Phosphorylation of Claudin-3 at Threonine 192 by cAMP-dependent Protein Kinase Regulates Tight Junction Barrier Function in Ovarian Cancer Cells

Abstract

Claudins are integral membrane proteins essential in the formation and function of tight junctions (TJs). Disruption of TJs, which have essential roles in cell permeability and polarity, is thought to contribute to epithelial tumorigenesis. Claudin-3 and -4 are frequently overexpressed in ovarian cancer, but the molecular pathways involved in the regulation of these proteins are unclear. Interestingly, several studies have demonstrated a role for phosphorylation in the regulation of TJ complexes, although evidence for claudin phosphorylation is scarce. Here, we showed that claudin-3 and -4 can be phosphorylated in ovarian cancer cells. In vitro phosphorylation assays using glutathione S-transferase fusion constructs demonstrated that the C terminus of claudin-3 is an excellent substrate for cAMP-dependent protein kinase (PKA). Using site-directed mutagenesis, we identified a PKA phosphorylation site at amino acid 192 in the C terminus of claudin-3. Overexpression of the protein containing a T192D mutation, mimicking the phosphorylated state, resulted in a decrease in TJ strength in ovarian cancer cell line OVCA433. Our results suggest that claudin-3 phosphorylation by PKA, a kinase frequently activated in ovarian cancer, may provide a mechanism for the disruption of TJs in this cancer. In addition, our findings may have general implications for the regulation of TJs in normal epithelial cells. Claudins are integral membrane proteins essential in the formation and function of tight junctions (TJs). Disruption of TJs, which have essential roles in cell permeability and polarity, is thought to contribute to epithelial tumorigenesis. Claudin-3 and -4 are frequently overexpressed in ovarian cancer, but the molecular pathways involved in the regulation of these proteins are unclear. Interestingly, several studies have demonstrated a role for phosphorylation in the regulation of TJ complexes, although evidence for claudin phosphorylation is scarce. Here, we showed that claudin-3 and -4 can be phosphorylated in ovarian cancer cells. In vitro phosphorylation assays using glutathione S-transferase fusion constructs demonstrated that the C terminus of claudin-3 is an excellent substrate for cAMP-dependent protein kinase (PKA). Using site-directed mutagenesis, we identified a PKA phosphorylation site at amino acid 192 in the C terminus of claudin-3. Overexpression of the protein containing a T192D mutation, mimicking the phosphorylated state, resulted in a decrease in TJ strength in ovarian cancer cell line OVCA433. Our results suggest that claudin-3 phosphorylation by PKA, a kinase frequently activated in ovarian cancer, may provide a mechanism for the disruption of TJs in this cancer. In addition, our findings may have general implications for the regulation of TJs in normal epithelial cells. Claudin proteins are a large family of transmembrane proteins essential in the formation and maintenance of tight junctions (TJs). 1The abbreviations used are: TJ, tight junction; FSK, forskolin; GST, glutathione S-transferase; PKA, cAMP-dependent protein kinase; PMA, phorbol 12-myristate 13-acetate; TER, transepithelial electrical resistance; PBS, phosphate-buffered saline; WT, wild type; DMSO, dimethyl sulfoxide; PKAc, PKA catalytic subunit. Tight junctions in epithelial and endothelial cells provide a selective barrier and establish cellular polarity (1Gonzalez-Mariscal L. Betanzos A. Nava P. Jaramillo B.E. Prog. Biophys. Mol. Biol. 2003; 81: 1-44Crossref PubMed Scopus (922) Google Scholar, 2Mitic L.L. Van Itallie C.M. Anderson J.M. Am. J. Physiol. Gastrointest. Liver Physiol. 2000; 279: G250-G254Crossref PubMed Google Scholar, 3Tsukita S. Furuse M. Itoh M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 285-293Crossref PubMed Scopus (2041) Google Scholar, 4Matter K. Balda M.S. Nat. Rev. Mol. Cell. Biol. 2003; 4: 225-236Crossref PubMed Scopus (717) Google Scholar). These structures are typically lost in cancer, and this loss may contribute to the invasive and metastatic phenotype of tumor cells (5Langbein L. Pape U.F. Grund C. Kuhn C. Praetzel S. Moll I. Moll R. Franke W.W. Eur. J. Cell Biol. 2003; 82: 385-400Crossref PubMed Scopus (98) Google Scholar, 6Martin T.A. Jiang W.G. Histol. Histopathol. 