title: Threonine-290 regulates nuclear translocation of the human pregnane X receptor through its phosphorylation/dephosphorylation by Ca2+/calmodulin-dependent protein kinase II and protein phosphatase 1.
abstract: The human pregnane X receptor (hPXR) is recognized as a xenobiotic-sensing nuclear receptor that transcriptionally regulates the gene expression of drug-metabolizing enzymes and transporters. Our study elucidates the mechanism by which the localization of hPXR is regulated through threonine-290. A phosphomimetic mutation at threonine-290 (T290D) retained hPXR in the cytoplasm of HepG2, HuH6, and SW480 cells in vitro and the mouse liver in vivo even after treatment with rifampicin, and a phosphodeficient mutation (T290A) translocated from the cytoplasm to the nucleus as the wild-type hPXR. The amount of the unphosphorylated wild-type yellow fluorescent protein-hPXR fusion protein but not the T290A mutant increased on Phos-tag gels in response to stimulations with rifampicin and cyclin-dependent kinase 2 inhibitor roscovitine, and a marked increase was observed in the unphosphorylated levels of the T290A mutant in nontreated cells. The Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93 [2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine)] and transfection with anti-CaMKII small-interfering RNA (siRNA) enhanced the unphosphorylated levels of the wild-type protein. CaMKII directly phosphorylated the threonine-290 of hPXR, and the T290A mutant conferred resistance to CaMKII. The protein phosphatase (PP) inhibitor okadaic acid (100 nM) and transfection with anti-PP1 siRNA but not anti-PP2A siRNA led to reduced expression of CYP3A4 mRNA. After the rifampicin and roscovitine stimulations, PP1 was recruited to the wild-type hPXR but not the T290A mutant. These results suggest that phosphorylation at threonine-290 by CaMKII may impair the function of hPXR by repressing its translocation to the nucleus, and dephosphorylation by PP1 is necessary for the xenobiotic-dependent nuclear translocation of hPXR.
authors: Department of Pharmaco-Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan sugatani@u-shizuoka-ken.ac.jp.
authors: Junko
authors: J
authors: Sugatani
authors: Department of Pharmaco-Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan.
authors: Yoshiki
authors: Y
authors: Hattori
authors: Department of Pharmaco-Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan.
authors: Yuji
authors: Y
authors: Noguchi
authors: Department of Pharmaco-Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan.
authors: Masahiko
authors: M
authors: Yamaguchi
authors: Department of Pharmaco-Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan.
authors: Yasuhiro
authors: Y
authors: Yamazaki
authors: Department of Pharmaco-Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan.
authors: Akira
authors: A
authors: Ikari
mesh_terms: D000818:Animals; D001596:Benzylamines / Q000494:pharmacology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D014453:Glucuronosyltransferase / Q000096:biosynthesis; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D009154:Mutation; D019319:Okadaic Acid / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D000077297:Pregnane X Receptor; D054645:Protein Phosphatase 1 / Q000378:metabolism*; D021381:Protein Transport; D011687:Purines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000494:pharmacology; D011987:Receptors, Steroid / Q000737:chemistry* / Q000378:metabolism*; D012293:Rifampin / Q000494:pharmacology; D000077546:Roscovitine; D013449:Sulfonamides / Q000494:pharmacology; D013912:Threonine / Q000235:genetics / Q000378:metabolism*; D000099148:UGT1A1 Enzyme; D000096926:Benzenesulfonamides
chemical_list: D001596:Benzylamines; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D014453:Glucuronosyltransferase; D018841:HSP90 Heat-Shock Proteins; D019319:Okadaic Acid; D000077297:Pregnane X Receptor; D054645:Protein Phosphatase 1; D011687:Purines; D034741:RNA, Small Interfering; D011987:Receptors, Steroid; D012293:Rifampin; D000077546:Roscovitine; D013449:Sulfonamides; D013912:Threonine; D000099148:UGT1A1 Enzyme; C072105:KN 93; D000096926:Benzenesulfonamides