 h, then the medium was changed for DMEM containing only 0.2% fetal bovine serum. CM-INV was collected 48 h later, concentrated roughly 50-fold using Centricon plus-80 filters, and used within 24 h. Cells were seeded for 48 h at 1–3 × 105cells/100 mm plate for immunoprecipitation experiments and at 2 × 104 cells/35 mm plate on coverslips for immunofluorescence experiments and grown in DMEM-NaHCO3 under 5% CO2. Cells were rinsed two times with serum-free DMEM and stimulated with either 1–50 ng/ml rHGF for 10 min or with 50 ng/ml rHGF for 5 to 180 min at 37 °C under a CO2 atmosphere. Control cells (CTL) were rinsed and incubated for the indicated time in DMEM without serum. The tyrosine kinase inhibitor herbimycin diluted in Me2SO was added at 2 and 4 μm, and anti-HGFα antibody at 0.02–20 μg/ml, both for 24 h in fresh medium containing 5% FBS. For reversibility experiments following anti-HGF incubation, cells were rinsed two times, and fresh medium containing 10% FBS alone or with 50 ng/ml rHGF was added for another 24 h. Cells were fixed for 15 min at room temperature with 2% paraformaldehyde at 37 °C and then permeabilized with 0.075% saponin for 10 min. F-actin was labeled with phalloidin-Texas Red for 30 min, and phosphotyrosine with mouse anti-p-Tyr for 1 h followed by fluorescein isothiocyanate-coupled anti-mouse IgG antibodies for 30 min. Control was performed without primary antibody, and no signal was detected. After labeling, coverslips were mounted in a 100 mm propylgallate solution in 50% glycerol and 100 mm Tris-HCl, pH 8.0, and labeled cells were examined in a Zeiss AxioSkop fluorescent microscope equipped with a 63× Plan Apochromat objective and selective filters for fluorescein isothiocyanate and Texas Red. MDCK strain II, MSV-MDCK, and MSV-MDCK-INV cells were grown to 60–80% confluence on Petri dishes in DMEM. Cell monolayers were washed three times with ice-cold PBS/CM (140 mm NaCl, 2.7 mm KCl, 10 mm Na2HPO4, 1.8 mmKH2PO4, 0.1 mm CaCl2, 1.0 mm MgCl2), harvested, and centrifuged at low speed, and cell lysates were prepared (43Le P.U. Nguyen T.N. Drolet-Savoie P. Leclerc N. Nabi I.R. Cancer Res. 1998; 58: 1631-1635Google Scholar). Briefly, cell pellets were suspended in lysis buffer consisting of PBS containing 1% SDS, 1 mm EDTA, protease inhibitors (1 μg/ml aprotinin, 1 μg/ml pepstatin, 1 μg/ml leupeptin, and 0.1 mm PMSF), and 0.2 mm orthovanadate. Cells were lysed for 20 min on ice, and DNA was broken by sonication. 50 μg of protein of the cell lysates were separated by 7.5% SDS-PAGE gels and transferred to Hybond C extra nitrocellulose membranes. Phosphotyrosine residues, phospho-c-Met, ezrin, and β-actin were revealed by blotting with the indicated primary antibodies and corresponding secondary antibodies coupled to HRP, and protein bands were revealed by chemiluminescence (ECL reagent). Anti-phosphotyrosine immunoprecipitation was performed according to the protocol proposed by BD Transduction Laboratories using 1 μg of p-Tyr antibody and 200–500 μg of cell lysate. Briefly, cells rinsed with PBS/CM were lysed with 5 mm Tris-HCl, 150 mm NaCl, containing 1% Triton X-100, 0.5% Igepal, protease inhibitors, and 0.2 mm sodium orthovanadate. Phosphotyrosine immunoprecipitates were loaded onto a 7.5% SDS-PAGE, transferred onto a Hybond C extra nitrocellulose membrane, and immunoblotted with the p-Tyr antibody. When indicated, antibodies were stripped for blotting with polyclonal phospho-specific c-Met antibodies. Phosphotyrosine immunoprecipitates (see above) from 10 × 150-mm-diameter Petri dishes at 60–80% confluence were electrophoresed on a 7% SDS-PAGE gel (0.75 mm). Thioglycolic acid (11.4 μg/ml) was added to the upper electrophoresis buffer to prevent in-gel N-terminal blocking. After electrophoresis, the gel was soaked in transfer buffer (10 mm3-(cyclohexylamino)-1-propanesulfonic acid, 