EphA2 Phosphorylates the Cytoplasmic Tail of Claudin-4 and Mediates Paracellular Permeability

Eph receptors and ephrin ligands are widely expressed in epithelial cells and mediate cell-cell interaction. EphA2 is expressed in various cancer tissues and cell lines. Although the mechanism of action of EphA2 is unknown, its expression correlates with progression of the malignant phenotype of cancerous tissues. Here, we have shown that EphA2 modulates the localization and function of claudin-4, a constituent of tight junctions. EphA2 associates with claudin-4 via their extracellular domains. This association, in turn, leads to phosphorylation of the cytoplasmic carboxyl terminus of claudin-4 at Tyr-208. The tyrosine phosphorylation of claudin-4 attenuates association of claudin-4 with ZO-1, decreasing integration of claudin-4 into sites of cell-cell contact and enhancing paracellular permeability. These results indicate that EphA2 moderates the function of tight junctions via phosphorylation of claudin-4. Eph receptors and ephrin ligands are widely expressed in epithelial cells and mediate cell-cell interaction. EphA2 is expressed in various cancer tissues and cell lines. Although the mechanism of action of EphA2 is unknown, its expression correlates with progression of the malignant phenotype of cancerous tissues. Here, we have shown that EphA2 modulates the localization and function of claudin-4, a constituent of tight junctions. EphA2 associates with claudin-4 via their extracellular domains. This association, in turn, leads to phosphorylation of the cytoplasmic carboxyl terminus of claudin-4 at Tyr-208. The tyrosine phosphorylation of claudin-4 attenuates association of claudin-4 with ZO-1, decreasing integration of claudin-4 into sites of cell-cell contact and enhancing paracellular permeability. These results indicate that EphA2 moderates the function of tight junctions via phosphorylation of claudin-4. The members of the Eph receptor family can be classified into two groups based on their sequence similarity and their preferential binding to ligands tethered to the cell surface by a glycosylphosphatidyl inositol anchor (ephrin-A) or by a transmembrane domain (ephrin-B) (1Blits-Huizinga C. Nelersa C. Malhotra A. Liebl D. IUBMB Life. 2004; 56: 257-265Crossref PubMed Scopus (53) Google Scholar, 2Murai K.K. Pasquale E. J. Cell Sci. 2003; 116: 2823-2832Crossref PubMed Scopus (301) Google Scholar, 3Kullander K. Klein R. Nat. Rev. Mol. Cell. Biol. 2002; 3: 475-486Crossref PubMed Scopus (971) Google Scholar, 4Poliakov A. Cotrina M. Wilkinson D.G. Dev. Cell. 2004; 7: 465-480Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar). Although the studies of Eph receptors and ephrins have focused on their neuronal targeting and neural plasticity (5Huot J. Prog. Neuropsychopharmacol. Biol. Psychiatry. 2004; 28: 813-818Crossref PubMed Scopus (38) Google Scholar, 6Hinck L. Dev. 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Claudins are one of the most frequently overexpressed genes in various malignant cells (25Aldred M.A. Huang Y. Livanarachchi S. Pellegata N.S. Gimm O. Jhiang S. Davuluri R.V. de la Chapelle A. Eng C. J. Clin. Oncol. 2004; 22: 3531-3539Crossref PubMed Scopus (128) Google Scholar, 26Nicols L.S. Ashfag R. Lacobuzio-Donahue C.A. Am. J. Clin. Pathol. 2004; 121: 226-230Crossref PubMed Scopus (163) Google Scholar, 27Rangel L.B. Agarwal R. D'Souza T. Pizer E.S. Alo P.L. Lancaster W.D. Gregoire L. Schwartz D.R. Cho K.R. Morin P.J. Clin. Cancer Res. 2003; 9: 2567-2575PubMed Google Scholar). During the screening of cross-talk between Eph-ephrin family molecules and intercellular adhesion molecules, we found that EphA2 makes a complex with claudin-4, which led us to investigate whether claudin-4 is a biochemical target of EphA2. In this study, we have described the biological interaction of EphA2 with claudin-4. Upon cell-cell contact, a