Phosphorylation and Free Pool of β-Catenin Are Regulated by Tyrosine Kinases and Tyrosine Phosphatases during Epithelial Cell Migration
1999; Elsevier BV; Volume: 274; Issue: 15 Linguagem: Inglês
10.1074/jbc.274.15.10173
ISSN1083-351X
AutoresThomas Müller, Axel Choidas, Ernst Reichmann, Axel Ullrich,
Tópico(s)Protein Tyrosine Phosphatases
ResumoCell migration requires precise control, which is altered or lost when tumor cells become invasive and metastatic. Although the integrity of cell-cell contacts, such as adherens junctions, is essential for the maintenance of functional epithelia, they need to be rapidly disassembled during migration. The transmembrane cell adhesion protein E-cadherin and the cytoplasmic catenins are molecular elements of these structures. Here we demonstrate that epithelial cell migration is accompanied by tyrosine phosphorylation of β-catenin and an increase of its free cytoplasmic pool. We show further that the protein-tyrosine phosphatase LAR (leukocyte common antigen related) colocalizes with the cadherin-catenin complex in epithelial cells and associates with β-catenin and plakoglobin. Interestingly, ectopic expression of protein-tyrosine phosphatase (PTP) LAR inhibits epithelial cell migration by preventing phosphorylation and the increase in the free pool of β-catenin; moreover, it inhibits tumor formation in nude mice. These data support a function for PTP LAR in the regulation of epithelial cell-cell contacts at adherens junctions as well as in the control of β-catenin signaling functions. Thus PTP-LAR appears to play an important role in the maintenance of epithelial integrity, and a loss of its regulatory function may contribute to malignant progression and metastasis. Cell migration requires precise control, which is altered or lost when tumor cells become invasive and metastatic. Although the integrity of cell-cell contacts, such as adherens junctions, is essential for the maintenance of functional epithelia, they need to be rapidly disassembled during migration. The transmembrane cell adhesion protein E-cadherin and the cytoplasmic catenins are molecular elements of these structures. Here we demonstrate that epithelial cell migration is accompanied by tyrosine phosphorylation of β-catenin and an increase of its free cytoplasmic pool. We show further that the protein-tyrosine phosphatase LAR (leukocyte common antigen related) colocalizes with the cadherin-catenin complex in epithelial cells and associates with β-catenin and plakoglobin. Interestingly, ectopic expression of protein-tyrosine phosphatase (PTP) LAR inhibits epithelial cell migration by preventing phosphorylation and the increase in the free pool of β-catenin; moreover, it inhibits tumor formation in nude mice. These data support a function for PTP LAR in the regulation of epithelial cell-cell contacts at adherens junctions as well as in the control of β-catenin signaling functions. Thus PTP-LAR appears to play an important role in the maintenance of epithelial integrity, and a loss of its regulatory function may contribute to malignant progression and metastasis. epithelial-mesenchymal transition protein-tyrosine phosphatase human PTP tyrosine kinase adenomatous polyposis coli lymphoid enhancer factor 1 epidermal growth factor polymerase chain reaction polyacrylamide gel electrophoresis glutathione S-transferase phosphate-buffered saline leukocyte common antigen related The cadherins represent a family of transmembrane receptors that mediate homophilic, Ca2+-dependent cell-cell adhesion. In epithelial cells, the members of this family, such as the classical E-, N-, and P-cadherins, are primarily found at the adherens junctions of adjacent cells (1Yap A.S. Brieher W.M. Gumbiner B.M. Annu. Rev. Cell Dev. Biol. 1997; 13: 119-146Crossref PubMed Scopus (685) Google Scholar). β-Catenin as well as plakoglobin (γ-catenin) associate directly with the highly conserved cytoplasmic domain of classical cadherins in a mutually exclusive manner (2Ozawa M. Baribault H. Kemler R. EMBO J. 1989; 8: 1711-1718Crossref PubMed Scopus (1143) Google Scholar, 3Hinck L. Näthke I.S. Papkoff J. Nelson W.J. J. Cell Biol. 1994; 125: 1327-1340Crossref PubMed Scopus (556) Google Scholar). The cadherin-catenin complex is linked via α-catenin either directly (4Rimm D.L. Koslov E.R. Kebriaei P. Cianci C.D. Morrow J.