10% methanol, pH 11) for 5 min to reduce the amount of Tris and glycine, and proteins were transferred onto a polyvinylidene difluoride membrane. Proteins were revealed by staining the membrane with 0.1% Coomassie Blue R-250 diluted in 50% methanol. The 160-kDa band was cut from the blot and sequenced by Edman degradation (Biotechnology Research Institute, Montréal, Quebec, Canada). Cell surface biotinylation with sulfo-NHS-LC-biotin (0.5 mg/ml × 20 min; repeat twice) was performed at 4 °C in PBS/CM. Cells were then rinsed with PBS and lysed in Ripa buffer (25 mm Tris pH 7.4, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1% SDS, 150 mm NaCl, 10 mm sodium fluoride, 0.2 mm sodium orthovanadate, and protease inhibitors as followed, 0.1 mm PMSF, 1 μg/ml aprotinin, 1 μg/ml pepstatin, 1 μg/ml leupeptin, 1 mm phenanthrolin) as described (46Jeffers M. Rong S. Vande Woude G.F. J. Mol. Med. 1996; 74: 505-513Google Scholar). Immunoprecipitation of biotinylated canine HGF-R or Met was performed on 300 μg of cell lysate with 0.2 μg of the monoclonal anti-c-Met DO-24 antibody directed against the extracellular domain of the human receptor as described by Prat et al.(47Prat M. Crepaldi T. Pennacchietti S. Bussolino F. Comoglio P.M. J. Cell Sci. 1998; 111: 237-247Google Scholar). Met immunoprecipitates were dissociated with the sample buffer, submitted to a 7.5% SDS-PAGE and transferred onto a Hybond-C extra nitrocellulose membrane. Biotinylated Met proteins were detected with streptavidin-HRP reagent, and phosphotyrosine residues were detected as described above on parallel samples. To ensure equivalent sample loading, the membranes were stripped (63 mm Tris HCl, 2% SDS, 100 mmβ-mercaptoethanol, pH 6.7 at 50 °C for 30 min) and reprobed with anti-p-Tyr or streptavidin-HRP, respectively. Video microscopy was performed using a Zeiss Axiovert microscope equipped with a Princeton Microview video camera. Images were collected and analyzed using Northern Eclipse image analysis software (Empix Imaging, Mississauga, ON). Cells were plated on a 12-well plate at 4 × 104 cells/well for 2 h in DMEM under CO2 atmosphere to allow adhesion and spreading, after which the medium was replaced with 1.8 ml of DMEM-HEPES + 5% FBS and covered with paraffin oil to prevent evaporation. Following a 5-h incubation in HEPES medium at 37 °C in a CO2-free atmosphere, 200 μl of medium supplemented with rHGF or alone (CTL) was then added to the medium with gentle mixing. The cells were observed with a 10× objective on a microscope stage maintained at 37 °C, and images were collected every 15 min for 10 h. Cell motility was determined by tracking nuclear position of 10 cells over the 10 h recording period, and the experiment was performed three times for a total of 30 cells. To measure the effect of polyclonal anti-HGFα antibodies on cell motility and morphology, cells were plated as above, but at 1 × 105 cells/well. After adhesion, medium was changed for supplemented DMEM-HEPES medium containing 5% FBS and incubated at 37 °C for 5 h in a CO2-free atmosphere. Fresh medium, alone or containing 20 μg/ml anti-HGFα was added, and images were collected every 15 min for 48 h. The high cell density facilitated visualization of cell-cell interactions but prevented quantification of cell motility in this set of experiments. A video is used to demonstrate the effect of HGF titration on the motility of MSV-MDCK-INV cells and on pseudopodial protrusions. The combined video represents two different sets of conditions recorded on different days. Cells were two passages apart and placed together to facilitate visualization. The first part represents a control period, and the second part illustrates the situation in the presence of 20 μg/ml anti-HGF-α added at time 0 in fresh medium. Tyrosine phosphorylation is generally associated with cellular transformation and increased