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Phosphorylation of the Tight Junction Protein Occludin on Ser490 Regulates Barrier Function and Contributes to Growth Control

Occludin was the first transmembrane tight junction protein identified but its function remains unclear. Occludin content correlates with barrier properties but gene deletion studies demonstrate it is not essential for formation of tight junctions. Recent data suggest occludin contributes to growth control. Our laboratory has shown that growth factor treatment of vascular endothelial cells alters barrier properties and leads to phosphorylation and redistribution of occludin and occludin Ser490 has been identified as a specific growth factor‐induced phosphorylation site. Here, our goal was to test the hypothesis that occludin Ser490 phosphorylation regulates barrier function in epithelial cells and contributes to control of cell growth. MDCK cell lines that stably overexpress wild type, Ser490 mutated to Ala (S490A) to block or Asp (S490D) to mimic phosphorylation were characterized. Occludin S490D promoted while S490A reduced growth rates as determined by cell count and DNA synthesis. Occludin S490A reduced permeability to 467Da TAMRA and Arg and increased electrical resistance. Finally, in a Ca2+ switch assay, S490A enhanced junction recovery rates while S490D recovered similar to empty vector. These results were further supported using confocal microscopy. Thus, phosphorylation of occludin on Ser490 regulates both barrier function and contributes to growth control in MDCK cells.