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• W2109025098 abstract "Cadherins are calcium-dependent glycoproteins that function as cell-cell adhesion molecules and are linked to the actin cytoskeleton via catenins. Newly synthesized cadherins contain a prosequence that must be proteolytically removed to generate a functional adhesion molecule. The goal of this study was to examine the proteolytic processing of N-cadherin and the assembly of the cadherin-catenin complex in cells that express endogenous N-cadherin. A monoclonal antibody specific for the proregion of human N-cadherin was generated and used to examine N-cadherin processing. Our data show that newly synthesized proN-cadherin is phosphorylated and proteolytically processed prior to transport to the plasma membrane. In addition, we show that औ-catenin and plakoglobin associate only with phosphorylated proN-cadherin, whereas p120ctn can associate with both phosphorylated and non-phosphorylated proN-cadherin. Immunoprecipitations using anti-proN-cadherin showed that cadherin-catenin complexes are assembled prior to localization at the plasma membrane. These data suggest that a core N-cadherin-catenin complex assembles in the endoplasmic reticulum or Golgi compartment and is transported to the plasma membrane where linkage to the actin cytoskeleton can be established. Cadherins are calcium-dependent glycoproteins that function as cell-cell adhesion molecules and are linked to the actin cytoskeleton via catenins. Newly synthesized cadherins contain a prosequence that must be proteolytically removed to generate a functional adhesion molecule. The goal of this study was to examine the proteolytic processing of N-cadherin and the assembly of the cadherin-catenin complex in cells that express endogenous N-cadherin. A monoclonal antibody specific for the proregion of human N-cadherin was generated and used to examine N-cadherin processing. Our data show that newly synthesized proN-cadherin is phosphorylated and proteolytically processed prior to transport to the plasma membrane. In addition, we show that औ-catenin and plakoglobin associate only with phosphorylated proN-cadherin, whereas p120ctn can associate with both phosphorylated and non-phosphorylated proN-cadherin. Immunoprecipitations using anti-proN-cadherin showed that cadherin-catenin complexes are assembled prior to localization at the plasma membrane. These data suggest that a core N-cadherin-catenin complex assembles in the endoplasmic reticulum or Golgi compartment and is transported to the plasma membrane where linkage to the actin cytoskeleton can be established. maltose-binding protein Dulbecco's modified Eagle's medium 4,6-diamidino-2-phenylindole Cadherins comprise a family of calcium-dependent cell-cell adhesion proteins that play important roles in the embryonic development and maintenance of normal tissue architecture. As the transmembrane component of cellular junctions, the cadherins are composed of three segments,i.e. an extracellular domain comprised of five homologous repeats that mediates adhesion, a single pass transmembrane domain, and a conserved cytoplasmic domain that interacts with catenins to link cadherins to the actin cytoskeleton (1Wheelock M.J. Soler A.P. Knudsen K.A. J. Mammary Gland Biol. Neoplasia. 2001; 6: 275-285Crossref PubMed Scopus (65) Google Scholar, 2Wheelock M.J. Knudsen K.A. Johnson K.R. Curr. Top. Membr. 1996; 43: 169-185Crossref Scopus (43) Google Scholar, 3Gumbiner B.M. J. Cell Biol. 2000; 148: 399-404Crossref PubMed Scopus (687) Google Scholar, 4Nollet F. Kools P. van Roy F. J. Mol. Biol. 2000; 299: 551-572Crossref PubMed Scopus (581) Google Scholar, 5Yagi T. Takeichi M. Genes Dev. 2000; 14: 1169-1180PubMed Google Scholar). The catenins were first identified as proteins that co-immunoprecipitated with cadherins and were termed α-catenin, औ-catenin, and γ-catenin (plakoglobin) according to their mobility on SDS-PAGE. Either औ-catenin or plakoglobin binds directly to the cadherin and to α-catenin, whereas α-catenin associates directly and indirectly with actin filaments (6Stappert J. Kemler R. Cell Adhes. Commun. 