Protein kinase C regulates the phosphorylation and cellular localization of occludin

Occludin is an integral membrane phosphoprotein specifically associated wit h tight junctions, contributing to the structure and function of this inter cellular seal. Occludin function is thought to be regulated by phosphorylat ion, but no information is available on the molecular pathways involved. In the present study, the involvement of the protein kinase C pathway in the regulation of the phosphorylation and cellular distribution of occludin has been investigated. Phorbol 12-myristate 13-acetate and 1,2-dioctanoylglyce rol induced the rapid phosphorylation of occludin in Madin-Darby canine kid ney cells cultured in low extracellular calcium medium with a concomitant t ranslocation of occludin to the regions of cell-cell contact. The extent of occludin phosphorylation as well as its incorporation into tight junctions induced by protein kinase C activators or calcium switch were markedly dec reased by the protein kinase C inhibitor GF-109203X. In addition, in vitro experiments showed that the recombinant COOH-terminal domain of murine occl udin could be phosphorylated by purified protein kinase C. Ser(338) of occl udin was identified as an in vitro protein kinase C phosphorylation site us ing peptide mass fingerprint analysis and electrospray ionization tandem ma ss spectroscopy. These findings indicate that protein kinase C is involved in the regulation of occludin function at tight junctions.