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• W2151824014 abstract "Membrane type 1 matrix metalloproteinase (MT1-MMP) is a transmembrane MMP that plays important roles in migratory processes underlying tumor invasion and angiogenesis. In addition to its matrix degrading activity, MT1-MMP also contains a short cytoplasmic domain whose involvement in cell locomotion seems important but remains poorly understood. In this study, we show that MT1-MMP is phosphorylated on the unique tyrosine residue located within this cytoplasmic sequence (Tyr573) and that this phosphorylation requires the kinase Src. Using phosphospecific antibodies recognizing MT1-MMP phosphorylated on Tyr573, we observed that tyrosine phosphorylation of the enzyme is rapidly induced upon stimulation of tumor and endothelial cells with the platelet-derived chemoattractant sphingosine-1-phosphate, suggesting a role in migration triggered by this lysophospholipid. Accordingly, overexpression of a nonphosphorylable MT1-MMP mutant (Y573F) blocked sphingosine-1-phosphate-induced migration of Human umbilical vein endothelial cells and HT-1080 (human fibrosarcoma) cells and failed to stimulate migration of cells lacking the enzyme (bovine aortic endothelial cells). Altogether, these findings strongly suggest that the Src-dependent tyrosine phosphorylation of MT1-MMP plays a key role in cell migration and further emphasize the importance of the cytoplasmic domain of the enzyme in this process. Membrane type 1 matrix metalloproteinase (MT1-MMP) is a transmembrane MMP that plays important roles in migratory processes underlying tumor invasion and angiogenesis. In addition to its matrix degrading activity, MT1-MMP also contains a short cytoplasmic domain whose involvement in cell locomotion seems important but remains poorly understood. In this study, we show that MT1-MMP is phosphorylated on the unique tyrosine residue located within this cytoplasmic sequence (Tyr573) and that this phosphorylation requires the kinase Src. Using phosphospecific antibodies recognizing MT1-MMP phosphorylated on Tyr573, we observed that tyrosine phosphorylation of the enzyme is rapidly induced upon stimulation of tumor and endothelial cells with the platelet-derived chemoattractant sphingosine-1-phosphate, suggesting a role in migration triggered by this lysophospholipid. Accordingly, overexpression of a nonphosphorylable MT1-MMP mutant (Y573F) blocked sphingosine-1-phosphate-induced migration of Human umbilical vein endothelial cells and HT-1080 (human fibrosarcoma) cells and failed to stimulate migration of cells lacking the enzyme (bovine aortic endothelial cells). Altogether, these findings strongly suggest that the Src-dependent tyrosine phosphorylation of MT1-MMP plays a key role in cell migration and further emphasize the importance of the cytoplasmic domain of the enzyme in this process. The degradation of extracellular matrix (ECM) 3The abbreviations used are: ECM, extracellular matrix; MT1-MMP, membrane-type matrix metalloproteinase; S1P, sphingosine-1-phosphate; siRNA, small interfering RNA; BAEC, bovine aortic endothelial cells; HUVEC, human umbilical vein endothelial cells; HT-1080, human fibrosarcoma cells; FBS, fetal bovine serum; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MES, 4-morpholineethanesulfonic acid.3The abbreviations used are: ECM, extracellular matrix; MT1-MMP, membrane-type matrix metalloproteinase; S1P, sphingosine-1-phosphate; siRNA, small interfering RNA; BAEC, bovine aortic endothelial cells; HUVEC, human umbilical vein endothelial cells; HT-1080, human fibrosarcoma cells; FBS, fetal bovine serum; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MES, 4-morpholineethanesulfonic acid. proteins by members of the matrix metalloproteinases (MMPs) plays a crucial role in several biological processes, including cell attachment, cell migration, invasiveness, cell proliferation, apoptosis, and angiogenesis (1Egeblad M. Werb Z. Nat. Rev. Cancer. 