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• W2068994296 abstract "Overexpression and amplification of hepatocyte growth factor (HGF) receptor (Met) have been detected in many types of human cancers, suggesting a critical role for Met in growth and development of malignant cells. However, the molecular mechanism by which Met contributes to tumorigenesis is not well known. The tyrosine kinase c-Src has been implicated as a modulator of cell proliferation, spreading, and migration; these functions are also regulated by Met. To explore whether c-Src kinase is involved in HGF-induced cell growth, a mouse mammary carcinoma cell line (SP1) that co-expresses HGF and Met and a nonmalignant epithelial cell line (Mv1Lu) that expresses Met but not HGF were used. In this study, we have shown that c-Src kinase activity is constitutively elevated in SP1 cells and is induced in response to HGF in Mv1Lu cells. In addition, c-Src kinase associates with Met following stimulation with HGF. The enhanced activity of c-Src kinase also correlates with its ability to associate with Met. Expression of a dominant negative double mutant of c-Src (SRC-RF), lacking both kinase activity (K295R) and a regulatory tyrosine residue (Y527F), in SP1 cells significantly reduced c-Src kinase activity and strongly blocked HGF-induced motility and colony growth in soft agar. In contrast, expression of the dominant negative c-Src mutant had no effect on HGF-induced cell proliferation on plastic. Taken together, our data strongly suggest that HGF-induced association of c-Src with Met and c-Src activation play a critical role in HGF-induced cell motility and anchorage-independent growth of mammary carcinomas and further support the notion that the presence of paracrine and autocrine HGF loops contributes significantly to the transformed phenotype of carcinoma cells. Overexpression and amplification of hepatocyte growth factor (HGF) receptor (Met) have been detected in many types of human cancers, suggesting a critical role for Met in growth and development of malignant cells. However, the molecular mechanism by which Met contributes to tumorigenesis is not well known. The tyrosine kinase c-Src has been implicated as a modulator of cell proliferation, spreading, and migration; these functions are also regulated by Met. To explore whether c-Src kinase is involved in HGF-induced cell growth, a mouse mammary carcinoma cell line (SP1) that co-expresses HGF and Met and a nonmalignant epithelial cell line (Mv1Lu) that expresses Met but not HGF were used. In this study, we have shown that c-Src kinase activity is constitutively elevated in SP1 cells and is induced in response to HGF in Mv1Lu cells. In addition, c-Src kinase associates with Met following stimulation with HGF. The enhanced activity of c-Src kinase also correlates with its ability to associate with Met. Expression of a dominant negative double mutant of c-Src (SRC-RF), lacking both kinase activity (K295R) and a regulatory tyrosine residue (Y527F), in SP1 cells significantly reduced c-Src kinase activity and strongly blocked HGF-induced motility and colony growth in soft agar. In contrast, expression of the dominant negative c-Src mutant had no effect on HGF-induced cell proliferation on plastic. Taken together, our data strongly suggest that HGF-induced association of c-Src with Met and c-Src activation play a critical role in HGF-induced cell motility and anchorage-independent growth of mammary carcinomas and further support the notion that the presence of paracrine and autocrine HGF loops contributes significantly to the transformed phenotype of carcinoma cells. Evidence supports a role of hepatocyte growth factor (HGF) 1The abbreviations used are: HGF, hepatocyte growth factor; PI, phosphatidylinositol; FBS, fetal bovine serum; PAGE, polyacrylamide gel electrophoresis; PIPES, 1,4-piperazinediethanesulfonic acid; PDGF, platelet-derived growth factor. and its receptor, the product of the met protooncogene, in both normal (1Brinkmann V. Foroutan H. Sachs M. Weidner K.M. Birchmeier W. J. Cell Biol. 1995; 131: 1573-1586Crossref PubMed Scopus (296) Google Scholar, 2Niranjan B. Buluwela L. Yant J. Perusinghe N. Atherton A. Phippard D. Dale T. Gusterson B. Kamalati T. Development. 