PT  - JOURNAL ARTICLE
AU  - Sugatani, Junko
AU  - Hattori, Yoshiki
AU  - Noguchi, Yuji
AU  - Yamaguchi, Masahiko
AU  - Yamazaki, Yasuhiro
AU  - Ikari, Akira
TI  - Threonine-290 Regulates Nuclear Translocation of the Human Pregnane X Receptor through Its Phosphorylation/Dephosphorylation by Ca<sup>2+</sup>/Calmodulin-Dependent Protein Kinase II and Protein Phosphatase 1
AID  - 10.1124/dmd.114.059139
DP  - 2014 Oct 01
TA  - Drug Metabolism and Disposition
PG  - 1708--1718
VI  - 42
IP  - 10
4099  - http://dmd.aspetjournals.org/content/42/10/1708.short
4100  - http://dmd.aspetjournals.org/content/42/10/1708.full
SO  - Drug Metab Dispos2014 Oct 01; 42
AB  - 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 Ca2+/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.