2001; 2: 163-176Crossref Google Scholar, 2Edwards D.R. Murphy G. Nature. 1998; 394: 527-528Crossref PubMed Scopus (131) Google Scholar, 3Folgueras A.R. Pendas A.M. Sanchez L.M. Lopez-Otin C. Int. J. Dev. Biol. 2004; 48: 411-424Crossref PubMed Scopus (479) Google Scholar, 4van Hinsbergh V.W. Engelse M.A. Quax P.H. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 716-728Crossref PubMed Scopus (322) Google Scholar). Among the various MMPs described to date, there is now considerable evidence that MMPs that are intrinsically associated with the plasma membrane because of the presence of a transmembrane domain within their sequence, the so-called membrane type MMPs, represent key components involved in pericellular proteolysis and subsequent cell locomotion and invasion (5Itoh Y. Seiki M. J. Cell. Physiol. 2006; 206: 1-8Crossref PubMed Scopus (404) Google Scholar, 6Haas T.L. Can. J. Physiol. Pharmacol. 2005; 83: 1-7Crossref PubMed Scopus (61) Google Scholar). The prototypical member of this family, MT1-MMP, actively participates in the remodeling of the pericellular ECM by acting as a cellular receptor and activator of proMMP-2 (7Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2361) Google Scholar) and as a potent matrix-degrading protease that proteolyses a broad spectrum of ECM proteins (8Pei D. Weiss S.J. J. Biol. Chem. 1996; 271: 9135-9140Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 9d'Ortho M.P. Will H. Atkinson S. Butler G. Messent A. Gavrilovic J. Smith B. Timpl R. Zardi L. Murphy G. Eur. J. Biochem. 1997; 250: 751-757Crossref PubMed Scopus (382) Google Scholar, 10Hiraoka N. Allen E. Apel I.J. Gyetko M.R. Weiss S.J. Cell. 1998; 95: 365-377Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar) as well as a number of cell surface-associated adhesion receptors (11Kajita M. Itoh Y. Chiba T. Mori H. Okada A. Kinoh H. Seiki M. J. Cell Biol. 2001; 153: 893-904Crossref PubMed Scopus (608) Google Scholar, 12Belkin A.M. Akimovm S.S. Zaritskaya L.S. Ratnikov B.I. Deryugina E.I. Strongin A.Y. J. Biol. Chem. 2001; 276: 18415-18422Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). These events are likely to be important in vivo because MT1-MMP null mice fail to thrive and have a markedly reduced lifespan (13Holmbeck K. Bianco P. Caterina J. Yamada S. Kromer M. Kuznetsov S.A. Mankani M. Robey P.G. Poole A.R. Pidoux I. Ward J.M. Birkedal-Hansen H. Cell. 1999; 99: 81-92Abstract Full Text Full Text PDF PubMed Scopus (1095) Google Scholar, 14Zhou Z. Apte S.S. Soininen R. Cao R. Baaklini G.Y. Rauser R.W. Wang J. Cao Y. Tryggvason K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4052-4057Crossref PubMed Scopus (676) Google Scholar). In addition to its role in normal physiology, MT1-MMP is also overexpressed in many types of tumors (15Nakada M. Nakamura H. Ikeda E. Fujimoto N. Yamashita J. Sato H. Seiki M. Okada Y. Am. J. Pathol. 