1995; 121: 2897-2908Crossref PubMed Google Scholar) and malignant (3Fixman E.D. Naujokas M.A. Rodrigues G.A. Moran M.F. Park M. Oncogene. 1995; 10: 237-249PubMed Google Scholar, 4Bellusci S. Moens G. Gaudino G. Comoglio P. Nakamura T. Thiery J.P. Jouanneau J. Oncogene. 1994; 9: 1091-1099PubMed Google Scholar, 5Maggiora P. Gambarotta G. Olivero M. Giordano S. Di Renzo M.F. Comoglio P.M. J. Cell. Physiol. 1997; 173: 183-186Crossref PubMed Scopus (40) Google Scholar) epithelial cell development. In addition, a majority of human breast cancers show increased expression of HGF and Met (6Tuck A.B. Park M. Sterns E.E. Boag A. Elliott B.E. Am. J. Pathol. 1996; 148: 225-232PubMed Google Scholar, 7Wang Y. Selden A.C. Morgan N. Stamp G.W. Hodgson H.J. Am. J. Pathol. 1994; 144: 675-682PubMed Google Scholar, 8Jin L. Fuchs A. Schnitt S. Yao Y. Joseph A. Lamszus K. Park M. Goldberg I. Rosen E. Cancer. 1997; 79: 749-760Crossref PubMed Scopus (151) Google Scholar), and this high level of HGF expression correlates with recurrence and poor patient survival (9Yamashita J. Ogawa M. Yamashita S. Nomura K. Kuramoto M. Saishoji T. Shin S. Cancer Res. 1994; 54: 1630-1633PubMed Google Scholar). Met is also overexpressed in several other human cancers, including ovarian (10Di Renzo M.F. Olivero M. Katsaros D. Crepaldi T. Gaglia P. Zola P. Sismondi P. Comoglio P.M. Int. J. Cancer. 1994; 58: 658-662Crossref PubMed Scopus (201) Google Scholar), melanoma (11Natali P.G. Nicotra M.R. Di Renzo M.F. Prat M. Bigotti A. Cavaliere R. Comoglio P.M. Br. J. Cancer. 1993; 68: 746-750Crossref PubMed Scopus (177) Google Scholar), colon carcinomas (12Di Renzo M.F. Olivero M. Giacomini A. Porte H. Chastre E. Mirossay L. Nordlinger B. Bretti S. Bottardi S. Giordano S. Plebani M. Gespach C. Comoglio P.M. Clin. Cancer Res. 1995; 1: 147-154PubMed Google Scholar), and osteosarcomas (13Ferracini R. Di Renzo M.F. Scotlandi K. Baldini N. Olivero M. Lollini P. Cremona O. Campanacci M. Comoglio P.M. Oncogene. 1995; 10: 739-749PubMed Google Scholar). Collectively, these observations suggest that activation of Met by overexpression, gene amplification, or establishment of an HGF autocrine loop may contribute to growth and development of mammary carcinomas. Previous studies demonstrated that co-expression of HGF and Met (4Bellusci S. Moens G. Gaudino G. Comoglio P. Nakamura T. Thiery J.P. Jouanneau J. Oncogene. 1994; 9: 1091-1099PubMed Google Scholar, 14Rong S. Bodescot M. Blair D. Dunn J. Nakamura T. Mizuno K. Park M. Chan A. Aaronson S. Vande Woude G.F. Mol. Cell. Biol. 1992; 12: 5152-5158Crossref PubMed Scopus (291) Google Scholar), as well as expression of a constitutively active Met (Tpr-Met) in NIH-3T3 fibroblasts (15Rong S. Segal S. Anver M. Resau J.H. Vande Woude G.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4731-4735Crossref PubMed Scopus (346) Google Scholar, 16Cooper C.S. Park M. Blair D. Tainsky M.A. Huebner K. Croce C.M. Vande Woude G.F. Nature. 