Protein kinase C regulates the phosphorylation and cellular localization of occludin.
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.
Andreeva
A Y
AY
Forschungsinstitut für Molekulare Pharmakologie, 13125 Berlin-Buch and Charité, Humboldt Universität Berlin, 13092 Berlin, Germany.
Krause
E
E
Müller
E C
EC
Blasig
I E
IE
Utepbergenov
D I
DI
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002273:Carcinogens; D002460:Cell Line; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004075:Diglycerides / Q000494:pharmacology; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry*; D051379:Mice; D008856:Microscopy, Fluorescence; D062793:Occludin; D017830:Octoxynol / Q000494:pharmacology; D010766:Phosphorylation; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019108:Tight Junctions; D013997:Time Factors
D002273:Carcinogens; D003902:Detergents; D004075:Diglycerides; D004791:Enzyme Inhibitors; D007211:Indoles; D008301:Maleimides; D008565:Membrane Proteins; D062793:Occludin; C568986:Ocln protein, mouse; D011994:Recombinant Proteins; C044295:1,2-dioctanoylglycerol; D012694:Serine; D017830:Octoxynol; D011493:Protein Kinase C; D000469:Alkaline Phosphatase; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium