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J. Biol. Chem., Vol. 280, Issue 28, 26233-26240, July 15, 2005

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

Theresa D'Souza, Rachana Agarwal, and Patrice J. Morin¶

From the Laboratory of Cellular and Molecular Biology, Gerontology Research Center, NIA, National Institutes of Health, Baltimore, Maryland 21224 and Department of Pathology, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21287

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.

Received for publication, February 22, 2005 , and in revised form, April 28, 2005.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Laboratory of Cellular and Molecular Biology, Gerontology Research Center, NIA, National Institutes of Health, 5600 Nathan Shock Dr., Baltimore, MD 21224. Tel.: 410-558-8506; Fax: 410-558-8386; E-mail: morinp{at}grc.nia.nih.gov.

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