1994; 311: 29-33Crossref Scopus (782) Google Scholar) directly leads to cell transformation and tumorigenicity. However, the molecular mechanism by which HGF binding to its receptor elicits cell transformation is not fully understood. A number of cytoplasmic signaling proteins, such as phosphatidylinositol (PI) 3-kinase, Grb2, Shc, Ras, and c-Src, have been shown to be involved in Met-dependent signal transduction pathways (17Zhu H. Naujokas M.A. Fixman E.D. Torossian K. Park M. J. Biol. Chem. 1994; 269: 29943-29948Abstract Full Text PDF PubMed Google Scholar, 18Ponzetto C. Bardelli A. Zhen Z. Maina F. dalla Zonca P. Giordano S.A.U. Panayotou G. Comoglio P.M. Cell. 1994; 77: 261-271Abstract Full Text PDF PubMed Scopus (896) Google Scholar). It is important to establish which of these signaling proteins regulate Met-dependent steps in tumor progression, because different signaling proteins may regulate various HGF-induced cellular functions, including mitogenic, motogenic, and morphogenic signals in target cells (18Ponzetto C. Bardelli A. Zhen Z. Maina F. dalla Zonca P. Giordano S.A.U. Panayotou G. Comoglio P.M. Cell. 1994; 77: 261-271Abstract Full Text PDF PubMed Scopus (896) Google Scholar, 19Tajima H. Matsumoto K. Nakamura T. FEBS Lett. 1991; 291: 229-232Crossref PubMed Scopus (206) Google Scholar, 20Rubin J.S. Chan A.M. Bottaro D.P. Burgess W.H. Taylor W.G. Cech A.C. Hirschfield D.W. Wong J. Miki T. Finch P.W. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 415-419Crossref PubMed Scopus (471) Google Scholar, 21Rosen E.M. Knesel J. Goldberg I.D. Jin L. Bhargava M. Joseph A. Zitnik R. Wines J. Kelley M. Rockwell S. Int. J. Cancer. 1994; 57: 706-714Crossref PubMed Scopus (84) Google Scholar, 22Schmidt C. Bladt F. Goedecke S. Brinkmann V. Zschiesche W. Sharpe M. Gherardi E. Birchmeier C. Nature. 1995; 373: 699-702Crossref PubMed Scopus (1231) Google Scholar). The HGF-mediated signaling pathway is further complicated by the observation that the majority of SH2-containing cytoplasmic effectors bind to a single multifunctional docking site on the cytoplasmic domain of Met, whereas a second site is required for Grb2 binding (17Zhu H. Naujokas M.A. Fixman E.D. Torossian K. Park M. J. Biol. Chem. 1994; 269: 29943-29948Abstract Full Text PDF PubMed Google Scholar, 18Ponzetto C. Bardelli A. Zhen Z. Maina F. dalla Zonca P. Giordano S.A.U. Panayotou G. Comoglio P.M. Cell. 1994; 77: 261-271Abstract Full Text PDF PubMed Scopus (896) Google Scholar). Recent findings using a mutational approach demonstrated that different HGF-induced effects are regulated by these separate Met binding sites for cytoplasmic transducers (23Ponzetto C. Zhen Z. Audero E. Maina F. Bardelli A. Basile M.L. Giordano S. Narsimhan R. Comoglio P. J. Biol. Chem. 1996; 271: 14119-14123Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 24Fixman E.D. Fournier T.M. Kamikura D.M. Naujokas M.A. Park M. J. Biol. Chem. 1996; 271: 13116-13122Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 25Giordano S. Bardelli A. Zhen Z. Menard S. Ponzetto C. Comoglio P.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13868-13872Crossref PubMed Scopus (82) Google Scholar) and that complementation intrans between these two binding sites is required for the invasive-metastatic phenotype (25Giordano S. Bardelli A. Zhen Z. Menard S. Ponzetto C. Comoglio P.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13868-13872Crossref PubMed Scopus (82) Google Scholar). However, to study the role of specific SH2-containing cytoplasmic effectors in HGF receptor function, approaches to target individual cytoplasmic effectors are required. Recently, we (26Rahimi N. Tremblay E. Elliott B. J. Biol. Chem. 1996; 271: 24850-24855Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) and others (27Royal I. Park M. J. Biol. Chem. 1995; 270: 27780-27787Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar) have demonstrated that PI 3-kinase activity is required for HGF-induced mitogenic (26Rahimi N. Tremblay E. Elliott B. J. Biol. Chem. 1996; 271: 24850-24855Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) and