2001; 16: 1183-1195PubMed Google Scholar, 7Itoh M. Bissell M.J. J. Mammary Gland Biol. Neoplasia. 2003; 8: 449-462Crossref PubMed Scopus (108) Google Scholar, 8Mullin J.M. Sci. STKE 2004. 2004; : pe2Google Scholar). Using serial analysis of gene expression, we have previously shown that claudin-3 and -4 are among the most highly up-regulated genes in ovarian cancer (9Hough C.D. Sherman-Baust C.A. Pizer E.S. Montz F.J. Im D.D. Rosenshein N.B. Cho K.R. Riggins G.J. Morin P.J. Cancer Res. 2000; 60: 6281-6287PubMed Google Scholar). The high expression of these claudins in ovarian cancer has been confirmed by our group and others using a variety of approaches such as microarrays, tissue arrays, and reverse transcription-PCR (10Rangel 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, 11Santin A.D. Zhan F. Bellone S. Palmieri M. Cane S. Bignotti E. Anfossi S. Gokden M. Dunn D. Roman J.J. O'Brien T.J. Tian E. Cannon M.J. Shaughnessy Jr., J. Pecorelli S. Int. J. Cancer. 2004; 112: 14-25Crossref PubMed Scopus (253) Google Scholar, 12Hibbs K. Skubitz K.M. Pambuccian S.E. Casey R.C. Burleson K.M. Oegema Jr., T.R. Thiele J.J. Grindle S.M. Bliss R.L. Skubitz A.P. Am. J. Pathol. 2004; 165: 397-414Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 13Heinzelmann-Schwarz V.A. Gardiner-Garden M. Henshall S.M. Scurry J. Scolyer R.A. Davies M.J. Heinzelmann M. Kalish L.H. Bali A. Kench J.G. Edwards L.S. Vanden Bergh P.M. Hacker N.F. Sutherland R.L. O'Brien P.M. Clin. Cancer Res. 2004; 10: 4427-4436Crossref PubMed Scopus (177) Google Scholar, 14Lu K.H. Patterson A.P. Wang L. Marquez R.T. Atkinson E.N. Baggerly K.A. Ramoth L.R. Rosen D.G. Liu J. Hellstrom I. Smith D. Hartmann L. Fishman D. Berchuck A. Schmandt R. Whitaker R. Gershenson D.M. Mills G.B. Bast Jr., R.C. Clin. Cancer Res. 2004; 10: 3291-3300Crossref PubMed Scopus (377) Google Scholar). Considering that TJs are typically lost in neoplasia, these findings are somewhat surprising, and the exact role of these proteins in ovarian cancer remains unclear. There is, however, some evidence that claudin overexpression may actually lead to increased cell survival and invasion. 2R. Agarwal, T. D'Souza, and P. J. Morin, unpublished observations. A better understanding of the regulation of claudin proteins in ovarian cancer cells may help clarify their roles in this disease. Several lines of evidence suggest that TJs are involved in various cell signaling pathways, providing a possible link between cell polarity/cell contact and downstream signaling events (4Matter K. Balda M.S. Nat. Rev. Mol. Cell. Biol. 2003; 4: 225-236Crossref PubMed Scopus (717) Google Scholar, 15Schneeberger E.E. Lynch R.D. Am. J. Physiol. Cell Physiol. 2004; 286: C1213-C1228Crossref PubMed Scopus (1113) Google Scholar). Furthermore, the involvement of kinases in the biogenesis and regulation of several tight junction components has been established (16Kale G. Naren A.P. Sheth P. Rao R.K. Biochem. Biophys. Res. Commun. 2003; 302: 324-329Crossref PubMed Scopus (165) Google Scholar, 17Clarke H. Marano C.W. Peralta Soler A. Mullin J.M. Adv. Drug Delivery Rev. 2000; 41: 283-301Crossref PubMed Scopus (83) Google Scholar, 18Wang Y. Zhang J. Yi X.J. Yu F.S. Exp. Eye Res. 2004; 78: 125-136Crossref PubMed Scopus (103) Google Scholar, 19Eckert J.J. McCallum A. Mears A. Rumsby M.G. Cameron I.T. Fleming T.P. Reproduction. 2004; 127: 653-667Crossref PubMed Scopus (44) Google Scholar, 20Karczewski J. Groot J. Am. J. Physiol. Gastrointest. Liver Physiol. 