1994; 2: 319-327Crossref PubMed Scopus (199) Google Scholar, 7Knudsen K.A. Soler A.P. Johnson K.R. Wheelock M.J. J. Cell Biol. 1995; 130: 67-77Crossref PubMed Scopus (560) Google Scholar, 8Rimm 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, 9Nieset J.E. Redfield A.R. Jin F. Knudsen K.A. Johnson K.R. Wheelock M.J. J. Cell Sci. 1997; 110: 1013-1022Crossref PubMed Google Scholar). The ability of cadherins to simultaneously self-associate and link to the actin cytoskeleton allows strong cell-cell adhesion.In addition to catenins, p120ctn, which was originally identified as a Src substrate, binds to the cytoplasmic domain of cadherins and has been suggested to play a role in regulating the adhesive activity of cadherins (10Shibamoto S. Hayakawa M. Takeuchi K. Hori T. Miyazawa K. Kitamura N. Johnson K.R. Wheelock M.J. Matsuyoshi N. Takeichi M. Ito F. J. Cell Biol. 1995; 128: 949-957Crossref PubMed Scopus (243) Google Scholar, 11Reynolds A.B. Daniel J. McCrea P.D. Wheelock M.J. Wu J. Zhang Z. Mol. Cell. Biol. 1994; 14: 8333-8342Crossref PubMed Google Scholar, 12Daniel J.M. Reynolds A.B. Mol. Cell. Biol. 1995; 15: 4819-4824Crossref PubMed Google Scholar). p120ctn binds to the juxtamembrane domain of cadherins, a domain that has been implicated in cadherin clustering and cell motility (13Finnemann S. Mitrik I. Hess M. Otto G. Wedlich D. J. Biol. Chem. 1997; 272: 11856-11862Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 14Navarro P. Ruco L. Dejana E. J. Cell Biol. 1998; 140: 1475-1484Crossref PubMed Scopus (245) Google Scholar, 15Chen H. Paradies N.E. Fedor-Chaiken M. Brackenbury R. J. Cell Sci. 1997; 110: 345-356PubMed Google Scholar, 16Yap A.S. Niessen C.M. Gumbiner B.M. J. Cell Biol. 1998; 141: 779-789Crossref PubMed Scopus (462) Google Scholar). It is thought that p120ctn influences the strength of cadherin-mediated adhesion, perhaps by influencing the organization of the actin cytoskeleton (17Aono S. Nakagawa S. Reynolds A.B. Takeichi M. J. Cell Biol. 1999; 145: 551-562Crossref PubMed Scopus (200) Google Scholar, 18Ohkubo T. Ozawa M. J. Biol. Chem. 1999; 274: 21409-21415Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 19Thoreson M.A. Anastasiadis P.Z. Daniel J.M. Ireton R.C. Wheelock M.J. Johnson K.R. Hummingbird D.K. Reynolds A.B. J. Cell Biol. 2000; 148: 189-202Crossref PubMed Scopus (385) Google Scholar). The goal of the present study was to further understand the sequence of events that leads to the formation of a functional cadherin-catenin-p120ctn complex.Cadherins are synthesized as precursor proteins that must be proteolytically cleaved to generate functional, mature proteins (20Posthaus H. Dubois C.M. Laprise M.H. Grondin F. Suter M.M. Muller E. FEBS Lett. 1998; 438: 306-310Crossref PubMed Scopus (59) Google Scholar,21Ozawa M. Kemler R. J. Cell Biol. 1990; 111: 1645-1650Crossref PubMed Scopus (182) Google Scholar). All of the classical cadherins have similar proteolytic cleavage sites within the proregion, suggesting that each is processed by proteases with similar specificities. Ozawa and Kemler (21Ozawa M. Kemler R. J. Cell Biol. 1990; 111: 1645-1650Crossref PubMed Scopus (182) Google Scholar) showed that mutant forms of E-cadherin missing the proteolytic cleavage sites were transported to the cell surface when transfected into cadherin-negative cells but were not active in cell-cell adhesion. These precursor forms could be converted to active molecules by exogenous cleavage of the proregion at the cell surface. An emerging idea in the cadherin field is that cadherin family members promote cell type-specific phenotypes. For example, we have presented evidence suggesting that N-cadherin expression can promote motility in epithelial cells, whereas E-cadherin suppresses motility in the same cells (22Islam S. Carey T.E. Wolf G.T. Wheelock M.J. Johnson K.R. J. Cell Biol. 1996; 135: 1643-1654Crossref PubMed Scopus (272) Google Scholar, 23Kim J.B. Islam S. Kim Y.J. Prudoff R.S. Sass K.M. Wheelock M.J. Johnson K.R. J. Cell Biol. 2000; 151: 1193-1206Crossref PubMed Scopus (177) Google Scholar, 24Nieman M.T. Prudoff R.S. Johnson K.R. Wheelock M.J. J. Cell Biol. 1999; 147: 631-644Crossref PubMed Scopus (641) Google Scholar). Thus, it is important to examine the activity and processing of cadherins in cells that endogenously express these proteins. To facilitate these studies, we developed monoclonal antibodies against the proregion of N-cadherin that would allow us to define the sequence of events that occur during the synthesis, processing, and transport to the cell surface of an endogenous N-cadherin-catenin-p120ctn complex.DISCUSSIONThe goal of the present study was to investigate endogenous N-cadherin processing and cadherin-catenin complex formation. We chose to use HeLa cells that endogenously express N-cadherin and catenins and form N-cadherin-containing cell-cell junctions. To monitor the processing of N-cadherin, we generated monoclonal antibodies specific for the proregion of N-cadherin. Immunoblot analysis of HeLa cell extracts with the proN-cadherin antibodies revealed two major bands. Immunoblots of extracts prepared from other cells expressing endogenous N-cadherin (HT1080 and VA13) gave similar results (data not shown). Metabolic labeling of HeLa cell cultures with 33P identified the faster migrating form of proN-cadherin as a non-phosphorylated form and the slower migrating form as a phosphorylated form. In pulse-chase experiments, the earliest (non-phosphorylated) form of proN-cadherin that we could detect rapidly chased into the slower migrating (phosphorylated) form. It was this slower migrating form that co-immunoprecipitated with औ-catenin and plakoglobin. In addition to the experiments with HeLa cells, the experiments with the N/E5a-myc chimeric cadherin showed that catenins could associate with cadherins that are in the endoplasmic reticulum. Taken together, these data suggest that the phosphorylation of proN-cadherin and the subsequent assembly of the cadherin-catenin complex occurs while the cadherin is in the endoplasmic reticulum and Golgi complex. Fig. 8 presents a model depicting the sequence of events leading to N-cadherin-catenin complex localization at the plasma membrane.The proregion of cadherins must be removed in order to generate functional adhesion molecules (21Ozawa M. Kemler R. J. Cell Biol. 1990; 111: 1645-1650Crossref PubMed Scopus (182) Google Scholar). Recently, Ozawa (32Ozawa M. J. Biol. Chem. 2002; 277: 19600-19608Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) used L cells to show that the proregion of E-cadherin not only prevented cell aggregation (which requires the formation of adhesion dimers) but also prevented the formation of lateral dimers. The fact that we found catenins complexed with proN-cadherin suggests that the catenins can load onto the monomeric form of this cadherin. Because our pulse-chase experiments showed that the earliest detectible form of proN-cadherin chases into a slower migrating form that co-immunoprecipitates all the catenins, it is likely that the proN-cadherin-catenin complex forms in the endoplasmic reticulum.The furin subgroup of subtilisin-like proprotein convertases is thought to be responsible for cadherin processing. E-cadherin has been shown to be a furin protease substrate in a baculovirus system, and the consensus sequence for furin protease in E-cadherin is identical to that of N-cadherin (20Posthaus H. Dubois C.M. Laprise M.H. Grondin F. Suter M.M. Muller E. FEBS Lett. 1998; 438: 306-310Crossref PubMed Scopus (59) Google Scholar). The furin convertases have been shown to be proteolytically active in the trans-Golgi network (33Nakayama K. Biochem. J. 1997; 327: 625-635Crossref PubMed Scopus (701) Google Scholar). Interestingly, our immunofluorescence experiments using anti-proN-cadherin antibodies revealed a staining pattern consistent with proN-cadherin localization to the ER and Golgi network. In addition, immunoprecipitation experiments showed that a 15-kDa fragment, corresponding to the cleaved proregion, was present in HeLa cell extracts, suggesting that the proregion of N-cadherin can be removed in one step, most likely by the furin convertases. The detection of the 15-kDa pro-peptide raises the possibility that it could be secreted and may have some yet to be identified function outside the cell. In permeabilization experiments, proN-cadherin was not present at the plasma membrane of HeLa cells, consistent with the proregion of N-cadherin being removed prior to transport to the plasma membrane.Studies focusing on E-cadherin-catenin complex formation in L cells suggested that α-catenin was added to the cadherinऔ-catenin complex after the cadherin proregion was removed (30Ozawa M. Kemler R. J. Cell Biol. 1992; 116: 989-996Crossref PubMed Scopus (324) Google Scholar). In addition, studies on the assembly of E-cadherin complexes in Madin-Darby canine kidney (MDCK) cells suggested that α-catenin was added to the adherens junction complex after localization at the plasma membrane (31Hinck L. Nathke I.S. Papkoff J. Nelson W.J. J. Cell Biol. 1994; 125: 1327-1340Crossref PubMed Scopus (556) Google Scholar). Our results demonstrate that this is not the case for N-cadherin in HeLa cells. Immunoprecipitations showed that औ-catenin, plakoglobin, p120ctn, and α-catenin were all found in a complex with proN-cadherin. The studies with E-cadherin raise the possibility that, although we could find the catenins associated with proN-cadherin, only a small fraction of the total proN-cadherin is actually complexed with the catenins. However, the experiment shown in Fig. 5 demonstrates that the association of α-catenin with proN-cadherin is not a rare event; the α-catenin-proN-cadherin complex has the same stoichiometry as the α-catenin-mature N-cadherin complex. The