motogenic functions (27Royal I. Park M. J. Biol. Chem. 1995; 270: 27780-27787Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). These findings strongly argue that PI 3-kinase may play an important role in HGF-mediated growth of mammary carcinomas. The tyrosine kinase c-Src is activated in response to HGF (17Zhu H. Naujokas M.A. Fixman E.D. Torossian K. Park M. J. Biol. Chem. 1994; 269: 29943-29948Abstract Full Text PDF PubMed Google Scholar, 18Ponzetto C. Bardelli A. Zhen Z. Maina F. dalla Zonca P. Giordano S.A.U. Panayotou G. Comoglio P.M. Cell. 1994; 77: 261-271Abstract Full Text PDF PubMed Scopus (896) Google Scholar) and other growth factors such as platelet-derived growth factor (PDGF) (28Oude Weernink P.A. Rijksen G. J. Biol. Chem. 1995; 270: 2264-2267Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 29Courtneidge S.A. Kypta R.M. Cooper J.A. Kazlauskas A. Cell Growth Differ. 1991; 2: 483-486PubMed Google Scholar, 30Kypta R.M. Goldberg Y. Ulug E.T. Courtneidge S.A. Cell. 1990; 62: 481-492Abstract Full Text PDF PubMed Scopus (480) Google Scholar), fibroblast growth factor (31Zhan X. Plourde C. Hu X. Friesel R. Maciag T. J. Biol. Chem. 1994; 269: 20221-20224Abstract Full Text PDF PubMed Google Scholar), and epidermal growth factor (32Muthuswamy S.K. Muller W.J. Oncogene. 1995; 11: 271-279PubMed Google Scholar). c-Src kinase activity is known to modulate cell proliferation (33Barone M.V. Courtneidge S.A. Nature. 1995; 378: 509-512Crossref PubMed Scopus (283) Google Scholar, 34Broome M.A. Hunter T. J. Biol. Chem. 1996; 271: 16798-16806Crossref PubMed Scopus (117) Google Scholar), spreading (35Kaplan K.B. Swedlow J.R. Morgan D.O. Varmus H.E. Genes Dev. 1995; 9: 1505-1517Crossref PubMed Scopus (295) Google Scholar, 36Rodier J-M. Vallés A.M. Denoyelle M. Thiery J.P. Boyer B. J. Cell Biol. 1995; 131: 761-773Crossref PubMed Scopus (72) Google Scholar), and migration (36Rodier J-M. Vallés A.M. Denoyelle M. Thiery J.P. Boyer B. J. Cell Biol. 1995; 131: 761-773Crossref PubMed Scopus (72) Google Scholar, 37Hansen K. Johnell M. Siegbahn A. Rorsman C. Engstrom U. Wernstedt C. Heldin C.H. Ronnstrand L. EMBO J. 1996; 15: 5299-5313Crossref PubMed Scopus (107) Google Scholar, 38Hall C.L. Lange L.A. Prober D.A. Zhang S. Turley E.A. Oncogene. 1996; 13: 2213-2224PubMed Google Scholar) in many cell types; these functions are also regulated by HGF (19Tajima H. Matsumoto K. Nakamura T. FEBS Lett. 1991; 291: 229-232Crossref PubMed Scopus (206) Google Scholar, 20Rubin J.S. Chan A.M. Bottaro D.P. Burgess W.H. Taylor W.G. Cech A.C. Hirschfield D.W. Wong J. Miki T. Finch P.W. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 415-419Crossref PubMed Scopus (471) Google Scholar, 21Rosen E.M. Knesel J. Goldberg I.D. Jin L. Bhargava M. Joseph A. Zitnik R. Wines J. Kelley M. Rockwell S. Int. J. Cancer. 1994; 57: 706-714Crossref PubMed Scopus (84) Google Scholar, 22Schmidt C. Bladt F. Goedecke S. Brinkmann V. Zschiesche W. Sharpe M. Gherardi E. Birchmeier C. Nature. 1995; 373: 699-702Crossref PubMed Scopus (1231) Google Scholar, 23Ponzetto C. Zhen Z. Audero E. Maina F. Bardelli A. Basile M.L. Giordano S. Narsimhan R. Comoglio P. J. Biol. Chem. 1996; 271: 14119-14123Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). c-Src kinase activity is increased 4-fold in human breast cancer (39Rosen N. Bolen J.B. Schwartz A.M. Cohen P. DeSeau V. Israel M.A. J. Biol. Chem. 1986; 261: 13754-13759Abstract Full Text PDF PubMed Google Scholar, 40Ottenhoff-Kalff A.E. Rijksen G.A.U. Hennipman A. Michels A.A. Staal G.E. Cancer Res. 1992; 52: 4773-4778PubMed Google Scholar) and is also elevated in Neu-induced mouse mammary carcinomas in transgenic mice (41Muthuswamy S.K. Siegel P.M. Dankort D.L. Webster M.A. Muller