tyrosine residue located in the carboxyl-terminal region of claudin-4 was phosphorylated by activated EphA2. This phosphorylation event lead to reduced association of claudin-4 with ZO-1 and decreased integration of claudin-4 into sites of cell-cell contact. Analysis of paracellular flux indicated that activation of EphA2 delayed assembly of tight junctions in Madin-Darby canine kidney (MDCK) 2The abbreviations used are: MDCKMadin-Darby canine kidneyGSTglutathione S-transferaseECDextracellular domainGFPgreen fluorescent proteinFITCfluorescein isothiocyanate. cells, and this depended on EphA2 kinase activity. These results show that, by regulating the localization and function of claudin-4, EphA2 moderates tight junction permeability. Madin-Darby canine kidney glutathione S-transferase extracellular domain green fluorescent protein fluorescein isothiocyanate. Plasmids and Antibodies—Plasmids encoding full-length human EphA2 and a mutant EphA2K646M were previously described (28Wang Y. Ota S. Kataoka H. Kanamori M. Li Z. Band H. Tanaka M. Sugimura H. Biochem. Biophys. Res. Commun. 2002; 296: 214-220Crossref PubMed Scopus (59) Google Scholar). The Fc fusion protein expression constructs EphA2, ephrin-A1, EphB2, and ephrin-B1 were constructed using PCR-generated amplicons of the entire extracellular domain of each protein as previously described (28Wang Y. Ota S. Kataoka H. Kanamori M. Li Z. Band H. Tanaka M. Sugimura H. Biochem. Biophys. Res. Commun. 2002; 296: 214-220Crossref PubMed Scopus (59) Google Scholar, 29Tanaka M. Ohashi R. Nakamura R. Shinmura K. Kamo T. Sakai R. Sugimura H. EMBO J. 2004; 23: 1075-1088Crossref PubMed Scopus (88) Google Scholar). Mutants of EphA2, lacking either the cytoplasmic domain (EphA2-(1-563)) or the extracellular domain (EphA2-(541-977)) were constructed by tagging PCR-generated amplicons with GFP at the carboxyl or amino terminus, respectively. The plasmids encoding claudin-4 and N-ZO-1 (amino acids 1-862) were donated by S. Tsukita. Mutants of claudin-4 (Y193F, Y197F, Y208F, Y193/197F, and Y193/197/208F) and EphA2 (I94N) were generated using the Altered Sites mutagenesis system (Promega). The truncated mutants of claudin-4 shown in Fig. 2a were constructed by cloning PCR-generated amplicons into pEBB with the addition of the FLAG epitope tag at the carboxyl terminus. The antibodies for the FLAG (M2) and hemagglutinin (Y-11) tags were obtained from Sigma and Santa Cruz, respectively. The monoclonal antibodies for EphA2 and phosphotyrosine (4G10) were purchased from Upstate Biotechnology. The antibodies for claudin-4 and ZO-1 were purchased from ZyMed. Anti-GFP was from NacalaiTesque. Alexa Fluor-labeled secondary antibodies of anti-goat IgG, anti-rabbit IgG, and anti-mouse IgG were purchased from Molecular Probes. Fusion proteins of Eph and ephrin with the Fc region of immunoglobulin were purified by passing the culture medium of COS1 cells transfected with plasmids encoding the Fc fusion proteins through a protein A-Sepharose column as previously described (29Tanaka M. Ohashi R. Nakamura R. Shinmura K. Kamo T. Sakai R. Sugimura H. EMBO J. 2004; 23: 1075-1088Crossref PubMed Scopus (88) Google Scholar). Cell Culture and Transfection—HT29 colon carcinoma cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum. MDCK cells and COS1 cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. For transient expression assays, COS1 cells were transfected with plasmid DNA using FuGENE 6 reagent (Roche Applied Science). Generation of Adenoviruses and Adenoviral Infection—To generate recombinant adenoviruses, cDNAs encoding wild type, the K646M mutant of EphA2, or EphA2-(1-563)-GFP were subcloned into the vector pShuttle-CMV (Stratagene). They were transformed into an Escherichia coli strain containing the Ad5-based adenovirus vector pADEasy-1 (Stratagene). Transposition of the EphA2 cDNAs from the pShuttle-CMV into