2000; 279: G660-G665Crossref PubMed Google Scholar). However, only a few studies have clearly demonstrated phosphorylation of claudins. For example, phosphorylation of claudin-1 by mitogen-activated protein kinases (21Fujibe M. Chiba H. Kojima T. Soma T. Wada T. Yamashita T. Sawada N. Exp. Cell Res. 2004; 295: 36-47Crossref PubMed Scopus (121) Google Scholar) and protein kinase C (22Nunbhakdi-Craig V. Machleidt T. Ogris E. Bellotto D. White III, C.L. Sontag E. J. Cell Biol. 2002; 158: 967-978Crossref PubMed Scopus (223) Google Scholar), as well as phosphorylation of claudin-5 by cAMP-dependent protein kinase (PKA) (23Ishizaki T. Chiba H. Kojima T. Fujibe M. Soma T. Miyajima H. Nagasawa K. Wada I. Sawada N. Exp. Cell Res. 2003; 290: 275-288Crossref PubMed Scopus (164) Google Scholar, 24Soma T. Chiba H. Kato-Mori Y. Wada T. Yamashita T. Kojima T. Sawada N. Exp. Cell Res. 2004; 300: 202-212Crossref PubMed Scopus (104) Google Scholar), has been reported. Also, WNK4 kinase has been shown to phosphorylate claudin-3 and -4 (25Yamauchi K. Rai T. Kobayashi K. Sohara E. Suzuki T. Itoh T. Suda S. Hayama A. Sasaki S. Uchida S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4690-4694Crossref PubMed Scopus (228) Google Scholar). To address the issue of claudin regulation in ovarian cancer, we have investigated phosphorylation as a possible modulatory mechanism of claudin function. We first show that both claudin-3 and -4 are phosphorylated in ovarian cancer cells, although probably by different kinases. Indeed, whereas claudin-3 is a target of PKA, claudin-4 appears to be phosphorylated by protein kinase C. For this report, we have focused on PKA-mediated phosphorylation of claudin-3. We show that claudin-3 is phosphorylated at threonine 192 in the cytoplasmic C-terminal region. Mimicking the phosphorylated state by mutating this site to an aspartic acid decreases TJ strength in ovarian cancer cells. Our data provide the first evidence for the phosphorylation of claudin-3 and -4 in ovarian cancer cells and suggest that phosphorylation of claudin-3 by PKA, a kinase frequently activated in ovarian cancer, may provide a mechanism for the disruption of TJs in this cancer. Cell Culture—Ovarian cell lines BG-1, CAOV3, HEY, IGROV-1, UCI101, 2008, A2780, OVCA420, OVCA429, OVCA432, OVCA433, OVCAR-2, OVCAR-3, OVCAR-4, and OVCAR-5 were cultured in McCoy's 5A growth medium (Invitrogen) supplemented with 10% fetal bovine serum and antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin). HOSE-B, an ovarian surface epithelial cell line immortalized with E6 and E7 (26Gregoire L. Munkarah A. Rabah R. Morris R.T. Lancaster W.D. In Vitro Cell. Dev. Biol. Anim. PubMed Scopus Google Scholar), in medium supplemented with 10% fetal serum and μg/ml cells were in McCoy's growth medium with the To establish of OVCA433, were using to the were and in McCoy's growth medium containing μg/ml claudin-3 and claudin-4 were of a and were and were were of claudin-3 and by of normal ovarian tissue and an ovarian cancer tissue in our (10Rangel 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). using For of in showed were by on a cell were by and to were with in 100 and and with PKAc, the in were with used for of the In and cancer cell lines HEY, OVCA432, and were at in were for and with in medium for at were with and phorbol 12-myristate for at to the of cells were at in and with with a claudin-3 the using of cells were with were in phosphate-buffered and with of 10% for at The by at for For the with of and of protein were with the with the in for and on The were to for and as of Claudin-3 and -4 and of and by ovarian cancer cell line and expression using the and of the fusion constructs were by using the claudin fusion proteins were with the C-terminal of claudin-3 and claudin-4 The were to to be with the in of the threonine in the PKA site were using the site-directed constructs were confirmed by and of constructs of and were in for protein to the with some were and protein expression with at for at for at were in with and the by at for proteins were by using The were with PBS, and the fusion protein with glutathione In Vitro GST, and claudin-3 and proteins were with kinase containing 100 and of of PKA to the to a of and at for The with The proteins on and for and were used to of and cells were at a of on using a The were for the of the and of and For with and were at with For the the in the medium using at a of S. J. Cell Biol. PubMed Scopus Google Scholar). were