W.J. Mol. Cell. Biol. 1994; 14: 735-743Crossref PubMed Google Scholar, 42Guy C.T. Muthuswamy S.K. Cardiff R.D. Soriano P. Muller W.J. Genes Dev. 1994; 8: 23-32Crossref PubMed Scopus (187) Google Scholar). Activation of c-Src tyrosine kinase in transgenic mice induces mammary epithelial hyperplasias and is required, but is not sufficient, for induction of mammary tumors in polyoma virus middle T-transgenic mice (42Guy C.T. Muthuswamy S.K. Cardiff R.D. Soriano P. Muller W.J. Genes Dev. 1994; 8: 23-32Crossref PubMed Scopus (187) Google Scholar, 43Webster M.A. Cardiff R.D. Muller W.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7849-7853Crossref PubMed Scopus (71) Google Scholar). Altogether, these observations support the notion that increased c-Src kinase activity in mammary carcinomas plays an important role in mammary tumor growth and development. However, the role of c-Src kinase in HGF-induced functions in mammary carcinoma cells is not clearly known. To analyze whether c-Src kinase is involved in HGF-induced mammary carcinoma cell growth, we used a mouse mammary carcinoma cell line, SP1, which expresses HGF and tyrosine-phosphorylated Met, thereby generating an autocrine HGF loop in these cells (44Rahimi N. Tremblay E. McAdam L. Park M. Schwall R. Elliott B. Cell Growth & Differ. 1996; 7: 263-270PubMed Google Scholar). Our current results demonstrate that c-Src kinase activity is elevated in SP1 cells, compared with nonmalignant Mv1Lu epithelial cells. The increased activity of c-Src kinase correlates with its ability to associate with tyrosine-phosphorylated Met. We therefore examined the effect of expressing a dominant negative mutant form of c-Src on c-Src kinase activity and HGF-induced cell motility and anchorage-independent growth of SP1 carcinoma cells. Taken together, our findings show that c-Src kinase activation plays a significant role in HGF-induced cell motility and anchorage-independent growth, characteristics of the transformed phenotype. Rabbit anti-sheep IgG conjugated to horseradish peroxidase was from Jackson ImmunoResearch Laboratories (Westgrove, PN). Mouse anti-phosphotyrosine (PY20) monoclonal antibody was purchased from Transduction Laboratories (Lexington, KT). Rabbit anti-c-Src IgG, anti-Met (mouse) IgG, and anti-PLC-γ1 IgG were obtained from Santa Cruz Biotechnology (San Diego, CA). Mv1Lu cells are members of a mink lung epithelial cell line obtained from ATCC (Rockville, MA). Maintenance medium for Mv1Lu cells was Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% FBS. The SP1 tumor cell line is derived from a spontaneous poorly metastatic murine mammary intraductal adenocarcinoma and expresses HGF and Met. The characteristics of the SP1 cell line have been described elsewhere (45Rahimi N. Saulnier R. Nakamura T. Park M. Elliott B. DNA Cell Biol. 1994; 13: 1189-1197Crossref PubMed Scopus (98) Google Scholar,46Elliott B.E. Tam S.P. Dexter D. Chen Z.Q. Int. J. Cancer. 1992; 51: 416-424Crossref PubMed Scopus (80) Google Scholar). Maintenance medium for SP1 cells was RPMI 1640 (Life Technologies, Inc.) supplemented with 7% FBS (Life Technologies, Inc.). cDNAs encoding wild typec-src (SRC) and a dominant negative double mutant ofc-src (SRC-RF) with loss-of-function mutations in the kinase domain (K295R) and a regulatory tyrosine residue (Y527F) ligated into the pRc/CMV plasmid (Invitrogen, San Diego, CA) carrying the neomycin resistance marker were obtained from Dr. J. Brugge (47Mukhoupadhyay D. Tsiokas L. Zhou X.M. Foster D. Brugge J.S. Sukhatme V.P. Nature. 