pADEasy-1 created the adenoviral vectors pAD-EphA2 (wild type, K646M, and EphA2-(1-563)-GFP) where the transgenes were under the control of the cytomegalovirus promoter. Recombinant adenoviral DNA was transfected into 293 human embryonic kidney cells to allow production of adenoviral particles. The titer of adenovirus stocks was determined by Adeno-X rapid titer kit (Clontech) according to the manufacturer's instructions. Confluent MDCK cells grown on Transwell filters or glass coverslips were infected with adenoviruses at a multiplicity of infection of 5 in medium containing 10% fetal bovine serum. After incubation for 12 h the virus-containing medium was removed and fresh medium containing 10% fetal bovine serum was added. The infected cells were used for permeability assays or immunostaining 48 h after the infection. Immunoprecipitation and Immunoblotting—Transfected cells were harvested 48 h after transfection, and cell lysates were prepared with protease inhibitors in PLC buffer (50 mm Hepes (pH 7.5), 150 mm NaCl, 1.5 mm MgCl2, 1 mm EGTA, 10% glycerol, 100 mm NaF, 1 mm Na3VO4, and 1% Triton X-100). The lysates were precleared by incubation with protein G-agarose (Roche Applied Science) for 1 h at 4°C. To purify target proteins, 1 μg of monoclonal or affinity-purified polyclonal antibody was incubated with 500 μg of precleared cell lysate for 2 h at 4°C and then precipitated with protein G-agarose for 1 h at 4°C. Immunoprecipitates were extensively washed with PLC buffer, separated by SDS-PAGE, and subjected to immunoblotting. After blocking, blots were incubated with appropriate primary antibodies. Blots were then washed four times with TBST (150 mm NaCl, 10 mm Tris (pH 8.0), and 0.05% Tween20), incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit whole IgG antibodies (Amersham Biosciences) for 30 min, washed, and visualized by autoradiography using chemiluminescence reagent (Western Lighting; PerkinElmer). Cell Staining—Cells were fixed for 5 min at room temperature with 4% paraformaldehyde in phosphate-buffered saline and permeabilized for 10 min with 0.2% Triton X-100. The cells were preincubated in 2% bovine serum albumin with 5% normal serum for 0.5 h and incubated with specific primary antibodies for 1 h at room temperature. After washing, cells were incubated with Alexa-conjugated secondary antibodies (Molecular Probes) for 0.5 h at room temperature. In some experiments, membrane-bound Fc fusion proteins were stained with Alexa488-conjugated anti-mouse IgGFc for 0.5 h. Photos were taken with a Radiance 2100 confocal microscope (Bio-Rad). In Vitro Binding Assay—The recombinant GST-tagged claudin-4 containing the second cytoplasmic domain was prepared in TKX1-competent cells (Stratagene) by transformation with pGEX4T claudin-4. Tyrosine-phosphorylated GST-claudin-4 was prepared by induction of a nonspecific tyrosine kinase in TKX1 cells according to the manufacturer's instructions. GST-tagged proteins (2.5 were purified using GST-tagged protein (2.5 was incubated with lysate prepared from COS1 cells transfected with N-ZO-1 in PLC buffer for h at 4°C. The were washed four times with the buffer, and proteins were separated by N-ZO-1 was by with cells were into the of After the cells and tight the medium of the and was with containing 2 mm The of cell-cell contact after with was by The medium was from to normal medium with fetal bovine serum and were to for the indicated or was to the at a of 10 and incubated for 30 were then taken from the of the The of in the was determined using with an of and of at with the of association between EphA2 and claudin-4 was in COS1 cells EphA2 and claudin-4. In these cells, EphA2 with claudin-4 with antibodies of an complex was by using antibodies EphA2 also with claudin-4 in of the colon cancer cell EphA2 and claudin-4 in colon cancer of EphA2 with of through the of receptors A. M. A. R. U. Klein