1999; 154: 417-428Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 16Zhai Y. Hotary K.B. Nan B. Bosch F.X. Munoz N. Weiss S.J. Cho K.R. Cancer Res. 2005; 65: 6543-6550Crossref PubMed Scopus (97) Google Scholar), and this overexpression appears crucial for tumor cell migration and invasion. For example, MT1-MMP-mediated degradation of some ECM proteins, such as the laminin-5γ2 chain (17Koshikawa N. Minegishim T. Sharabi A. Quaranta V. Seiki M. J. Biol. Chem. 2005; 280: 88-93Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), stimulates migration, whereas proteolysis of the dense, cross-linked meshwork of type I collagen fibrils by the enzyme confers neoplastic cells with tissue-invasive activity (18Sabeh F. Ota I. Holmbeck K. Birkedal-Hansen H. Soloway P. Balbin M. Lopez-Otin C. Shapiro S. Inada M. Krane S. Allen E. Chung D. Weiss S.J. J. Cell Biol. 2004; 167: 769-781Crossref PubMed Scopus (476) Google Scholar) and sustains tumor cell growth in otherwise growth-restrictive three-dimensional matrices (19Hotary K.B. Allen E.D. Brooks P.C. Datta N.S. Long M.W. Weiss S.J. Cell. 2003; 114: 33-45Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar). Despite its importance to normal physiology and in the development of malignancy, the mechanisms underlying MT1-MMP-mediated cell invasion remain incompletely understood. During cell migration, MT1-MMP localizes predominantly to the cell adherent edge at the migration front, an appropriate location for the degradation of the ECM barrier (20Sato T. del Carmen Ovejero M. Hou P. Heegaard A.M. Kumegawa M. Foged N.T. Delaisse J.M. J. Cell Sci. 1997; 110: 589-596Crossref PubMed Google Scholar). In addition, we and others have shown that MT1-MMP is preferentially localized into caveolae, specialized domains of the plasma membrane (21Annabi B. Lachambre M-P. Bousquet-Gagnon N. Pagé M. Gingras D. Béliveau R. Biochem. J. 2001; 353: 547-553Crossref PubMed Scopus (133) Google Scholar, 22Puyraimond A. Fridman R. Lemesle M. Arbeille B. Menashi S. Exp. Cell Res. 2001; 262: 28-36Crossref PubMed Scopus (152) Google Scholar), and this localization may contribute to the spatiotemporal regulation of its proteolytic activity by controlling adequate endocytosis and recycling of the enzyme (23Galvez B.G. Matias-Roman S. Yanez-Mo M. Vicente-Manzanares M. Sanchez-Madrid F. Arroyo A.G. Mol. Biol. Cell. 2004; 15: 678-687Crossref PubMed Scopus (146) Google Scholar, 24Remacle A. Murphy G. Roghi C. J. Cell Sci. 2003; 116: 3905-3916Crossref PubMed Scopus (209) Google Scholar). In addition to the importance of MT1-MMP-mediated proteolytic breakdown of ECM proteins for the induction of cell migration, recent studies suggested that the cytoplasmic domain of the enzyme may also play a role in this process. For example, MT1-MMP mutants lacking the cytoplasmic domain remain localized at the cell surface and failed to induce migration, suggesting an important role for the enzyme cytoplasmic sequence in the regulation of its activity (25Lethi K. Valtanen H. Wickstrom S. Lohi J. Keski-Oja J. J. Biol. Chem. 2000; 275: 15006-15013Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 26Jiang A. Lethi K. Wang X. Weiss S.J. Keski-Oja J. Pei D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13693-13698Crossref PubMed Scopus (221) Google Scholar, 27Ukeita T. Itoh Y. Yana I. Ohno H. Seiki M. J. Cell Biol. 2001; 155: 1345-1356Crossref PubMed Scopus (213) Google Scholar). In this respect, the cytoplasmic domain of MT1-MMP has been shown to be involved in several aspects of enzyme activity, including the formation of oligomers (28Lethi K. Lohi J. Juntunen M.M. Pei D. Keski-Oja J. J. Biol. Chem. 2002; 277: 8440-8448Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 29Rozanov D.V. Deryugina E.I. Ratnikov B.I. Monosov E.Z. Marchenko G.N. Quigley J.P. Strongin A.Y. J. Biol. Chem. 2001; 276: 25705-25714Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), the localization of the enzyme to invadopodia (30Nakahara H. Howard L. Thompson E.W. Sato H. Seiki M. Yeh Y. Chen W.