1995; 375: 577-581Crossref PubMed Scopus (539) Google Scholar). SP1 cells expressing the mutant c-Src and wild type c-Src were established using the stable transfection LipofectAMINE (Life Technologies, Inc.) method (48Seth P. Brinkmann U. Schwartz G.N. Katayose D. Gress R. Pastan I. Cowan K. Cancer Res. 1996; 56: 1346-1351PubMed Google Scholar). Briefly, SP1 cells were grown to 80% confluence. The DNA (1 μg) was mixed with LipofectAMINE reagent (9 μl) in 200 μl of serum-free medium and was incubated for 15 min at room temperature. Before transfection, cells were washed once with 2 ml of serum-free medium. For each transfection, the mixed DNA and LipofectAMINE were combined with 0.8 ml of serum-free RPMI 1640 medium, and the cells were incubated with this transfection mixture. After 5 h of incubation, an equal volume of RPMI/14% FBS was added to the transfection medium, and incubation proceeded for an additional 24 h. For most experiments, pooled transfected cells selected with G418 (450 μg/ml) were used. In one experiment, SP1 cells were transfected with SRC-RF or SRC, and clones were isolated and tested for Src kinase activity and colony forming efficiency. Cell proliferation was carried out as described elsewhere (45Rahimi N. Saulnier R. Nakamura T. Park M. Elliott B. DNA Cell Biol. 1994; 13: 1189-1197Crossref PubMed Scopus (98) Google Scholar). Briefly, SP1 carcinoma cells and Mv1Lu cells were plated at 104cells/well in 24-well plates under the various conditions indicated. DNA synthesis was measured by adding 0.2 μCi of [3H]thymidine (Amersham Pharmacia Biotech, Oakville, ON, Canada) at 24 h. After an additional 24 h, cells were harvested with trypsin/EDTA. Aliquots of cells were placed in 96-well microtiter plates and transferred to filters using a Titertek cell harvester (ICN, Costa Mesa, CA), and [3H]thymidine incorporation was measured in a scintillation counter (Beckman, Mississauga, ON, Canada). Results are expressed as the mean cpm/well ± S.D. of triplicates. Colony growth assays were performed as described previously (49Saulnier R. Bhardwaj B. Klassen J. Leopold D. Rahimi N. Tremblay E. Mosher D. Elliott B. Exp. Cell Res. 1996; 222: 360-369Crossref PubMed Scopus (32) Google Scholar). Briefly, a solution of 1.2% Bactoagar (Difco Lab) was mixed (1:1) with 2× RPMI 1640, supplemented with FBS at final concentrations of 7 or 1% alone or with HGF as indicated, and layered onto 60 × 15-mm tissue culture plates. SP1 cells (103/2.5 ml) were mixed in a 0.36% Bactoagar solution prepared in a similar way and layered (2.5 ml/plate) on top of the 0.6% Bactoagar layer. Plates were incubated at 37 °C in 5% CO2 for 8–10 days. Colonies were fixed with methanol, stained with Giemsa, and counted manually. Results are expressed as mean number of colonies per dish ± S.D. of quadruplicates. To measure cell motility, Transwell culture inserts (8-μm pore size) (Costar, Toronto, ON, Canada) were coated uniformly with gelatin (0.25% w/v, Sigma, Oakville, ON, Canada) on both sides for 2 min at room temperature (50Boyer B. Roche S. Denoyelle M. Thiery J.P. EMBO J. 1997; 16: 5904-5913Crossref PubMed Scopus (124) Google Scholar). Membranes were washed twice with serum-free RPMI 1640 medium and inserted into a 24-well culture plate (Costar, Toronto, ON, Canada) with 1 ml of RPMI 1640 containing 0.5 mg/ml bovine serum albumin (Life Technologies, Inc.). Cells were grown to 50% confluence, serum-starved overnight, and harvested in 5 mm EDTA. Cells (2 × 104/100 μl) were plated in the insert and incubated for 6–8 h at 37 °C. Following the incubation, excess medium was removed, and cells were fixed in 1% paraformaldehyde (Sigma) for 15 min and stained with hematoxylin (Fisher, Oakville, ON, Canada). Cells on the upper side of the membrane were removed by wiping with cotton. Cells on the under side of the membrane were counted using an inverted microscope with phase contrast illumination. Cell motility is expressed as the number of migrating cells per well. In a