cadherin-औ-catenin-α-catenin complex has been estimated to have molar ratios of 1:1:1 (30Ozawa M. Kemler R. J. Cell Biol. 1992; 116: 989-996Crossref PubMed Scopus (324) Google Scholar), a conclusion that is supported by recent structural studies (34Huber A.H. Weis W.I. Cell. 2001; 105: 391-402Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar, 35Pokutta S. Weis W.I. Mol. Cell. 2000; 5: 533-543Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). It is likely that proN-cadherin binds the catenins in the same ratios as does mature N-cadherin.To date, p120ctn association with unprocessed cadherin has not been investigated. Our data show that p120ctn, but not औ-catenin or plakoglobin, can bind to non-phosphorylated proN-cadherin, which is the earliest form of N-cadherin we can detect. Taken together, our results suggest a model where, following synthesis, proN-cadherin associates immediately with p120ctn. Following phosphorylation of the cadherin, possibly by casein kinase II (36Lickert H. Bauer A. Kemler R. Stappert J. J. Biol. Chem. 2000; 275: 5090-5095Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar), औ-catenin or plakoglobin then associates with the cadherin (Fig. 8). Our data also show that औ-catenin-α-catenin complexes can form in the cytosol, raising the possibility that औ-catenin and α-catenin may simultaneously load onto proN-cadherin. The complex is then transported to the plasma membrane where linkage to the actin cytoskeleton occurs. The mechanism of cadherin-catenin complex transport to the plasma membrane is unknown, although recent work suggests a microtubule-dependent mechanism for the formation of N-cadherin cell-cell contacts (37Mary S. Charrasse S. Meriane M. Comunale F. Travo P. Blangy A. Gauthier-Rouviere C. Mol. Biol. Cell. 2002; 13: 285-301Crossref PubMed Scopus (120) Google Scholar). Understanding the sequence of events leading to the formation of the N-cadherin-catenin complex and its localization at the plasma membrane will help clarify the regulation of N-cadherin mediated cell-cell adhesion and identify new mechanisms for controlling N-cadherin-mediated cell-cell adhesion. Cadherins comprise a family of calcium-dependent cell-cell adhesion proteins that play important roles in the embryonic development and maintenance of normal tissue architecture. As the transmembrane component of cellular junctions, the cadherins are composed of three segments,i.e. an extracellular domain comprised of five homologous repeats that mediates adhesion, a single pass transmembrane domain, and a conserved cytoplasmic domain that interacts with catenins to link cadherins to the actin cytoskeleton (1Wheelock M.J. Soler A.P. Knudsen K.A. J. Mammary Gland Biol. Neoplasia. 2001; 6: 275-285Crossref PubMed Scopus (65) Google Scholar, 2Wheelock M.J. Knudsen K.A. Johnson K.R. Curr. Top. Membr. 1996; 43: 169-185Crossref Scopus (43) Google Scholar, 3Gumbiner B.M. J. Cell Biol. 2000; 148: 399-404Crossref PubMed Scopus (687) Google Scholar, 4Nollet F. Kools P. van Roy F. J. Mol. Biol. 2000; 299: 551-572Crossref PubMed Scopus (581) Google Scholar, 5Yagi T. Takeichi M. Genes Dev. 2000; 14: 1169-1180PubMed Google Scholar). The catenins were first identified as proteins that co-immunoprecipitated with cadherins and were termed α-catenin, औ-catenin, and γ-catenin (plakoglobin) according to their mobility on SDS-PAGE. Either औ-catenin or plakoglobin binds directly to the cadherin and to α-catenin, whereas α-catenin associates directly and indirectly with actin filaments (6Stappert J. Kemler R. Cell Adhes. Commun. 1994; 2: 319-327Crossref PubMed Scopus (199) Google Scholar, 7Knudsen K.A. Soler A.P. Johnson K.R. Wheelock M.J. J. Cell Biol. 1995; 130: 67-77Crossref PubMed Scopus (560) Google Scholar, 8Rimm 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, 9Nieset J.E. Redfield A.R. Jin F. Knudsen K.A. Johnson K.R. Wheelock M.J. J. Cell Sci. 1997; 110: 1013-1022Crossref PubMed Google Scholar). The ability of cadherins to simultaneously self-associate and link to the actin cytoskeleton allows strong cell-cell adhesion. In addition to catenins, p120ctn, which was originally identified as a Src substrate, binds to the cytoplasmic domain of cadherins and has been suggested to play a role in regulating the adhesive activity of cadherins (10Shibamoto S. Hayakawa M. Takeuchi K. Hori T. Miyazawa K. Kitamura N. Johnson K.R. Wheelock M.J. Matsuyoshi N. Takeichi M. Ito F. J. Cell Biol. 1995; 128: 949-957Crossref PubMed Scopus (243) Google Scholar, 11Reynolds A.B. Daniel J. McCrea P.D. Wheelock M.J. Wu J. Zhang Z. Mol. Cell. Biol. 1994; 14: 8333-8342Crossref PubMed Google Scholar, 12Daniel J.M. Reynolds