the an were with and with normal medium were for by of medium with normal and were the were at and for to and with an molecular of in McCoy's to a of to the The with the and were the and by using at and at The for used for and at were with PBS, by with for cells with were in a cells were with and in bovine serum by with claudin-3 at for with PBS, the cells were with for are as S.E. analysis by using analysis of and Scholar) with Claudin-3 and -4 in Cancer Cell first to claudin-3 and -4 be phosphorylated in ovarian cancer cells. We used in phosphorylation assays in which we ovarian cancer cell lines with and PMA, a PKA and a protein kinase C with cells, claudin-3 phosphorylation increased in both and cells. In addition, some of the claudin-3 protein to be phosphorylated in the cells. cells, which were as a and the phosphorylated in these cells were with molecular different of the claudin results were with claudin-4 Indeed, of cells with the phosphorylation of claudin-4 in both and cell The in claudin-4 phosphorylation is in cells, in which phosphorylation is but highly cells were used as claudin-4 protein as To which of the increased the phosphorylation of claudin-3 and -4 an in phosphorylation in cells in the of that claudin-3 phosphorylated whereas increased the phosphorylation of claudin-4 in cells. results were in cells These results in these cells, claudin-3 is phosphorylated by PKA, whereas claudin-4 is phosphorylated by protein kinase C. phosphorylation of claudin-3 by a PKA to phosphorylation our that claudin-3 is phosphorylated in by PKA in these cells. of PKA and Claudin-3 in Cancer Cell and Cancer of the findings that claudin-3 is a target for PKA in ovarian cancer cells, we to protein of in a of ovarian cancer cells. in cell lines HEY, which to claudin-3 expression of the catalytic with the various can various in different cells K. Physiol. Rev. 2004; PubMed Scopus Google Scholar). of the cell lines in the of PKA in these cells. is in with a of PKA in ovarian cancer Atkinson S. P. J. Cancer. PubMed Scopus Google Scholar, Hacker N.F. PubMed Scopus Google Scholar). Claudin-3 expression in cells but in most of the ovarian cancer of cell lines both claudin-3 and is with our expression of claudin-3 protein in the of ovarian (10Rangel 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). of normal ovarian tissue with and claudin-3 the of in the and of the but claudin-3 clearly a and to we for the expression of these proteins in the ovarian epithelial cells of our ovarian cancer tissue with showed that this protein in the ovarian cancer and that the of with of claudin-3 and These proteins were both at the membrane and The of and which are shown at the of and claudin-3. These results are with our that claudin-3 may be a target for PKA phosphorylation in ovarian cancer cells. Claudin-3 in Vitro by to the exact PKA phosphorylation in claudin-3. We an in vitro phosphorylation with fusion proteins containing the C terminus of claudin-3 and -4 PKA phosphorylated the claudin-3 C-terminal whereas claudin-4 phosphorylated with claudin-3 The protein claudin phosphorylated in these These in vitro findings are with the in that claudin-3 is phosphorylated by a PKA with claudin-4 To which of the in the C-terminal of claudin-3 is we site-directed on that the for PKA that a threonine to at 192 of the C-terminal of claudin-3 the phosphorylation at phosphorylation although to a in phosphorylation of the claudin-3 C In addition, various with of claudin-3 and transmembrane were phosphorylated in these in vitro assays Claudin-3 in Vitro and in by PKA at claudin-3 be phosphorylated in vitro and in at a analysis of claudin-3 showed that the containing be of the for we to approaches to this claudin-3 and were overexpressed in cell and both of which were of claudin-3. proteins were with with for a kinase of claudin-3 both and phosphorylated whereas phosphorylation in cells the In addition, we claudin-3 with in cells. in the of the PKAc, claudin-3 whereas the Interestingly, the phosphorylated claudin-3 in the in the Claudin-3 the role of PKA phosphorylation in the barrier function of