parallel study, a wounding assay was performed, as described previously (36Rodier J-M. Vallés A.M. Denoyelle M. Thiery J.P. Boyer B. J. Cell Biol. 1995; 131: 761-773Crossref PubMed Scopus (72) Google Scholar). Briefly, monolayers of each cell type were “wounded” by scraping with an Eppendorf yellow tip, washed, and incubated alone or with HGF for varying times. Migration was assessed visually by the ability of cells to close the wounded area. Cells were grown to confluence and serum-starved for 24 h. Cells were rinsed with cold phosphate-buffered saline three times and lysed in a lysis buffer containing 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, 1 mm Na3VO4, 50 mm NaF, 2 mm EGTA, 2 μg/ml aprotinin, 2 μg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride. Lysates were centrifuged for 10 min at 14,000 rpm in an IEC/Micromax centrifuge at 4 °C. Protein concentration of supernatants was determined using a bicinchoninic acid protein assay (Pierce). Equal protein amounts from each cell lysate were incubated with the indicated antibodies at 4 °C for 2 h or overnight. Immunoprecipitates were collected on protein A-Sepharose (Amersham Pharmacia Biotech), washed three times with lysis buffer, separated by SDS-PAGE, and transferred to a nitrocellulose membrane. The membrane was blocked for 15 min with 3% skimmed milk in TBST (10 mm Tris-HCl, pH 8.0, 150 mm NaCl, 0.1% Tween 20), and probed for 1 h with the indicated antibodies. The membrane was washed three times for 5 min each with TBST buffer, incubated with horseradish peroxidase-labeled secondary anti-rabbit or anti-mouse antibodies for 15 min, and washed three times with TBST for 10 min each time. Immune complexes were detected using ECL (Amersham). In most experiments, anin vitro c-Src kinase assay using enolase as a substrate was performed as described previously (51Atfi A. Drobetsky E. Boissonneault M. Chapdelaine A. Chevalier S. J. Biol. Chem. 1994; 269: 30688-30693Abstract Full Text PDF PubMed Google Scholar). Briefly, lysates from SP1 and Mv1Lu cells were prepared, and equal protein amounts from each cell lysate were immunoprecipitated with anti-c-Src IgG (Santa Cruz Biotechnology) as described above. The amount of anti-c-Src IgG was pre-determined to be in excess over c-Src protein, indicating that the majority of c-Src protein in cell lysates is immunoprecipitated (data not shown). One-half of each immunoprecipitate was subjected to SDS-PAGE under nonreducing conditions and Western blot analysis to confirm the amount of c-Src protein present. The other half of each immunoprecipitate was assayed for c-Src kinase activity, by incubating with 10 μl of reaction buffer (20 mm PIPES, pH 7.0, 10 mm MnCl2, 10 μmNa3VO4), 1 μl of freshly prepared acid-denatured enolase (Sigma) (5 μg of enolase + 1 μl of 50 mm HCl incubated at 30 °C for 10 min then neutralized with 1 μl of 1 m PIPES, pH 7.0), and 10 μCi of [γ-32P]ATP. After 10 min of incubation at 30 °C, reactions were terminated by the addition of 2× SDS sample buffer, and samples were subjected to 8% SDS-PAGE. Serine and threonine phosphorylations were hydrolyzed by incubating the acrylamide gel in 1m KOH at 45 °C for 30 min, followed by fixing in 45% MeOH and 10% acetic acid for 30 min at room temperature and drying for 2 h at 80 °C under a vacuum. Autoradiograms were produced and quantitated using a Storm PhosphorImager (Molecular Dynamics, Sunnyvale, CA). In some experiments (see Fig. 3), c-Src kinase activity was assayed according to Cheng et al. (52Cheng H. Nishio H. Hatase O. Ralph S. Wang J. J. Biol. Chem. 1992; 267: 9248-9256Abstract Full Text PDF PubMed Google Scholar) using the c-Src tyrosine kinase family-specific cdc2 peptide substrate. Anti-c-Src or anti-Met immunoprecipitates prepared as