cellular motility of tumor cells. To determine whether protein phosphorylation on tyrosine is associated with MDCK cell transformation and acquisition of invasive capacities, MDCK, MSV-MDCK, and MSV-MDCK-INV cells were immunofluorescently double-labeled for F-actin and phosphotyrosine (Fig. 1). When compared with MSV-MDCK, MSV-MDCK-INV cells do not have actin stress fibers and accumulate F-actin at the tips of multiple pseudopodia (Fig. 1, E and F). Specific phosphotyrosine labeling localized with F-actin in both MDCK, MSV-MDCK, and MSV-MDCK-INV cells and was particularly concentrated at the tips of the multiple pseudopodia of MSV-MDCK-INV cells (Fig. 1, Cand F). To identify tyrosine phosphorylated proteins specifically expressed or selectively phosphorylated in MSV-MDCK-INV cells, the tyrosine phosphorylation pattern of MDCK, MSV-MDCK and MSV-MDCK-INV cells was compared. An anti-phosphotyrosine immunoblot of total cell lysates shows that for the same amount of protein, reflected by the similar amount of ezrin (Fig. 2a, middle panel), the overall tyrosine phosphorylation state of numerous proteins is significantly increased in MSV-MDCK-INV cells compared with MSV-MDCK and wild-type MDCK cells (Fig. 2a, upper panel). The major tyrosine-phosphorylated 160-kDa (157.2 ± 1.2 kDa (n = 12)) protein that is selectively phosphorylated in MSV-MDCK-INV cells relative to both MDCK and MSV-MDCK cells exhibited the same increase in tyrosine phosphorylation after immunoprecipitation with the anti-p-Tyr antibody (Fig. 2b) as observed by immunoblot on total cell lysates (Fig. 2a,upper panel). After an anti-p-Tyr immunoprecipitation from large quantities of MSV-MDCK-INV cells, the Coomassie Blue-labeled band corresponding to the 160-kDa protein was N-terminally sequenced. The N-terminal sequence of the indicated band representing a molecular mass of 160 kDa (TREEVFNILQAAYV) identified this protein, with 100% identity, as HGF-R (no. p16056, Blast data base) or the c-Met oncogene (no. CAA65582, Blast data base). Immunoprecipitation of biotinylated cell surface HGF-R/c-MET from MDCK, MSV-MDCK, and MSV-MDCK-INV cells followed by anti-phosphotyrosine immunoblot revealed that cell surface associated HGF-R is specifically tyrosine phosphorylated in MSV-MDCK-INV cells (Fig. 3, upper panel). The increased detection of phosphorylated HGF-R/c-MET is not related to expression levels of the receptor due to observation of essentially equivalent cell surface expression of HGF-R/c-MET for the three cell lines (Fig. 3, lower panel). Constitutive HGF-R/c-Met phosphorylation is therefore associated not with MSV transformation of MDCK cells but more particularly with the invasive phenotype of MSV-MDCK-INV cells.Figure 3HGF-R/Met phosphorylation is specifically increased in MSV-MDCK-INV cells. Cell surface biotinylation (upper panel) and tyrosine phosphorylation (lower panel) of immunoprecipitated HGF-R/Met was determined for MDCK, MSV-MDCK, and MSV-MDCK-INV cells. Total biotinylated receptors were revealed by streptavidin-HRP (upper panel) and phosphorylation on tyrosine residues (middle panel) was revealed on parallel samples (shown here) or stripped blots. The lower panel shows the quantification of n = 4 similar experiments. Similar amounts of HGF-R/Met are found on the plasma membrane of these three cell lines but HGF-R/Met is highly phosphorylated only in MSV-MDCK-INV cells.View Large Image Figure ViewerDownload (PPT) A critical role for tyrosine phosphorylation in the acquisition of the motile phenotype of MSV-MDCK-INV cells was determined using the tyrosine kinase inhibitor, herbimycin A (Fig. 4). Tyrosine phosphorylation of the 160-kDa protein identified as HGF-R/c-Met, decreased significantly on herbimycin A treatment, reaching negligible levels in the presence of 4 μm herbimycin. Treatment of MSV-MDCK-INV cells with 2 and 4 μm