PKCη regulates occludin phosphorylation and epithelial tight junction integrity

• Published in: Proceedings of the National Academy of Sciences - PNAS 2009-01, Vol.106 (1), p.61-66
• Main Authors: Suzuki, Takuya, Elias, Bertha C, Seth, Ankur, Shen, Le, Turner, Jerrold R, Giorgianni, Francesco, Desiderio, Dominic, Guntaka, Ramareddy, Rao, Radhakrishna
• Format: Article
• Language: English
• Subjects: Animals; Antibodies; Binding Sites; Biological Sciences; Caco 2 cells; Calcium; Cell Line; Dogs; Epithelium - metabolism; Epithelium - ultrastructure; Goods and services tax; Humans; Intercellular junctions; Membrane Proteins - genetics; Membrane Proteins - metabolism; Mutation; Occludin; Phosphorylation; Physiological regulation; Protein isoforms; Protein Kinase C - metabolism; Protein Kinase C - physiology; Proteins; Tight junctions; Tight Junctions - metabolism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0802741106

PKCη is expressed predominantly in the epithelial tissues; however, its role in the regulation of epithelial tight junctions (TJs) is unknown. We present evidence that PKCη phosphorylates occludin on threonine residues (T403 and T404) and plays a crucial role in the assembly and/or maintenance of TJs in Caco-2 and MDCK cell monolayers. Inhibition of PKCη by specific pseudo substrate inhibitor or knockdown of PKCη by specific shRNA disrupts the junctional distribution of occludin and ZO-1 and compromises the epithelial barrier function. Expression of dominant negative, PKCηK₃₉₄R disrupts the TJ and barrier function, whereas wild-type PKCη and constitutively active PKCηA₁₆₁E enhance the TJ integrity. Inhibition and knockdown of PKCη or expression of PKCηK₃₉₄R induce dephosphorylation of occludin on threonine residues, whereas active PKCη elevates occludin phosphorylation. PKCη directly interacts with the C-terminal domain of occludin and phosphorylates it on highly conserved T403 and T404. T403/404A mutations result in the loss of occludin's ability to localize at the TJs, whereas T403/404D mutations attenuates the PKCη inhibitor-mediated redistribution of occludin from the intercellular junctions. These results reveal an important mechanism of epithelial TJ regulation by PKCη.