above were incubated with 40 μl of a reaction buffer (100 mm Tris-HCl, pH 7.0, 0.4 mm EGTA, 0.4 mm Na3VO4, 40 mm Mg(OAc)2), 5 μl of cdc2 peptide (Life Technologies, Inc., 250 μm/assay), 5 μl of cold ATP (25 μm), and 2.5 μCi of [γ-32P]ATP. A control consisting of immunoprecipitation with anti-Met IgG under more stringent conditions with RIPA buffer (150 mm NaCl, 1.0% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mm Tris, pH 8.0) where c-Src would not be co-precipitated was also carried out. After 15 min of incubation at 37 °C, reactions were terminated by the addition of 20 μl of 40% trichloroacetic acid and incubated for an additional 5 min. Aliquots subsequently were blotted on to p81 paper (Whatman, Fisher, Ottawa, ON, Canada). The p81 paper was washed three times (5 min/wash) with 0.75% phosphoric acid and once with acetone at room temperature, and the radiolabeled c-Src kinase substrate was counted in a liquid scintillation counter. Cell lysates from SP1 and Mv1Lu cells were prepared, and equal protein amounts of each lysate were immunoprecipitated with anti-Met IgG as described above. Immunoprecipitates were washed twice with cold lysis buffer and once with cold kinase buffer (20 mm PIPES, pH 7.0, 10 mm MnCl2, 10 μmNa3VO4). In vitro Met kinase activity was determined by incubating immunoprecipitates with 20 μl of kinase buffer containing 10 μCi of [γ-32P]ATP at 30 °C for 10 min. The reaction was stopped by addition of 2× SDS sample buffer containing 5% β-mercaptoethanol. Samples were boiled for 3 min and subjected to 7% SDS-PAGE. Serine and threonine phosphorylations were hydrolyzed by incubating the acrylamide gel in 1m KOH at 45 °C for 30 min, followed by fixing and drying as described above. Autoradiograms were produced and quantitated using a Storm PhosphorImager (Molecular Dynamics). SP1 carcinoma cells express HGF and tyrosine-phosphorylated Met, consistent with an HGF autocrine loop in these cells (44Rahimi N. Tremblay E. McAdam L. Park M. Schwall R. Elliott B. Cell Growth & Differ. 1996; 7: 263-270PubMed Google Scholar). To test the possibility that activation of c-Src kinase may be involved in Met-induced signaling pathways, we measured the kinase activity of c-Src in SP1 carcinoma cells and an HGF-sensitive epithelial cell line, Mv1Lu. c-Src kinase activity was measured by the capacity of c-Src immunoprecipitates from these cells to tyrosine phosphorylate the substrate, enolase. c-Src immunoprecipitates from serum-starved SP1 cells showed a pronounced elevated kinase activity, which increased only slightly following treatment with exogenous HGF (Fig. 1). In contrast, c-Src kinase activity in Mv1Lu cells was highly dependent on stimulation of cells with exogenous HGF (Fig. 1). The levels of c-Src kinase activity observed correlated with the constitutive tyrosine phosphorylation of Met (44Rahimi N. Tremblay E. McAdam L. Park M. Schwall R. Elliott B. Cell Growth & Differ. 1996; 7: 263-270PubMed Google Scholar) and in vitro Met kinase activity (data not shown) in SP1 cells, and the HGF-induced tyrosine phosphorylation of Met in Mv1Lu cells (Ref. 26Rahimi N. Tremblay E. Elliott B. J. Biol. Chem. 1996; 271: 24850-24855Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar and data not shown). It is conceivable that the high level of c-Src kinase activity in SP1 cells, could have resulted from interaction of c-Src with activated Met due to an autocrine HGF loop in these cells (44Rahimi N. Tremblay E. McAdam L. Pa" @default.
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• W2068994296 title "c-Src Kinase Activity Is Required for Hepatocyte Growth Factor-induced Motility and Anchorage-independent Growth of Mammary Carcinoma Cells" @default.
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