herbimycin for 24 h resulted in a gradual disappearance of the β-actin-rich pseudopodia and the expression of multiple actin stress fibers (Fig. 4b, B andC). This in turn resulted in increased spreading and formation of cell-cell contacts by the treated cells. The activity of cellular tyrosine kinases is therefore required for pseudopodial protrusion found in MSV-MDCK-INV cells, supporting a role for HGF-R/c-Met tyrosine phosphorylation in this process. Phosphorylation of Met on tyrosine residues 1234 and 1235 in its tyrosine kinase domain occurs upon ligand interaction. This interaction in turn activates its intrinsic kinase activity (24Bottaro D.P. Rubin J.S. Faletto D.L. Chan A.M.-L. Kmiecik T.E. Vande Woude G.F. Aaronson S.A. Science. 1991; 251: 802-804Google Scholar, 48Naldini L. Weidner K.M. Vigna E. Gaudino G. Bardelli A. Ponzetto C. Narsimhan R.P. Hartmann G. Zarnegar R. Michalopoulos G.K. Birchmeier W. Comoglio P.M. EMBO J. 1991; 10: 2867-2878Google Scholar) such that increased tyrosine auto-phosphorylation of receptor tyrosine kinases is an indication of its enzymatic activity (49Jeffers M. Schmidt L. Nakaigawa N. Webb C.P. Weirich G. Kishida T. Zbar B. Vande Woude G.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11445-11450Google Scholar). Subsequent phosphorylation of tyrosines at positions 1349, 1356, and 1365 located in the C-terminal region promotes binding of multiple SH2-containing transducers. It is for this reason that this region is called the multifunctional docking site (see Ref. 50Furge K.A. Zhang Y.-W. Vande Woude G.F. Oncogene. 2000; 19: 5582-5589Google Scholar for an integrated view). To confirm that the 160-kDa protein identified as HGF-R by protein sequencing exhibits HGF-dependent tyrosine kinase activity, MDCK strain I, MSV-MDCK, and MSV-MDCK-INV cells cultured in serum-containing medium were stimulated with rHGF as reported (51Webb C.P. Lane K. Dawson A.P. Vande Woude G.F. Warn R.M. J. Cell Sci. 1996; 109: 2371-2381Google Scholar). Cells were rinsed rapidly with serum-free medium and then stimulated with 50 ng/ml rHGF for 10 min (Fig. 5a). HGF substantially increased (6.1 ± 0.4-fold, n = 3) the overall tyrosine phosphorylation of the 160-kDa protein in MSV-MDCK cells. The effect of HGF on c-Met phosphorylation in MDCK (1.6 ± 0.4-fold,n = 4) and MSV-MDCK-INV (2.0 ± 0.9-fold,n = 4) cells was much weaker. Similar results were obtained using phospho-specific c-Met antibodies following antibody stripping. In the absence of serum and HGF, c-Met from MDCK and MSV-MDCK cells is very lightly phosphorylated on tyrosines 1230, 1234, and 1235 of the tyrosine kinase domain, and on tyrosines 1349 and 1365 of the docking site. In the case of MSV-MDCK-INV cells, however, these tyrosines are highly phosphorylated. After rHGF stimulation, receptor auto-phosphorylation on tyrosines 1230, 1234, and 1235, as well as tyrosines 1349 and 1365 is strongly increased in MSV-MDCK cells but not in MDCK nor in MSV-MDCK-INV cells. These results confirm that HGF-R/c-Met from MSV-MDCK-INV cells is constitutively phosphorylated and illustrate for the first time the combined tyrosine phosphorylation motif characteristic of an active c-Met receptor. Moreover, these results indicate that c-Met from MSV-MDCK cells is highly responsive to rHGF stimulation. The dramatic increase in HGF-R phosphorylation in MSV-MDCK cells led us to perform kinetic analysis of HGF stimulation. Incubation with 50 ng/ml HGF at 37 °C led to a maximal tyrosine phosphorylation signal between 10–30 min, which decreased as a function of time reaching" @default.
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• W2023007685 title "Autocrine Activation of the Hepatocyte Growth Factor Receptor/Met Tyrosine Kinase Induces Tumor Cell Motility by Regulating Pseudopodial Protrusion" @default.
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