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7959-7964Crossref PubMed Scopus (357) Google Scholar), the activation of the extracellular signal-regulated protein kinase signaling pathway (31Gingras D. Bousquet-Gagnon N. Langlois S. Lachambre M-P. Annabi B. Béliveau R. FEBS Lett. 2001; 507: 231-236Crossref PubMed Scopus (102) Google Scholar), and its interaction with a number of intracellular proteins such as cupin (32Uekita T. Gotoh I. Kinoshita T. Itoh Y. Sato H. Shiomi T. Okada Y. Seiki M. J. Biol. Chem. 2004; 279: 12734-12743Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), the μ2 subunit of adaptor protein-2 (27Ukeita T. Itoh Y. Yana I. Ohno H. Seiki M. J. Cell Biol. 2001; 155: 1345-1356Crossref PubMed Scopus (213) Google Scholar), and tyrosine-phosphorylated caveolin-1 (33Labrecque L. Nyalendo C. Langlois S. Durocher Y. Roghi C. Murphy G. Gingras D. Béliveau R. J. Biol. Chem. 2004; 279: 52132-52140Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Although the mechanisms by which the MT1-MMP cytoplasmic sequence is implicated in these processes remain poorly understood, recent observations indicate that it could involve an important function of this domain in the cooperation of the enzyme with serum-derived chemoattractant molecules such as sphingosine-1-phosphate (S1P), a bioactive lipid secreted by activated platelets (34Langlois S. Gingras D. Béliveau R. Blood. 2004; 103: 3020-3028Crossref PubMed Scopus (85) Google Scholar). In this study, we report that MT1-MMP is tyrosine-phosphorylated within its cytoplasmic domain, in a Src-dependent manner. This tyrosine phosphorylation is observable upon stimulation of endothelial and tumor cells with S1P and seems important for both tumor and endothelial cell migration triggered by this lipid. Reagents and Antibodies—Cell culture media, antibiotics (penicillin and streptomycin) and glutamine were purchased from Invitrogen. Trypsin was obtained from Sigma. Basic fibroblast growth factor was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). PolyFect and HiPerFect transfection reagents, control, and caveolin-1 siRNAs were purchased from Qiagen. The TransPass D2 transfection reagent was from New England BioLabs Inc. (Ipswitch, MA). Antibodies against caveolin (610059 and 610406) and caveolin pY14 (611338) were obtained from BD Transduction Laboratories (Mississauga, Canada); anti-MT1-MMP (AB815 and MAB3328) were from Chemicon International (Temecula, CA); anti-Tyr(P) (pY99) and c-Myc (clone 9E10) were from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Src (clone GD11) was from Upstate Biotechnology, Inc.. Mouse and rabbit horseradish peroxidase-conjugated secondary antibodies were purchased from Jackson ImmunoResearch (Mississauga, Canada). Mouse Alexa 488-conjugated (A21202) and rabbit rhodamine-conjugated (T2769) antibodies were from Molecular Probes (Invitrogen). Protein G- and A-coupled Sepharose, immobilized pH gradient strips, and apparatus for isoelectric focusing were from Amersham Biosciences. Electrophoresis apparatus and reagents were purchased from Bio-Rad. Polyvinylidene difluoride transfer membranes and Western Lightning Chemiluminescence Reagent Plus were obtained from PerkinElmer Life Sciences. Cell Culture—Bovine aortic endothelial cells (BAEC), human umbilical vein endothelial cells (HUVEC), human fibrosarcoma cells (HT-1080), and monkey kidney cells (COS-7) were purchased from Clonetics and were cultured at 37 °C in a humidified atmosphere containing 5% CO2. BAEC were cultured in Dulbecco's modified Eagle's medium with low glucose supplemented with 10% bovine calf serum, 10 ng/ml basic fibroblast growth factor, 100 IU/ml penicillin, 100 mg/ml streptomycin, and 4 mm glutamine. HUVEC were maintained in endothelial cell growth medium BulletKit (EGM-2) supplemented with 2% fetal bovine serum (FBS), human epidermal growth factor, hydrocortisone, vascular endothelial growth factor, human