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8813-8817Crossref PubMed Scopus (631) Google Scholar) or indirectly to the actin filament network via the actin-binding proteins α-actinin or vinculin (5Knudsen K.A. Soler A.P. Johnson K.R. Wheelock M.J. J. Cell Biol. 1995; 130: 67-77Crossref PubMed Scopus (560) Google Scholar, 6Weiss E.E. Kroemker M. Rudiger A.H. Jockusch B.M. Rudiger M. J. Cell Biol. 1998; 141: 755-764Crossref PubMed Scopus (214) Google Scholar). The association of the cadherin-catenin complex with the cytoskeleton is essential for tight cell-cell interaction.Nevertheless, cadherin/catenin-mediated cell-cell contacts have to be highly dynamic because, particularly during embryonic development or wound healing, adherens junctions have to be rapidly disassembled and reassembled (7Marrs J.A. Nelson W.J. Int. Rev. Cytol. 1996; 165: 159-205Crossref PubMed Google Scholar). Down-regulation of cadherins results in the separation of neighboring cells, a phenomenon that is observed during embryonic development at the epithelial-mesenchymal transition (EMT)1 of forming mesoderm (8Huber O. Bierkamp C. Kemler R. Curr. Opin. Cell Biol. 1996; 8: 685-691Crossref PubMed Scopus (308) Google Scholar) as well as in tumor cells, allowing their invasion and dissemination throughout the body (9Becker K.F. Atkinson M.J. Reich U. Becker I. Nekarda H. Siewert J.R. Höfler H. Cancer Res. 1994; 54: 3845-3852PubMed Google Scholar). During epithelial-mesenchymal transition, cells transiently lose their epithelial features and acquire a fibroblastoid morphology (10Thiery J.P. Duband J.L. Tucker G.C. Annu. Rev. Cell Biol. 1985; 1: 91-113Crossref PubMed Scopus (118) Google Scholar). The critical importance of an intact cadherin-catenin complex is underscored by the observation that down-regulation of any of its components resulting in the loss of the tumor-suppressive actions of adherens junctions correlates with tumor invasion and metastasis (11Birchmeier W. Hülsken J. Behrens J. CIBA Found. Symp. 1995; 189: 124-141PubMed Google Scholar). Moreover, the integrity of adherens junctions appears to be dynamically regulated by tyrosine phosphorylation. Transfection of a v-src oncogene (12Behrens J. Vakaet L. Friis R. Winterhager E. Van Roy F. Mareel M.M. Birchmeier W. J. Cell Biol. 1993; 120: 757-766Crossref PubMed Scopus (838) Google Scholar, 13Hamaguchi M. Matsuyoshi N. Ohnishi Y. Gotoh B. Takeichi M. Nagai Y. EMBO J. 1993; 12: 307-314Crossref PubMed Scopus (386) Google Scholar) or treatment with growth factors (14Shibamoto S. Hayakawa M. Takeuchi K. Hori T. Oku N. Miyazawa K. Kitamura N. Takeichi M. Ito F. Cell Adhes. Commun. 1994; 1: 295-305Crossref PubMed Scopus (401) Google Scholar, 15Fujii K. Furukawa F. Matsuyoshi N. Exp. Cell Res. 1996; 223: 50-62Crossref PubMed Scopus (48) Google Scholar) causes unstable cell-cell adhesion and migration of cells, and inhibition of PTPs enhances this destabilizing effect (16Volberg T. Zick Y. Dror R. Sabany I. Gilon C. Levitzki A. Geiger B. EMBO J. 1992; 11: 1733-1742Crossref PubMed Scopus (269) Google Scholar). The model in which reversible tyrosine phosphorylation serves to regulate cadherin-mediated cell-cell adhesion is further supported by the demonstration of cadherin-catenin complex association with the receptor tyrosine kinases EGF receptor and human EGF receptor 2/Neu (17Hoschuetzky H. Aberle H. Kemler R. J. Cell Biol. 1994; 127: 1375-1380Crossref PubMed Scopus (670) Google Scholar, 18Ochiai A. Akimoto S. Kanai Y. Shibata T. Oyama T. Hirohashi S. Biochem. Biophys. Res. Commun. 