A.B. Mol. Cell. Biol. 1995; 15: 4819-4824Crossref PubMed Google Scholar). p120ctn binds to the juxtamembrane domain of cadherins, a domain that has been implicated in cadherin clustering and cell motility (13Finnemann S. Mitrik I. Hess M. Otto G. Wedlich D. J. Biol. Chem. 1997; 272: 11856-11862Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 14Navarro P. Ruco L. Dejana E. J. Cell Biol. 1998; 140: 1475-1484Crossref PubMed Scopus (245) Google Scholar, 15Chen H. Paradies N.E. Fedor-Chaiken M. Brackenbury R. J. Cell Sci. 1997; 110: 345-356PubMed Google Scholar, 16Yap A.S. Niessen C.M. Gumbiner B.M. J. Cell Biol. 1998; 141: 779-789Crossref PubMed Scopus (462) Google Scholar). It is thought that p120ctn influences the strength of cadherin-mediated adhesion, perhaps by influencing the organization of the actin cytoskeleton (17Aono S. Nakagawa S. Reynolds A.B. Takeichi M. J. Cell Biol. 1999; 145: 551-562Crossref PubMed Scopus (200) Google Scholar, 18Ohkubo T. Ozawa M. J. Biol. Chem. 1999; 274: 21409-21415Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 19Thoreson M.A. Anastasiadis P.Z. Daniel J.M. Ireton R.C. Wheelock M.J. Johnson K.R. Hummingbird D.K. Reynolds A.B. J. Cell Biol. 2000; 148: 189-202Crossref PubMed Scopus (385) Google Scholar). The goal of the present study was to further understand the sequence of events that leads to the formation of a functional cadherin-catenin-p120ctn complex. Cadherins are synthesized as precursor proteins that must be proteolytically cleaved to generate functional, mature proteins (20Posthaus H. Dubois C.M. Laprise M.H. Grondin F. Suter M.M. Muller E. FEBS Lett. 1998; 438: 306-310Crossref PubMed Scopus (59) Google Scholar,21Ozawa M. Kemler R. J. Cell Biol. 1990; 111: 1645-1650Crossref PubMed Scopus (182) Google Scholar). All of the classical cadherins have similar proteolytic cleavage sites within the proregion, suggesting that each is processed by proteases with similar specificities. Ozawa and Kemler (21Ozawa M. Kemler R. J. Cell Biol. 1990; 111: 1645-1650Crossref PubMed Scopus (182) Google Scholar) showed that mutant forms of E-cadherin missing the proteolytic cleavage sites were transported to the cell surface when transfected into cadherin-negative cells but were not active in cell-cell adhesion. These precursor forms could be converted to active molecules by exogenous cleavage of the proregion at the cell surface. An emerging idea in the cadherin field is that cadherin family members promote cell type-specific phenotypes. For example, we have presented evidence suggesting that N-cadherin expression can promote motility in epithelial cells, whereas E-cadherin suppresses motility in the same cells (22Islam S. Carey T.E. Wolf G.T. Wheelock M.J. Johnson K.R. J. Cell Biol. 1996; 135: 1643-1654Crossref PubMed Scopus (272) Google Scholar, 23Kim J.B. Islam S. Kim Y.J. Prudoff R.S. Sass K.M. Wheelock M.J. Johnson K.R. J. Cell Biol. 2000; 151: 1193-1206Crossref PubMed Scopus (177) Google Scholar, 24Nieman M.T. Prudoff R.S. Johnson K.R. Wheelock M.J. J. Cell Biol. 1999; 147: 631-644Crossref PubMed Scopus (641) Google Scholar). Thus, it is important to examine the activity and processing of cadherins in cells that endogenously express these proteins. To facilitate these studies, we developed monoclonal antibodies against the proregion of N-cadherin that would allow us to define the sequence of events that occur during the synthesis, processing, and transport to the cell surface of an endogenous N-cadherin-catenin-p120ctn complex. DISCUSSIONThe goal of the present study was to investigate endogenous N-cadherin processing and cadherin-catenin complex formation. We chose to use HeLa cells that endogenously express N-cadherin and catenins and form N-cadherin-containing cell-cell junctions. To monitor the processing of N-cadherin, we generated monoclonal antibodies specific for the proregion of N-cadherin. Immunoblot analysis of HeLa cell extracts with the proN-cadherin antibodies revealed two major bands. Immunoblots of extracts prepared from other cells expressing endogenous N-cadherin (HT1080 and VA13) gave similar results (data not shown). Metabolic labeling of HeLa cell cultures with 33P identified the faster migrating form of proN-cadherin as a non-phosphorylated form and the slower migrating form as a phosphorylated form. In pulse-chase experiments, the earliest (non-phosphorylated) form of proN-cadherin that we could detect rapidly chased into the slower migrating (phosphorylated) form. It was this slower migrating form that co-immunoprecipitated with औ-catenin and plakoglobin. In addition to the experiments with HeLa cells, the experiments with the