basic fibroblast growth factor, insulin-like growth factor 1, ascorbic acid, heparin, gentanamycin, and amphotericin-B. HT-1080 were grown in minimum essential medium supplemented with 1 mm pyruvate, 10% FBS, 100 IU/ml penicillin, 100 mg/ml streptomycin, and 4 mm glutamine. COS-7 were grown in Dulbecco's modified Eagle's medium with high glucose supplemented with 10% FBS, 100 IU/ml penicillin, 100 mg/ml streptomycin, and 4 mm glutamine. Transfection—The cDNAs encoding the full-length human MT1-MMP, its cytoplasmic domain-deleted (CΔ20), and catalytically inactive (E240A) mutants have been previously described (31Gingras D. Bousquet-Gagnon N. Langlois S. Lachambre M-P. Annabi B. Béliveau R. FEBS Lett. 2001; 507: 231-236Crossref PubMed Scopus (102) Google Scholar, 34Langlois S. Gingras D. Béliveau R. Blood. 2004; 103: 3020-3028Crossref PubMed Scopus (85) Google Scholar). The MT1-MMP cytoplasmic mutants L571A/L572A/Y573A and Y573F were produced by site-directed mutagenesis, as previously described (33Labrecque L. Nyalendo C. Langlois S. Durocher Y. Roghi C. Murphy G. Gingras D. Béliveau R. J. Biol. Chem. 2004; 279: 52132-52140Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The Myc-tagged full-length MT1-MMP (MycMT1), in which the Myc epitope was inserted in the hinge region of the enzyme between proline 312 and threonine 313 (PEQKLISEEDLT), was synthesized using the overlap extension method. The wild-type and dominant-negative Src plasmids were kindly provided by Dr. Isabelle Royal (Université de Montréal, Montréal, Canada). Transient transfection of COS-7 cells was performed using the PolyFect transfection reagent (Qiagen). HT-1080 cells were transiently transfected using the FuGENE 6 transfection reagent (Roche Applied Science). HUVEC and BAEC were transiently transfected with TransPass D2 transfection reagent (BioLabs). All of the transfections were performed according to the manufacturer's instructions. Production of Phospho-MT1-MMP(Y573) Antibodies—Polyclonal phosphospecific antibodies were produced by 21st Century Biochemicals (Marlboro, MA). Briefly, antigenic phosphopeptide (GTPRRLL[pY]CQRSL-amide) and nonphosphopeptide (GTPRRLLYCQRSL-amide) were synthesized based on the human MT1-MMP cytoplasmic sequence and purified by high pressure liquid chromatography. The sequences were verified by mass spectrometry. The rabbits were inoculated five times with the phosphopeptide conjugated with a immune carrier, and serum was collected and subjected to affinity depletion using the nonphosphopeptide, followed by affinity purification using a phosphopeptide affinity column. To verify the specificity of the antibodies against the phosphorylated peptide, pMT1-MMP(Y573) antibodies were preincubated for 45 min at 37 °C with a 5-fold molar excess of either the phosphopeptide or the nonphosphopeptide, and immunodetection was performed as described below. Two-dimensional Gel Electrophoresis—For the first dimension, immunoprecipitates bound to protein A-coupled Sepharose beads were solubilized in rehydration buffer (5 m urea, 2 m thiourea, 2% CHAPS, 2% SB3-10, 0.3% dithiothreitol, bromphenol blue) containing ampholytes (pH 4-7). Solubilized proteins were incubated with 7-cm IPG strips containing a linear pH gradient (4van Hinsbergh V.W. Engelse M.A. Quax P.H. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 716-728Crossref PubMed Scopus (322) Google Scholar, 5Itoh Y. Seiki M. J. Cell. Physiol. 2006; 206: 1-8Crossref PubMed Scopus (404) Google Scholar, 6Haas T.L. Can. J. Physiol. Pharmacol. 