1994; 205: 73-78Crossref PubMed Scopus (141) Google Scholar) as well as with the transmembrane PTPs μ, κ, and λ (19Brady-Kalnay S.M. Rimm D.L. Tonks N.K. J. Cell Biol. 1995; 130: 977-986Crossref PubMed Scopus (287) Google Scholar, 20Fuchs M. Müller T. Lerch M.M. Ullrich A. J. Biol. Chem. 1996; 271: 16712-17719Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 21Cheng J. Wu K. Armanini M. O'Rourke N. Dowbenko D. Lasky L.A. J. Biol. Chem. 1997; 272: 7264-7277Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar).β-Catenin and plakoglobin are mammalian homologues of theDrosophila protein Armadillo, whose function is critical for normal segmental pattern formation during development (22Riggleman B. Wieschaus E. Schedl P. Genes Dev. 1989; 3: 96-113Crossref PubMed Scopus (235) Google Scholar). The presence of a repeating 42-amino acid sequence motif defines members of the "armadillo family" (23Peifer M. Reynolds A.B. Cell. 1994; 76: 789-791Abstract Full Text PDF PubMed Scopus (545) Google Scholar). Data obtained in theDrosophila and Xenopus systems suggest an additional function for β-catenin independent of cadherin-mediated cell adhesion. This involves translocation of β-catenin to the nucleus that is preceded by its accumulation in the cytoplasm. Thus, free β-catenin is involved in transcriptional regulation of specific genes that are essential for embryonic development (24Miller J.R. Moon R.T. Genes Dev. 1996; 10: 2527-2539Crossref PubMed Scopus (606) Google Scholar). The signals resulting in a free pool of β-catenin include the binding of Wingless/Wnt to its transmembrane receptor Frizzled and the inhibition of the serine/threonine kinase Zeste-White 3 (Shaggy) or its vertebrate homologue glycogen synthase kinase 3 (25Moon R.T. Miller J.R. Trends Genet. 1997; 13: 256-258Abstract Full Text PDF PubMed Scopus (41) Google Scholar). Further functions for β-catenin and plakoglobin are indicated by their association with the adenomatous polyposis coli (APC) tumor suppressor protein, which is thought to serve as a cytoplasmic effector of β-catenin, negatively regulating the accumulation of cytosolic β-catenin in concert with glycogen synthase kinase 3 (26Bullions L.C. Levine A.J. Curr. Opin. Oncol. 1998; 10: 81-87Crossref PubMed Scopus (185) Google Scholar) and axin/conductin (27Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1093) Google Scholar, 28Behrens J. Jerchow B.A. Wurtele M. Grimm J. Asbrand C. Wirtz R. Kuhl M. Wedlich D. Birchmeier W. Science. 1998; 280: 596-599Crossref PubMed Scopus (1104) Google Scholar, 29Sakanaka C. Weiss J.B. Williams L.T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3020-3023Crossref PubMed Scopus (282) Google Scholar) by inducing ubiquitin-dependent degradation of β-catenin (30Aberle H. Bauer A. Stappert J. Kispert A. Kemler R. EMBO J. 1997; 16: 3797-3804Crossref PubMed Scopus (2135) Google Scholar).In this report we show that during epithelial migration, β-catenin accumulates in the cytosol in a free, uncomplexed and tyrosine-phosphorylated form. However, in confluent cells, β-catenin maintains epithelial cell integrity as an essential part of the cadherin-catenin tumor suppressor system. Our findings suggest that tyrosine phosphorylation regulates the function of β-catenin as a signaling molecule during epithelial cell migration. We further demonstrate PTP LAR to be a modulator of epithelial cell migration, which strongly supports a function of this protein tyrosine phosphatase in the regulation of cell-cell contacts and epithelial cell integrity. Moreover, we show that ectopic expression of PTP LAR inhibits tumor formation in nude mice. A dysfunction of PTP LAR may therefore lead to tumor invasion and metastasis.DISCUSSIONCadherin-catenin complex-mediated cell-cell adhesion as well as adhesion-independent functions of catenins have been implicated in the modulation of multicellular differentiation, proliferation, and malignant transformation of epithelial cells (7Marrs J.A. Nelson W.J. Int. Rev. Cytol. 