Protein Kinase C Regulates the Phosphorylation and Cellular Localization of Occludin

Anna Y Andreeva,Eberhard Krause,Eva-Christina Müller,Ingolf E Blasig,Darkhan I Utepbergenov

doi:10.1074/jbc.m104923200

Abstract

Occludin is an integral membrane phosphoprotein specifically associated with tight junctions, contributing to the structure and function of this intercellular seal. Occludin function is thought to be regulated by phosphorylation, 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-dioctanoylglycerol induced the rapid phosphorylation of occludin in Madin-Darby canine kidney cells cultured in low extracellular calcium medium with a concomitant translocation 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 decreased by the protein kinase C inhibitor GF-109203X. In addition, in vitro experiments showed that the recombinant COOH-terminal domain of murine occludin could be phosphorylated by purified protein kinase C. Ser(338) of occludin was identified as an in vitro protein kinase C phosphorylation site using peptide mass fingerprint analysis and electrospray ionization tandem mass spectroscopy. These findings indicate that protein kinase C is involved in the regulation of occludin function at tight junctions.

Highlights

Tight junctions (TJs),1 the most apical component of the junctional complex of epithelial and endothelial cells, form a diffusion barrier limiting the flux of hydrophilic molecules through the paracellular pathway and maintain cell polarity by acting as a boundary between the apical and basolateral plasma membrane domains
We investigated the effect of PKC activators on the phosphorylation and cellular distribution of occludin in MDCK cells cultured in low calcium medium, which disrupts tight junctions
When phorbol 12myristate 13-acetate (PMA) at 2 nM was added for 2 h to the cells in Low Ca2ϩ medium (LC), a portion of the occludin was translocated to the plasma membrane at regions of cell-cell contact (Fig. 1C, arrows)

Summary

Introduction

Tight junctions (TJs), the most apical component of the junctional complex of epithelial and endothelial cells, form a diffusion barrier limiting the flux of hydrophilic molecules through the paracellular pathway and maintain cell polarity by acting as a boundary between the apical and basolateral plasma membrane domains (reviewed in Refs. 1 and 2). We analyzed the effect of phorbol 12myristate 13-acetate (PMA) and 1,2-dioctanoylglycerol (diC8), activators of protein kinase C, on the phosphorylation and cellular localization of occludin in monolayers of MDCK cells incubated in low extracellular Ca2ϩ medium. The phosphorylation and incorporation of occludin into tight junctions induced by PMA, This paper is available on line at http://www.jbc.org diC8, or calcium switch were inhibited by a PKC inhibitor. Ser338 of the recombinant COOH-terminal domain of murine occludin was found to be phosphorylated in vitro by purified protein kinase C. These findings suggest that the regulation of phosphorylation and cellular distribution of occludin are mediated by protein kinase C

Methods

Results

Conclusion