2005; 83: 1-7Crossref PubMed Scopus (61) Google Scholar, 7Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2361) Google Scholar) (Amersham Biosciences) at room temperature for 10 h, and isoelectric focusing was performed at 500 V for 30 min, 1000 V for 30 min, and then 5000 V for 13 h. For the second dimension, IPG strips were incubated for 15 min at room temperature in equilibration buffer (50 mm Tris-HCl, pH 6.8, 6 m urea, 30% glycerol, 2% SDS) containing 2% dithiothreitol and then for 15 min in equilibration buffer containing 5% iodoacetamide and bromphenol blue. IPG strips were loaded on 7.5% SDS-PAGE, and Western blotting was performed as described below. Caveolae Isolation—Caveolae were purified using a hyperosmotic carbonate method, as described previously (33Labrecque L. Nyalendo C. Langlois S. Durocher Y. Roghi C. Murphy G. Gingras D. Béliveau R. J. Biol. Chem. 2004; 279: 52132-52140Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Briefly, confluent COS-7 cells cultured in 100-mm2 dishes were scraped into 3 ml of 0.5 m sodium carbonate (pH 11) and homogenized extensively using a Polytron (three pulses of 15 s, at speed 4) followed by sonication (five pulses of 15 s, at 70% of maximal power). 2.5 ml of the resulting homogenate was brought to 45% sucrose by the addition of 2.5 ml of 90% sucrose in MES-buffered saline (25 mm MES, pH 6.5, 150 mm NaCl) and overlaid with two layers (6 ml each) of 35 and 5% sucrose in MES-buffered saline containing 0.25 m carbonate. The gradient was then centrifuged at 200,000 × g for 18 h using a Beckman SW41Ti rotor. For analysis of the resulting gradient, 1-ml fractions were collected from the top to the bottom of the gradient. Caveolae-enriched (5-8 fractions) or noncaveolae (12-15 fractions) fractions were pooled, diluted in 10 mm Tris-HCl, pH 7.5, and centrifuged at 100,000 × g for 1 h. Stimulation of HT1080 and HUVEC Cells with S1P—HT1080 and HUVEC cells grown to 90% confluence were serum-starved for 18 h in medium containing 0.5% serum. The cells were then preincubated 2 h at 37 °C with 1 μm PP2 (or an equivalent amount of vehicle), followed by incubation for 2-30 min with 1 μm S1P. The cells were then solubilized in SDS lysis buffer (10 mm Tris-HCl, pH 7.4, 1% SDS, 1 mm sodium orthovanadate), followed by boiling for 5 min at 100 °C and homogenization using a 26-gauge needle. Protein concentrations were determined by the bicinchoninic acid method (Pierce). Immunoprecipitation and Western Blotting—The procedures have been described elsewhere (33Labrecque L. Nyalendo C. Langlois S. Durocher Y. Roghi C. Murphy G. Gingras D. Béliveau R. J. Biol. Chem. 2004; 279: 52132-52140Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Briefly, equal amounts of proteins were incubated in lysis buffer (10 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Triton X-100, 0.5% Nonidet P-40, 1 mm EDTA, 1 mm EGTA, 1 mm sodium vanadate) over-night at 4 °C in the presence of 1-2 μg/ml of specific antibodies, and the immune complexes were collected by incubating the mixtures with protein A- or G-coupled Sepharose beads. Bound material was solubilized in Laemmli sample buffer, boiled for 5 min, and separated by SDS-PAGE. The proteins were transferred onto polyvinylidene difluoride membranes, blocked overnight at 4 °C with Tris buffer saline with 0.1% Tween 20 (TBST) buffer containing 3% bovine serum albumin, and incubated for 1 h at room temperature with the desired primary antibodies. Immunoreactive bands were revealed following 1 h of incubation with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies, and the signals were visualized by chemiluminescence. Densitometric analysis was performed using the IPLab Gel program. Immunofluorescence and Confocal Microscopy—Endothelial cells (HUVEC) were plated on cover glasses coated with 10 μg/ml fibronectin, serum-starved, and treated (or