1996; 165: 159-205Crossref PubMed Google Scholar, 8Huber O. Bierkamp C. Kemler R. Curr. Opin. Cell Biol. 1996; 8: 685-691Crossref PubMed Scopus (308) Google Scholar, 11Birchmeier W. Hülsken J. Behrens J. CIBA Found. Symp. 1995; 189: 124-141PubMed Google Scholar). Cell migration is an essential process during embryonic development and in epithelial regeneration of adult organisms, for example in wound healing, and requires precise control, which is altered or lost when tumor cells become invasive and metastatic. In this study we have defined at the cellular and biochemical level distinct mechanisms regulating epithelial cell migration. Furthermore, we have characterized in motile cells the impact of tyrosine phosphorylation of β-catenin and its regulation by tyrosine kinases and the protein-tyrosine phosphatase LAR.Growth factors such as EGF, acidic fibroblast growth factor, or hepatocyte growth factor/scatter factor have been shown to induce migration of epithelial cells (38Manske M. Bade E.G. Int. Rev. Cytol. 1994; 155: 49-96Crossref PubMed Scopus (43) Google Scholar). A correlation has been suggested between migration and tyrosine phosphorylation of β-catenin (12Behrens J. Vakaet L. Friis R. Winterhager E. Van Roy F. Mareel M.M. 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Cell Biol. 1995; 131: 761-773Crossref PubMed Scopus (71) Google Scholar). We demonstrate here that after induction of migration, the pool of free, uncomplexed β-catenin is increased and that this increase correlates with enhanced β-catenin tyrosine phosphorylation. Therefore, tyrosine phosphorylation may result in a reduced interaction of β-catenin with both E-cadherin and the actin-cytoskeleton. Because the integrity of the cadherin-catenin complex is essential for strong cell-cell adhesion, this reduced interaction may lead to an overall decrease in intercellular contacts. Interestingly, a fusion protein of E-cadherin and α-catenin was reported to be able of mediating the interaction of E-cadherin with the cytoskeleton independent of β-catenin. However, these cells were no longer capable of migration (48Nagafuchi A. Ishihara S. Tsukita S. J. Cell Biol. 1994; 127: 235-245Crossref PubMed Scopus (359) Google Scholar). 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During EMT or cell migration this common regulator would switch off expression of epithelial genes while switching on genes for the mesenchymal phenotype. The β-catenin·LEF1 complex may represent this common regulator, and molecules that control the free pool of β-catenin may be essential for proper development or wound healing.PTPs were proposed to play an important role in the regulation of cell-cell contacts because treatment with sodium orthovanadate, a potent inhibitor of phosphatase activity, diminished normal cell contact inhibition in epithelial cells and led to increased tyrosine phosphorylation at adherens junctions (16Volberg T. Zick Y. Dror R. Sabany I. Gilon C. Levitzki A. Geiger B. EMBO J. 1992; 11: 1733-1742Crossref PubMed Scopus (269) Google Scholar). PTPs may therefore act as steady state equilibrium antagonists of TKs for regulatory events at adherens junctions. Consistent with these findings we show that tyrosine phosphorylation of β-catenin and plakoglobin in migrating epithelial cells was significantly increased after pretreatment with sodium orthovanadate. Little is known about interacting proteins orin vivo substrates of transmembrane PTPs. PTP LAR was recently shown to interact and colocalize at focal adhesions with LAR interacting protein 1 (LIP-1) and the multidomain protein Trio. However, neither of these proteins appears to be a substrate for PTP LAR (45Serra-Pagès C. Kedersha N.L. Fazikas L. Medley Q. Debant A. Streuli M. EMBO J. 1995; 14: 2827-2838Crossref PubMed Scopus (290) Google Scholar, 67Debant A. Serra-Pagès C. Seipel K. O'Brien S. Tang M. Park S.