N/E5a-myc chimeric cadherin showed that catenins could associate with cadherins that are in the endoplasmic reticulum. Taken together, these data suggest that the phosphorylation of proN-cadherin and the subsequent assembly of the cadherin-catenin complex occurs while the cadherin is in the endoplasmic reticulum and Golgi complex. Fig. 8 presents a model depicting the sequence of events leading to N-cadherin-catenin complex localization at the plasma membrane.The proregion of cadherins must be removed in order to generate functional adhesion molecules (21Ozawa M. Kemler R. J. Cell Biol. 1990; 111: 1645-1650Crossref PubMed Scopus (182) Google Scholar). Recently, Ozawa (32Ozawa M. J. Biol. Chem. 2002; 277: 19600-19608Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) used L cells to show that the proregion of E-cadherin not only prevented cell aggregation (which requires the formation of adhesion dimers) but also prevented the formation of lateral dimers. The fact that we found catenins complexed with proN-cadherin suggests that the catenins can load onto the monomeric form of this cadherin. Because our pulse-chase experiments showed that the earliest detectible form of proN-cadherin chases into a slower migrating form that co-immunoprecipitates all the catenins, it is likely that the proN-cadherin-catenin complex forms in the endoplasmic reticulum.The furin subgroup of subtilisin-like proprotein convertases is thought to be responsible for cadherin processing. E-cadherin has been shown to be a furin protease substrate in a baculovirus system, and the consensus sequence for furin protease in E-cadherin is identical to that of N-cadherin (20Posthaus H. Dubois C.M. Laprise M.H. Grondin F. Suter M.M. Muller E. FEBS Lett. 1998; 438: 306-310Crossref PubMed Scopus (59) Google Scholar). The furin convertases have been shown to be proteolytically active in the trans-Golgi network (33Nakayama K. Biochem. J. 1997; 327: 625-635Crossref PubMed Scopus (701) Google Scholar). Interestingly, our immunofluorescence experiments using anti-proN-cadherin antibodies revealed a staining pattern consistent with proN-cadherin localization to the ER and Golgi network. In addition, immunoprecipitation experiments showed that a 15-kDa fragment, corresponding to the cleaved proregion, was present in HeLa cell extracts, suggesting that the proregion of N-cadherin can be removed in one step, most likely by the furin convertases. The detection of the 15-kDa pro-peptide raises the possibility that it could be secreted and may have some yet to be identified function outside the cell. In permeabilization experiments, proN-cadherin was not present at the plasma membrane of HeLa cells, consistent with the proregion of N-cadherin being removed prior to transport to the plasma membrane.Studies focusing on E-cadherin-catenin complex formation in L cells suggested that α-catenin was added to the cadherinऔ-catenin complex after the cadherin proregion was removed (30Ozawa M. Kemler R. J. Cell Biol. 1992; 116: 989-996Crossref PubMed Scopus (324) Google Scholar). In addition, studies on the assembly of E-cadherin complexes in Madin-Darby canine kidney (MDCK) cells suggested that α-catenin was added to the adherens junction complex after localization at the plasma membrane (31Hinck L. Nathke I.S. Papkoff J. Nelson W.J. J. Cell Biol. 1994; 125: 1327-1340Crossref PubMed Scopus (556) Google Scholar). Our results demonstrate that this is not the case for N-cadherin in HeLa cells. Immunoprecipitations showed that औ-catenin, plakoglobin, p120ctn, and α-catenin were all found in a complex with proN-cadherin. The studies with E-cadherin raise the possibility that, although we could find the catenins associated with proN-cadherin, only a small fraction of the total proN-cadherin is actually complexed with the catenins. However, the experiment shown in Fig. 5 demonstrates that the association of α-catenin with proN-cadherin is not a rare event; the α-catenin-proN-cadherin complex has the same stoichiometry as the α-catenin-mature N-cadherin complex. The cadherin-औ-catenin-α-catenin complex has been estimated to have molar ratios of 1:1:1 (30Ozawa M. Kemler R. J. Cell Biol. 1992; 116: 989-996Crossref PubMed Scopus (324) Google Scholar), a conclusion that is supported by recent structural studies (34Huber A.H. Weis W.I. Cell. 2001; 105: 391-402Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar, 35Pokutta S. Weis W.I. Mol. Cell. 2000; 5: 533-543Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). It is likely that proN-cadherin binds the catenins in the same ratios as does mature N-cadherin.To date, p120ctn association with