not) with 1 μm S1P for 15 min. After 5 min of incubation with Hoechst 20 μm for nuclei staining, the cells were fixed with 3.7% formaldehyde for 15 min, permeabilized with 0.2% Triton X-100 for 5 min, blocked (1% bovine serum albumin in Tris-buffered saline containing 0.1% Tween) for 30 min, and stained with specific primary antibodies against phopho-MT1-MMP (1/50 dilution), phospho-caveolin-1, and MT1-MMP (1/100 dilution). The cells were incubated with Alexa 488- or rhodamine-conjugated secondary antibodies. The slides were mounted with Immuno-Fluore mounting medium (MP Biomedicals). Immunostaining was visualized and photographed using a Zeiss LSM 510 Meta confocal microscope. Cell Migration—Migration assays of BAEC, HUVEC, or HT1080 cells transfected with either pcDNA3.1, WT, or Y573F forms of MT1-MMP, were performed on transwells precoated with 10 μg/ml fibronectin. The transwells were assembled in 24-well plates, and the lower chambers were filled with serumfree media with or without 1 μm S1P or 10% FBS. Transfected cells were harvested, resuspended in 100 μl of fresh cell media at a density of 5 × 105 cells/ml, and inoculated into the upper chamber of each transwell. The plates were then placed at 37 °C in 5% CO2, 95% air for 3 h, and cells that had migrated were quantified using computer-assisted imaging (Northern Eclipse 6.0; Empix Imaging, Mississauga, Canada). The data are expressed as the average density of migrated cells/4 fields (original magnification, 50×) (34Langlois S. Gingras D. Béliveau R. Blood. 2004; 103: 3020-3028Crossref PubMed Scopus (85) Google Scholar). Statistical analysis was performed by two-way analysis of variance followed by the Bonferroni post-tests. p < 0.05 was considered statistically significant. For the measurement of transfection efficiencies, the total membranes were isolated from HUVEC and BAEC as described (34Langlois S. Gingras D. Béliveau R. Blood. 2004; 103: 3020-3028Crossref PubMed Scopus (85) Google Scholar), and MT1-MMP levels were monitored by immunoblotting. For HT-1080 cells, transfection efficiencies were monitored by zymographic analysis of MT1-MMP-dependent activation of proMMP-2 in as described (21Annabi B. Lachambre M-P. Bousquet-Gagnon N. Pagé M. Gingras D. Béliveau R. Biochem. J. 2001; 353: 547-553Crossref PubMed Scopus (133) Google Scholar). MT1-MMP Is Phosphorylated on Cytoplasmic Tyrosine 573: Involvement of Src Kinase—We previously showed that overexpression of Src induces the tyrosine phosphorylation of caveolin-1 and its subsequent association with MT1-MMP (33Labrecque L. Nyalendo C. Langlois S. Durocher Y. Roghi C. Murphy G. Gingras D. Béliveau R. J. Biol. Chem. 2004; 279: 52132-52140Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Because this interaction required the cytoplasmic domain of MT1-MMP, we hypothesized that the stimulatory effect of Src could also involve the phosphorylation of the unique tyrosine residue located in the cytoplasmic sequence of MT1-MMP. As a first step to examine this possibility, COS-7 cells were transiently transfected with a Myc-tagged version of MT1-MMP along with Src or a kinase-inactive dominant-negative form of Src. MycMT1 was immunoprecipitated using an anti-Myc monoclonal antibody, and the extent of tyrosine phosphorylation was determined by immunoblotting using an anti-phosphotyrosine antibody. As shown in Fig. 1A, immunoprecipitation of MT1-MMP from cells overexpressing MT1-MMP and Src resulted in the appearance of two tyrosine-phosphorylated bands in the 63-66-kDa range, whereas co-expression of MT1-MMP with an inactive Src mutant failed to induce the tyrosine phosphorylation of these proteins. The upper phosphorylated band (66 kDa) shows an