-H. Streuli M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5466-5471Crossref PubMed Scopus (393) Google Scholar). Moreover, a PTP LAR-like PTP was reported to interact with the cadherin-catenin complex at adherens junctions of neurosecretory PC12 cells (68Kypta R.M. Su H. 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In contrast, during wound healing, cells at the wound edge are in a subconfluent situation, where PTP action is reduced. This could favor tyrosine kinase signaling through stimulation by growth factors such as EGF, transforming growth factor α, and keratinocyte growth factor, which results in an increase in cell migration and proliferation and, thus, to accelerated wound healing, because the expression of these growth factors was increased during a wound situation (74Buckley A. Davidson J.M. Kamerath C.D. Wolt T.B. Woodward S.C. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7340-7344Crossref PubMed Scopus (273) Google Scholar, 75Rappolee D.A. Mark D. Banda M.J. Werb Z. Science. 1988; 241: 708-712Crossref PubMed Scopus (902) Google Scholar, 76Werner S. Peters K.G. Longaker M.T. Fuller-Pace F. Banda M.J. Williams L.T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6896-6900Crossref PubMed Scopus (531) Google Scholar).The importance of free and tyrosine-phosphorylated β-catenin during epithelial cell migration is underscored by our observations that interfering with this parameter by overexpressing hPTP LAR inhibits epithelial cell motility. Furthermore, expression of hPTP LAR in NBT II cells inhibited tumor formation in nude mice, although the growth characteristics of these cells in vitro were not altered. Ectopic expression of hPTP LAR to about twice the endogenous level was sufficient to result in the biological effects observed. Such modest ectopic expression of hPTP LAR appears to be critical, because high overexpression results in completely altered growth characteristics and apoptosis of the cells (77Weng L.P. Yuan J. Yu Q. Curr. Biol. 1998; 8: 247-256Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The data presented in this report strongly support an important role for PTP LAR in the regulation of cell-cell adhesion and epithelial cell migration as well as tumorigenesis by controlling the free pool of signaling β-catenin. Because mutations or deletions in either APC or β-catenin leading to a stabilized pool of free β-catenin have been correlated with tumor formation (61Korinek V. Barker N. Morin P.J. van Wichen D. de Weger R. Kinzler K.W. Vogelstein B. Clevers H. Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2911) Google Scholar, 62Morin P.J. Sparks A.B. Korinek V. Barker N. Clevers H. Vogelstein B. Kinzler K.W. Science. 1997; 275: 1787-1790Crossref PubMed Scopus (3480) Google Scholar,78Rubinfeld B. Robbins P. El-Gamil M. Albert I. Porfiri E. Polakis P. Science. 1997; 275: 1790-1792Crossref PubMed Scopus (1134) Google Scholar), loss of PTP LAR function may also contribute to tumor formation and metastasis. As a potential tumor suppressor gene product, PTP LAR could therefore serve as a prognostic marker for human cancer. The cadherins represent a family of transmembrane receptors that mediate homophilic, Ca2+-dependent cell-cell adhesion. In epithelial cells, the members of this family, such as the classical E-, N-, and P-cadherins, are primarily found at the adherens junctions of adjacent cells (1Yap A.S. Brieher W.M. Gumbiner B.M. Annu. Rev. Cell Dev. Biol. 1997; 13: 119-146Crossref PubMed Scopus (685) Google Scholar). β-Catenin as well as plakoglobin (γ-catenin) associate directly with the highly conserved cytoplasmic domain of classical cadherins in a mutually exclusive manner (2Ozawa M. Baribault H. Kemler R. EMBO J. 1989; 8: 1711-1718Crossref PubMed Scopus (1143) Google Scholar, 3Hinck L. Näthke I.S. Papkoff J. Nelson W.J. J. Cell Biol. 1994; 125: 1327-1340Crossref PubMed Scopus (556) Google Scholar). The cadherin-catenin complex is linked via α-catenin either directly (4Rimm D.L. 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