unprocessed cadherin has not been investigated. Our data show that p120ctn, but not औ-catenin or plakoglobin, can bind to non-phosphorylated proN-cadherin, which is the earliest form of N-cadherin we can detect. Taken together, our results suggest a model where, following synthesis, proN-cadherin associates immediately with p120ctn. Following phosphorylation of the cadherin, possibly by casein kinase II (36Lickert H. Bauer A. Kemler R. Stappert J. J. Biol. Chem. 2000; 275: 5090-5095Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar), औ-catenin or plakoglobin then associates with the cadherin (Fig. 8). Our data also show that औ-catenin-α-catenin complexes can form in the cytosol, raising the possibility that औ-catenin and α-catenin may simultaneously load onto proN-cadherin. The complex is then transported to the plasma membrane where linkage to the actin cytoskeleton occurs. The mechanism of cadherin-catenin complex transport to the plasma membrane is unknown, although recent work suggests a microtubule-dependent mechanism for the formation of N-cadherin cell-cell contacts (37Mary S. Charrasse S. Meriane M. Comunale F. Travo P. Blangy A. Gauthier-Rouviere C. Mol. Biol. Cell. 2002; 13: 285-301Crossref PubMed Scopus (120) Google Scholar). Understanding the sequence of events leading to the formation of the N-cadherin-catenin complex and its localization at the plasma membrane will help clarify the regulation of N-cadherin mediated cell-cell adhesion and identify new mechanisms for controlling N-cadherin-mediated cell-cell adhesion. The goal of the present study was to investigate endogenous N-cadherin processing and cadherin-catenin complex formation. We chose to use HeLa cells that endogenously express N-cadherin and catenins and form N-cadherin-containing cell-cell junctions. To monitor the processing of N-cadherin, we generated monoclonal antibodies specific for the proregion of N-cadherin. Immunoblot analysis of HeLa cell extracts with the proN-cadherin antibodies revealed two major bands. Immunoblots of extracts prepared from other cells expressing endogenous N-cadherin (HT1080 and VA13) gave similar results (data not shown). Metabolic labeling of HeLa cell cultures with 33P identified the faster migrating form of proN-cadherin as a non-phosphorylated form and the slower migrating form as a phosphorylated form. In pulse-chase experiments, the earliest (non-phosphorylated) form of proN-cadherin that we could detect rapidly chased into the slower migrating (phosphorylated) form. It was this slower migrating form that co-immunoprecipitated with औ-catenin and plakoglobin. In addition to the experiments with HeLa cells, the experiments with the N/E5a-myc chimeric cadherin showed that catenins could associate with cadherins that are in the endoplasmic reticulum. Taken together, these data suggest that the phosphorylation of proN-cadherin and the subsequent assembly of the cadherin-catenin complex occurs while the cadherin is in the endoplasmic reticulum and Golgi complex. Fig. 8 presents a model depicting the sequence of events leading to N-cadherin-catenin complex localization at the plasma membrane. The proregion of cadherins must be removed in order to generate functional adhesion molecules (21Ozawa M. Kemler R. J. Cell Biol. 1990; 111: 1645-1650Crossref PubMed Scopus (182) Google Scholar). Recently, Ozawa (32Ozawa M. J. Biol. Chem. 2002; 277: 19600-19608Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) used L cells to show that the proregion of E-cadherin not only prevented cell aggregation (which requires the formation of adhesion dimers) but also prevented the formation of lateral dimers. The fact that we found catenins complexed with proN-cadherin suggests that the catenins can load onto the monomeric form of this cadherin. Because our pulse-chase experiments showed that the earliest detectible form of proN-cadherin chases into a slower migrating form that co-immunoprecipitates all the catenins, it is likely that the proN-cadherin-catenin complex forms in the endoplasmic reticulum. The furin subgroup of subtilisin-like proprotein convertases is thought to be responsible for cadherin processing. E-cadherin has been shown to be a furin protease substrate in a baculovirus system, and the consensus sequence for furin protease in E-cadherin is identical to that of N-cadherin (20Posthaus H. Dubois C.M. Laprise M.H. Grondin F. Suter M.M. Muller E. FEBS Lett. 1998; 438: 306-310Crossref PubMed Scopus (59) Google Scholar). The furin convertases have been shown to be proteolytically active in the trans-Golgi network (33Nakayama K. Biochem. J. 1997; 327: 625-635Crossref PubMed Scopus (701) Google Scholar). Interestingly, our immunofluorescence experiments using anti-proN-cadherin antibodies revealed a staining pattern consistent with proN-cadherin localization to the ER and Golgi network. In addition, immunoprecipitation experiments showed that a 15-kDa fragment, corresponding to the cleaved proregion, was present in HeLa cell extracts, suggesting that the proregion of N-cadherin can be removed in one step, most likely by the furin convertases. The detection of the 15-kDa pro-peptide raises the possibility that it could be secreted and may have some yet to be identified function outside the cell. In permeabilization experiments, proN-cadherin was not present at the plasma membrane of HeLa cells, consistent with the proregion of N-cadherin being removed prior to transport to the plasma membrane. Studies focusing on E-cadherin-catenin complex formation in L cells suggested that α-catenin was added to the cadherinऔ-catenin complex after the cadherin proregion was removed (30Ozawa M. Kemler R. J. Cell Biol. 1992; 116: 989-996Crossref PubMed Scopus (324) Google Scholar). In addition, studies on the assembly of E-cadherin complexes in Madin-Darby canine kidney (MDCK) cells suggested that α-catenin was added to the adherens junction complex after localization at the plasma membrane (31Hinck L. Nathke I.S. Papkoff J. Nelson W.J. J. Cell Biol. 1994; 125: 1327-1340Crossref PubMed Scopus (556) Google Scholar). Our results demonstrate that this is not the case for N-cadherin in HeLa cells. Immunoprecipitations showed that औ-catenin, plakoglobin, p120ctn, and α-catenin were all found in a complex with proN-cadherin. The studies with E-cadherin raise the possibility that, although we could find the catenins associated with proN-cadherin, only a small fraction of the total proN-cadherin is actually complexed with the catenins. However, the experiment shown in Fig. 5 demonstrates that the association of α-catenin with proN-cadherin is not a rare event; the α-catenin-proN-cadherin complex has the same stoichiometry as the α-catenin-mature N-cadherin complex. The cadherin-औ-catenin-α-catenin complex has been estimated to have molar ratios of 1:1:1 (30Ozawa M. Kemler R. J. Cell Biol. 1992; 116: 989-996Crossref PubMed Scopus (324) Google Scholar), a conclusion that is supported by recent structural studies (34Huber A.H. Weis W.I. Cell. 2001; 105: 391-402Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar, 35Pokutta S. Weis W.I. Mol. Cell. 2000; 5: 533-543Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). It is likely that proN-cadherin binds the catenins in the same ratios as does mature N-cadherin. To date, p120ctn association with unprocessed cadherin has not been investigated. Our data show that p120ctn, but not औ-catenin or plakoglobin, can bind to non-phosphorylated proN-cadherin, which is the earliest form of N-cadherin we can detect. Taken together, our results suggest a model where, following synthesis, proN-cadherin associates immediately with p120ctn. Following phosphorylation of the cadherin, possibly by casein kinase II (36Lickert H. Bauer A. Kemler R. Stappert J. J. Biol. Chem. 2000; 275: 5090-5095Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar), औ-catenin or plakoglobin then associates with the cadherin (Fig. 8). Our data also show that औ-catenin-α-catenin complexes can form in the cytosol, raising the possibility that औ-catenin and α-catenin may simultaneously load onto proN-cadherin. The complex is then transported to the plasma membrane where linkage to the actin cytoskeleton occurs. The mechanism of cadherin-catenin complex transport to the plasma membrane is unknown, although recent work suggests a microtubule-dependent mechanism for the formation of N-cadherin cell-cell contacts (37Mary S. Charrasse S. Meriane M. Comunale F. Travo P. Blangy A. Gauthier-Rouviere C. Mol. Biol. Cell. 2002; 13: 285-301Crossref PubMed Scopus (120) Google Scholar). Understanding the sequence of events leading to the formation of the N-cadherin-catenin complex and its localization at the plasma membrane will help clarify the regulation of N-cadherin mediated cell-cell adhesion and identify new mechanisms for controlling N-cadherin-mediated cell-cell adhesion. We thank Jill Nieset and Jennifer Oiler for expert technical assistance in preparing proN-cadherin fusion proteins and generating the constructs used in this study." @default.
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• W2109025098 title "N-cadherin-Catenin Complexes Form Prior to Cleavage of the Proregion and Transport to the Plasma Membrane" @default.
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