electrophoretic mobility similar to that of Myc-tagged MT1-MMP and was also observed upon reverse immunoprecipitation with anti-phosphotyrosine antibody, strongly suggesting it represents a tyrosine-phosphorylated form of MT1-MMP. To further establish whether MT1-MMP is indeed tyrosine-phosphorylated in Src-expressing cells and whether this phosphorylation occurs at an intracellular location, we next examined the effect of various mutants of the enzyme on this event. As shown in Fig. 1B, immunoprecipitation of both Myc-tagged (MycMT1) or WT MT1-MMP resulted in the appearance of tyrosine-phosphorylated bands corresponding to the molecular masses of both forms of the enzyme, indicating that the observed phosphorylation was not restricted to the epitope-tagged version of MT1-MMP. We next examined the requirement for the catalytic activity and cytoplasmic sequence of the enzyme using various mutants. The catalytically inactive mutant (E240A) was also phosphorylated but to a lower extent than the WT enzyme. Because this mutation impairs the refolding of a recombinant MT1-MMP polypeptide (35Koo H.M. Kim J-H. Hwang I.K. Lee S.J. Kim T.-H. Rhee K.H. Lee S.-T. Mol. Cells. 2002; 13: 118-124PubMed Google Scholar), it is possible that the E240A mutation could have induced a global conformational change in the enzyme structure, thus partially preventing MT1-MMP tyrosine phosphorylation. However, removal of the cytoplasmic domain (CΔ20) decreased almost completely tyrosine phosphorylation (Fig. 1B). Because the MT1-MMP cytoplasmic sequence contains only one tyrosine residue at position 573 of the protein, we next examined the effect of mutated versions of MT1-MMP lacking this residue. Interestingly, overexpression of the Y573F and L571A/L572A/Y573A mutants abolished tyrosine phosphorylation, further suggesting that the intracellular tyrosine residue of MT1-MMP is the site of phosphorylation (Fig. 1B). To unambiguously establish that MT1-MMP is phosphorylated on tyrosine 573, we next analyzed immunoprecipitates from cells expressing WT or Y573F forms of MT1-MMP along with Src, using two-dimensional gel electrophoresis. As shown in Fig. 1C, immunoprecipitation of MT1-MMP from cells co-expressing Src resulted in the appearance of at least four well defined tyrosine-phosphorylated spots (vertical arrows). We observed that the more acidic forms migrated at a position identical to immunoreactive MT1-MMP (63 kDa) (horizontal arrow), whereas the more basic forms, with slightly lower molecular masses (60 kDa), were not associated with detectable levels of the enzyme. These 60-kDa forms are, however, likely to represent low abundant tyrosine-phosphorylated MT1-MMP because overexpression of the Y573F mutant completely abolished the tyrosine phosphorylation of all MT1-MMP isoforms (Fig. 1C); these forms were also recognized by phosphospecific MT1-MMP antibodies (see Fig. 3D). Overall, these results indicate that MT1-MMP is tyrosine-phosphorylated in Src-expressing cells and that this event involves its unique cytoplasmic tyrosine 573 residue. Caveolin-1 and Caveolae Are Not Necessary for MT1-MMP Phosphorylation—Based on our observation that tyrosine-phosphorylated caveolin-1 interacts with MT1-MMP, we next examined whether the tyrosine phosphorylation of MT1-MMP is involved in its interaction with caveolin-1 (33Labrecque L. Nyalendo C. Langlois S. Durocher Y. Roghi C. Murphy G. Gingras D. Béliveau R. J. Biol. Chem. 2004; 279: 52132-52140Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). First, COS-7 cells were transfected with either wild-type MT1-MMP or the nonphosphorylable MT1-MMP mutant (Y573F), and the presenc" @default.
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