title: Threonine-408 Regulates the Stability of Human Pregnane X Receptor through Its Phosphorylation and the CHIP/Chaperone-Autophagy Pathway.
abstract: The human pregnane X receptor (hPXR) is a xenobiotic-sensing nuclear receptor that transcriptionally regulates drug metabolism-related genes. The aim of the present study was to elucidate the mechanism by which hPXR is regulated through threonine-408. A phosphomimetic mutation at threonine-408 (T408D) reduced the transcriptional activity of hPXR and its protein stability in HepG2 and SW480 cells in vitro and mouse livers in vivo. Proteasome inhibitors (calpain inhibitor I and MG132) and Hsp90 inhibitor geldanamycin, but not Hsp70 inhibitor pifithrin-μ, increased wild-type (WT) hPXR in the nucleus. The translocation of the T408D mutant to the nucleus was significantly reduced even in the presence of proteasome inhibitors, whereas the complex of yellow fluorescent protein (YFP)-hPXR T408D mutant with heat shock cognate protein 70/heat shock protein 70 and carboxy terminus Hsp70-interacting protein (CHIP; E3 ligase) was similar to that of the WT in the cytoplasm. Treatment with pifithrin-μ and transfection with anti-CHIP small-interfering RNA reduced the levels of CYP3A4 mRNA induced by rifampicin, suggesting the contribution of Hsp70 and CHIP to the transactivation of hPXR. Autophagy inhibitor 3-methyladenine accumulated YFP-hPXR T408D mutant more efficiently than the WT in the presence of proteasome inhibitor lactacystin, and the T408D mutant colocalized with the autophagy markers, microtubule-associated protein 1 light chain 3 and p62, which were contained in the autophagic cargo. Lysosomal inhibitor chloroquine caused the marked accumulation of the T408D mutant in the cytoplasm. Protein kinase C (PKC) directly phosphorylated the threonine-408 of hPXR. These results suggest that hPXR is regulated through its phosphorylation at threonine-408 by PKC, CHIP/chaperone-dependent stability check, and autophagic degradation pathway.
authors.affiliation: Department of Pharmaco-Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Japan sugatani@u-shizuoka-ken.ac.jp.
authors.forename: Junko
authors.initials: J
authors.lastname: Sugatani
authors.affiliation: Department of Pharmaco-Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Noguchi
authors.affiliation: Department of Pharmaco-Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Japan.
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Hattori
authors.affiliation: Department of Pharmaco-Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Japan.
authors.forename: Masahiko
authors.initials: M
authors.lastname: Yamaguchi
authors.affiliation: Department of Pharmaco-Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Yamazaki
authors.affiliation: Department of Pharmaco-Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Ikari
mesh_terms: D021581:Active Transport, Cell Nucleus; D000818:Animals; D001343:Autophagy; D051544:Cytochrome P-450 CYP3A / Q000096:biosynthesis / Q000235:genetics; D004790:Enzyme Induction; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D008297:Male; D008813:Mice, Inbred ICR; D009154:Mutation; D010766:Phosphorylation; D000077297:Pregnane X Receptor; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism*; D061988:Proteasome Inhibitors / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D055550:Protein Stability; D059748:Proteolysis; D034622:RNA Interference; D011987:Receptors, Steroid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013912:Threonine; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*
chemical_list: D018840:HSP70 Heat-Shock Proteins; D000077297:Pregnane X Receptor; D061988:Proteasome Inhibitors; D011987:Receptors, Steroid; D013912:Threonine; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; C467728:STUB1 protein, human; D044767:Ubiquitin-Protein Ligases; D011493:Protein Kinase C; D046988:Proteasome Endopeptidase Complex