Phosphorylation of claudin-3 at threonine 192 by cAMP-dependent protein kinase regulates tight junction barrier function in ovarian cancer 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.
D'Souza
Theresa
T
Laboratory of Cellular and Molecular Biology, Gerontology Research Center, NIA, National Institutes of Health, Baltimore, Maryland 21224, USA.
Agarwal
Rachana
R
Morin
Patrice J
PJ
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D062465:Claudin-3; D062506:Claudin-4; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D017097:Electric Impedance; D004591:Electrophoresis, Polyacrylamide Gel; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005982:Glutathione Transferase / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D009154:Mutation; D010051:Ovarian Neoplasms / Q000378:metabolism*; D010539:Permeability; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D013912:Threonine / Q000737:chemistry*; D019108:Tight Junctions / Q000737:chemistry*; D013997:Time Factors; D014162:Transfection
C568122:CLDN3 protein, human; C568157:CLDN4 protein, human; D062465:Claudin-3; D062506:Claudin-4; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013912:Threonine; D005982:Glutathione Transferase; D017868:Cyclic AMP-Dependent Protein Kinases; D002118:Calcium