R. Mol. Cell. 2002; 9: Full Text Full Text PDF PubMed Scopus Google Scholar, C.A. M. 2001; PubMed Scopus Google Scholar, C.A. M. Cell Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). in the localization of claudin-4 after of EphA2 with For binding of the Fc fusion protein to the cells were at In cells, claudin-4 is expressed on the cell and in the after to claudin-4 as in the that 1 h after of claudin-4 with was as in the The of EphA2 and claudin-4 association of these two a of mutants of claudin-4 were generated to the region required for interaction with EphA2 the mutants of claudin-4, the one containing the region including the extracellular domain to EphA2 mutants of EphA2 were to the region involved in the interaction with claudin-4 The extracellular the cytoplasmic domain of EphA2, associates with claudin-4 The region of EphA2 (amino acids to its K.R. M. C.A. M. 2001; PubMed Scopus Google Scholar). to which the region for the interaction with its the a specific EphA2 which was found to binding with Tanaka and R. EphA2 binding to claudin-4 These that and claudin-4 to the region of EphA2 (amino acids in in the with is to activation by overexpressed in COS1 was phosphorylated on tyrosine residue with wild EphA2, with a EphA2 mutant EphA2K646M tyrosine phosphorylation of claudin-4 by wild EphA2 in a was also that the claudin-4 mutant that the (amino acids claudin-4 was phosphorylated These results indicate that association of EphA2 with claudin-4 and EphA2 kinase is required for phosphorylation of claudin-4. has tyrosine in the second cytoplasmic is tyrosine in the cytoplasmic the tyrosine in the second cytoplasmic the one located at the carboxyl terminus is conserved in most claudin families (22Itoh M. Furuse M. Morita K. Kubota K. Saitou M. Tsukita S. J. Cell Biol. 1999; 147: 1351-1363Crossref PubMed Scopus (934) Google Scholar). phosphorylation of claudin-4 was the tyrosine at the carboxyl terminus was by of tyrosine the phosphorylation of claudin-4 was determined whether cell-cell contact tyrosine phosphorylation of claudin-4 by activation of EphA2. cells were used they the which can EphA2 cell-cell adhesion L. D. P. Cancer 2002; PubMed Scopus Google Scholar). In cells, EphA2 and claudin-4 were phosphorylated on tyrosine the cells were at high their phosphorylation was cell-cell interaction was by of in the medium with a of on the of tyrosine phosphorylation of claudin-4 and EphA2 the that a of by the phosphorylation of of cells with phosphorylation of claudin-4 phosphorylation of claudin-4 in cells was by expression of which the cytoplasmic region of EphA2 These results that claudin-4 is phosphorylated in cells EphA2 is activated cell-cell interaction. EphA2, with claudin-4 after of cell-cell adhesion after the cells were as a cell through incubation with the the of association between EphA2 and claudin-4 on the cell of the cells in than on two cells in of with carboxyl-terminal YV sequence of claudin associates with the PDZ domain of ZO-1 (22Itoh M. Furuse M. Morita K. Kubota K. Saitou M. Tsukita S. J. Cell Biol. 1999; 147: 1351-1363Crossref PubMed Scopus (934) Google Scholar). whether tyrosine phosphorylation of claudin-4 at the cytoplasmic interaction between claudin-4 and and of recombinant GST-tagged which the cytoplasmic were purified using the TKX1 E. coli expression system the region of ZO-1 expressed in COS1 the phosphorylated claudin-4 precipitated of N-ZO-1 1 and tyrosine we also generated recombinant in which two were to that phosphorylation of of claudin-4 is for of its binding with wild and the mutant of ZO-1 1 and phosphorylated in which was to ZO-1 claudin-4 lacking the carboxyl-terminal YV sequence to ZO-1 5 and were to whether EphA2 activation leads to of association between claudin-4 and The of claudin-4 with ZO-1 was reduced in COS1 cells between claudin-4 and ZO-1 was also in MDCK cells EphA2, and this