TJs, we overexpressed claudin-3 WT, and T192D the phosphorylated state of the in ovarian cancer cells. For this we the cell line among the ovarian cancer cell lines these cells the TJ function as by transepithelial electrical cells, as well as cells claudin-3 the high a of However, expression of the T192D mimicking the phosphorylated state, to a of with at a with the of claudin-3 WT, at that the TJs The data shown are of but results were with of as well as with of We with cells, which have high and are used in TJ to we for cells, expression of the T192D in cells the with claudin-3 WT, We permeability of using the the established with cells, cells claudin-3 T192D increased of Indeed, the increased by of and by of The claudin-3 and permeability to the of is in of results in a disruption of TJs C. U. H. Cell Biol. 2000; PubMed Scopus (44) Google Scholar). PKA has been shown to the of TJs the of a substrate for PKA, in TJ regulation K.M. Am. J. Physiol. Cell Physiol. 2002; PubMed Scopus Google Scholar). We to TJs by of be by overexpression of claudin-3 and phosphorylation The cells claudin-3 were with normal medium medium the in the at the by the in The the were the as of TJ in cells, the TJ in cells claudin-3 the but in the cells, that phosphorylation of the to a decrease in the of the cells to In addition, with these of cells, which to claudin-3 phosphorylation resulted in a decrease in in a of of have shown that claudin-3 can be phosphorylated by PKA and that a mimicking the phosphorylated state can decrease TJ function To better the to this of TJs, we phosphorylation at of claudin-3. claudin-3 and were at the However, whereas claudin-3 T192D at the the to be and the to be These results show that phosphorylation may a of claudin-3 the TJs to membrane cytoplasmic and may provide a mechanism for the TJ strength In addition, the with with this Indeed, claudin-3 to the surface of the cells to FSK, however, claudin-3 and at the of the cells with the PKA by of in the of the of A of has confirmed the overexpression of claudin-3 and -4 in ovarian cancer (9Hough C.D. Sherman-Baust C.A. Pizer E.S. Montz F.J. Im D.D. Rosenshein N.B. Cho K.R. Riggins G.J. Morin P.J. Cancer Res. 2000; 60: 6281-6287PubMed Google Scholar, 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, 11Santin A.D. Zhan F. Bellone S. Palmieri M. Cane S. Bignotti E. Anfossi S. Gokden M. Dunn D. Roman J.J. O'Brien T.J. Tian E. Cannon M.J. Shaughnessy Jr., J. Pecorelli S. Int. J. Cancer. 2004; 112: 14-25Crossref PubMed Scopus (253) Google Scholar, 12Hibbs K. Skubitz K.M. Pambuccian S.E. Casey R.C. Burleson K.M. Oegema Jr., T.R. Thiele J.J. Grindle S.M. Bliss R.L. Skubitz A.P. Am. J. Pathol. 2004; 165: 397-414Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 13Heinzelmann-Schwarz V.A. Gardiner-Garden M. Henshall S.M. Scurry J. Scolyer R.A. Davies M.J. Heinzelmann M. Kalish L.H. Bali A. Kench J.G. Edwards L.S. Vanden Bergh P.M. Hacker N.F. Sutherland R.L. O'Brien P.M. Clin. Cancer Res. 2004; 10: 4427-4436Crossref PubMed Scopus (177) Google Scholar, 14Lu K.H. Patterson A.P. Wang L. Marquez R.T. Atkinson E.N. Baggerly K.A. Ramoth L.R. Rosen D.G. Liu J. Hellstrom I. Smith D. Hartmann L. Fishman D. Berchuck A. Schmandt R. Whitaker R. Gershenson D.M. Mills G.B. Bast Jr., R.C. Clin. Cancer Res. 2004; 10: 3291-3300Crossref PubMed Scopus (377) Google Scholar), but the role and regulation of claudin proteins in this cancer have been a first in the regulation of claudins in ovarian cancer, we have focused on the of phosphorylation of these Using PKA and protein kinase C we show in different cancer cell claudin-3 is target of PKA, whereas claudin-4 is phosphorylated by protein kinase C. The of the regulation of these claudins is at this but the of PKA on TJs have we have focused on the phosphorylation of claudin-3 by We clearly that with PKA of both and to increased claudin-3 However, the for the in the phosphorylation of claudin-3 between these cell lines is phosphorylated in with is may be by in the of PKA in these cell lines may be to kinases. the remains that claudin-3 phosphorylation is in these cells and that a PKA