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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.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2005 Jul 15; Vol. 280 (28), pp. 26233-40. Date of Electronic Publication: 2005 May 19. - Publication Year :
- 2005
-
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.
- Subjects :
- Binding Sites
Calcium metabolism
Cell Line
Cell Line, Tumor
Claudin-3
Claudin-4
Cyclic AMP-Dependent Protein Kinases chemistry
Cyclic AMP-Dependent Protein Kinases metabolism
Electric Impedance
Electrophoresis, Polyacrylamide Gel
Electrophysiology
Enzyme Inhibitors pharmacology
Female
Humans
Immunoblotting
Immunohistochemistry
Immunoprecipitation
Membrane Proteins metabolism
Microscopy, Fluorescence
Models, Genetic
Mutation
Permeability
Phosphorylation
Protein Structure, Tertiary
Recombinant Fusion Proteins chemistry
Recombinant Fusion Proteins metabolism
Recombinant Proteins chemistry
Time Factors
Transfection
Cyclic AMP-Dependent Protein Kinases physiology
Glutathione Transferase metabolism
Membrane Proteins chemistry
Membrane Proteins physiology
Ovarian Neoplasms metabolism
Threonine chemistry
Tight Junctions chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 280
- Issue :
- 28
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 15905176
- Full Text :
- https://doi.org/10.1074/jbc.M502003200