association was decreased by with these results we that EphA2 activation can association of claudin-4 with ZO-1 by phosphorylation of of claudin-4 in To the biological of of the binding between claudin-4 and ZO-1, the in localization of claudin-4 was the of of cell-cell or the mutant of EphA2 was expressed at high by in MDCK cells, which claudin-4. the cells were with ZO-1 and claudin-4 localization to cell-cell contact sites was 30 min after the medium with normal medium ZO-1 was found to to the cell-cell contact sites of the of or EphA2 the in cells EphA2, claudin-4 also to cell-cell contact sites 30 min after in cells EphA2, claudin-4 to these sites h after claudin-4 to cell-cell contact sites in cells EphA2 These results that EphA2 a in of claudin-4 to tight junctions has on EphA2 of on the biological of the interaction of EphA2 with claudin-4 on tight paracellular permeability was using in MDCK epithelial The of the flux through a of MDCK cells was EphA2 was overexpressed we to the of EphA2 on of the paracellular barrier after of EphA2 the of paracellular permeability through an MDCK after and this was cells were by the EphA2 paracellular permeability that the kinase of EphA2 permeability. In this the phosphorylation of claudin-4 h after decreased to the as in the cells 2 and The paracellular flux of was at shown in Fig. of in This is the tyrosine phosphorylation of claudin in with its biological EphA2 the specific phosphorylation of in the cytoplasmic of claudin-4 in COS1 cells, and claudin-4 was phosphorylated in to EphA2 activation in the kinase of EphA2 and association between EphA2 and claudin-4 are required for phosphorylation of claudin-4. that EphA2 and claudin-4 on the cell surface in claudin-4 is most a of EphA2. results indicate that activation of EphA2 by its decreases integration of claudin-4 into tight junctions and paracellular permeability. phosphorylation of claudin-4 in its carboxyl-terminal binding to is also reported that phosphorylation of the carboxyl-terminal of by also its association with ZO-1, ZO-2, and in A. P. Biochem. Biophys. Res. Commun. 2003; PubMed Scopus Google Scholar). tyrosine phosphorylation the PDZ is to interaction. results that activation of EphA2 integration of claudin-4 into the sites of cell-cell after the barrier function of tight junctions. Although the mechanism of assembly of and ZO-1 into tight junctions is ZO-1, which is to tight junctions B. A. M. H. J. Biol. 2002; PubMed Scopus Google Scholar), to phosphorylated claudin-4 to loss of This is with the that claudin-4, ZO-1, localization to cell is decreased after activation of EphA2 The of paracellular permeability was the of tight junction by the expression of EphA2 tight and permeability and claudin-4 localization to were after in MDCK cells claudin-4 was phosphorylated These results that phosphorylation of claudin-4 the of tight junctions the of tight junctions. is that the phosphorylation of claudin-4 in MDCK cells was and can be for by members of the claudin family or by some are screening cell in which claudin-4 is phosphorylated at tight junctions to this of in the of specific the of association between each of the Eph family and the claudin family is one Eph family is to be involved in this as claudin-4 was also tyrosine phosphorylated by with in COS1 cells the tyrosine residue located at the carboxyl terminus is conserved among at claudin family members, also have the to be phosphorylated by Eph receptors. are this In EphA2, assembly of tight junctions be via phosphorylation of This has the to the malignant phenotype of the such as loss of cell in EphA2 and claudin-4, colon cancer cells, phosphorylation of claudin-4 from tight junction are to of signaling through tyrosine phosphorylation of claudin-4 and their in the of epithelial cells and cancer S. Tsukita for the plasmids used in this B. J. of and D. B. of Molecular for this