title: Interaction of Mrp2 with radixin causes reversible canalicular Mrp2 localization induced by intracellular redox status.
abstract: Oxidative stress is a feature of cholestatic syndrome and induces multidrug resistance-associated protein 2 (Mrp2) internalization from the canalicular membrane surface. We have previously shown that the activation of a novel protein kinase C (nPKC) by oxidative stress regulates Mrp2 internalization. The internalized Mrp2 was recycled to the canalicular surface in a protein kinase A (PKA)-dependent manner after intracellular glutathione (GSH) levels were replenished. However, the putative phosphorylation targets of these protein kinases involved in reversible Mrp2 trafficking remain unclear. In this study, we investigated the effect of changing the intrahepatic redox status on the C-terminal phosphorylation status of radixin (p-radixin), which links Mrp2 to F-actin, and the interaction of p-radixin with Mrp2 in rat hepatocytes. We detected a significant decrease in the amount of p-radixin that co-immunoprecipitated with Mrp2 after tertiary-butylhydroperoxide (t-BHP) treatment. After treatment with GSH-ethylester (GSH-EE), the phosphorylation level became the same as that of the control. A PKC and protein phosphatase (PP)-1/2A inhibitor, but not a PP-2A selective inhibitor, prevented the t-BHP-induced decrease of p-radixin and subsequent canalicular Mrp2 localization. In contrast, a PKA inhibitor affected the recovery process facilitated by GSH-EE treatment. In conclusion, the interaction of p-radixin with Mrp2 was decreased by the activation of PKC and PP-1 under oxidative stress conditions which subsequently led to Mrp2 internalization, whereas the interaction of p-radixin and Mrp2 was increased by the activation of PKA during recovery from oxidative stress.
authors.affiliation: Laboratory of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan.
authors.forename: Shuichi
authors.initials: S
authors.lastname: Sekine
authors.forename: Kousei
authors.initials: K
authors.lastname: Ito
authors.forename: Junjiro
authors.initials: J
authors.lastname: Saeki
authors.forename: Toshiharu
authors.initials: T
authors.lastname: Horie
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000199:Actins / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism*; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D007124:Immunoenzyme Techniques; D047468:Immunoprecipitation; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D054645:Protein Phosphatase 1 / Q000037:antagonists & inhibitors; D051381:Rats; D017207:Rats, Sprague-Dawley; D020122:tert-Butylhydroperoxide / Q000494:pharmacology
chemical_list: D018528:ATP-Binding Cassette Transporters; C423013:Abcc2 protein, rat; D000199:Actins; D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; C071776:radixin; D020122:tert-Butylhydroperoxide; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D054645:Protein Phosphatase 1; D005978:Glutathione