[摘要]：Protein kinases play an important role in the regulation of epithelial tight junctions. In the present study, we investigated the role of PKC zeta (protein kinase C zeta) in tight junction regulation in Caco-2 and MDCK (Madin-Darby canine kidney) cell monolayers. Inhibition of PKC zeta by a specific PKC zeta pseudosubstrate peptide results in redistribution of occludin and ZO-1 (zona occludens 1) from the intercellular junctions and disruption of barrier function without affecting cell viability. Reduced expression of PKC zeta by antisense oligonucleotide or shRNA (short hairpin RNA) also results in compromised tight junction integrity. Inhibition or knockdown of PKC zeta delays calcium-induced assembly of tight junctions. Tight junction disruption by PKC zeta pseudosubstrate is associated with the dephosphorylation of occludin and ZO-1 on serine and threonine residues. PKC zeta directly binds to the C-terminal domain of occludin and phosphorylates it on threonine residues. Thr(403), Thr(404), Thr(424) and Thr(438) in the occludin C-terminal domain are the predominant sites of PKC zeta-dependent phosphorylation. A T424A or T438A mutation in full-length occludin delays its assembly into the tight junctions. Inhibition of PKC zeta also induces redistribution of occludin and ZO-1 from the tight junctions and dissociates these proteins from the detergent-insoluble fractions in mouse ileum. The present study demonstrates that PKC zeta phosphorylates occludin on specific threonine residues and promotes assembly of epithelial tight junctions.