this We identified as the PKA phosphorylation site in claudin-3. is demonstrated using in vitro phosphorylation assays of wild and We these findings in assays wild claudin-3 in cells claudin-3. To the possible of claudin-3 phosphorylation in ovarian cancer cells, we have the most function of claudin the to TJ for these studies TER, a used for the of TJ Interestingly, overexpression of a claudin-3 mimicking the phosphorylated a with the and the of the that phosphorylation at threonine 192 may with TJ In addition, we showed that expression of the T192D increased the permeability of cells using an In of both the and permeability appears that the TJ may be in the of overexpressed claudin-3 The exact to a decrease in TJ function by phosphorylated claudin-3 are but our data provide some Indeed, with the expression of the T192D to lead to at the that the phosphorylation may the of claudin-3 to in TJ of the cells with a of for claudin-3 and In addition, we that whereas claudin-3 in both and the phosphorylated in the is possible that phosphorylation the of the claudin these in with our suggest a in and molecular in the barrier function signaling of claudin These results are in with findings that signaling the of claudin-5 T. Chiba H. Kato-Mori Y. Wada T. Yamashita T. Kojima T. Sawada N. Exp. Cell Res. 2004; 300: 202-212Crossref PubMed Scopus (104) Google Scholar). However, is possible that different phosphorylation events may have different on claudin In addition, is to that of claudin-3 with and proteins and their by PKA may a has been shown to be phosphorylated by PKA, function at the A. E. J. S. Betanzos A. M. L. Biochem. J. 2001; PubMed Scopus Google Scholar). demonstrated that PKA junction M.J. P. N. J. Cell. Physiol. PubMed Scopus Google Scholar). be to the between claudin and TJ proteins and the of claudin phosphorylation on these In this we show in ovarian cancer cells, claudin-3 and -4 can be phosphorylated by PKA and protein kinase to our the first of claudin phosphorylation by these kinases in cell The of the by these kinases on these claudins to be but may contribute to ovarian tumorigenesis. shown that claudin-3 and -4 are for WNK4 kinase (25Yamauchi K. Rai T. Kobayashi K. Sohara E. Suzuki T. Itoh T. Suda S. Hayama A. Sasaki S. Uchida S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4690-4694Crossref PubMed Scopus (228) Google Scholar), which is to a with an in the exact molecular are that of claudin be to the in These data are with our that claudin-3 can be phosphorylated in and the that this phosphorylation with TJ function. have protein kinase C in the regulation of TJs phorbol H. Marano C.W. Peralta Soler A. Mullin J.M. Adv. Drug Delivery Rev. 2000; 41: 283-301Crossref PubMed Scopus (83) Google Scholar, C.M. V. M. Am. J. Physiol. Cell Physiol. 2001; PubMed Google Scholar), but regulation of TJs has been and is J. Groot J. Am. J. Physiol. Gastrointest. Liver Physiol. 2000; 279: G660-G665Crossref PubMed Google Scholar). PKA has been to have a role in TJ disruption C. U. H. Cell Biol. 2000; PubMed Scopus (44) Google Scholar), and claudin-3 phosphorylation may of the to this Interestingly, PKA is activated in ovarian cancer Atkinson S. P. J. Cancer. PubMed Scopus Google Scholar, Hacker N.F. PubMed Scopus Google Scholar). In addition, our unpublished that claudin overexpression in ovarian cancer cells may contribute to an in and survival of these cells, at in the of PKA, role in the phosphorylation of may contribute to the decrease in polarity and the invasive of ovarian cancer cells. In this this may an target for cancer and our provide an to PKA for M. R. Cho C. C. N. Y. Acad. Sci. 2002; PubMed Scopus Google Scholar). Our of claudin phosphorylation a mechanism for the regulation of claudin-3 in ovarian cancer. In addition, we are the of regulation of this protein in ovarian cancer. remains to be the mechanism in this is a general of epithelial cells is to the state of ovarian cancer. We Sherman-Baust and Roman and for on the We for the in analysis and on the We Sherman-Baust for The cell line OVCA420, OVCA429, OVCA432, OVCA433, and were Bast and and were C.