Abstract
The pregnane X receptor (PXR) plays crucial roles in multiple physiological processes. However, the signaling mechanisms responsible are not well defined; it is most likely that multiple functions of PXR are modulated by its phosphorylation. Therefore, we sought to determine whether mutation at a highly conserved Thr57 affects human PXR (hPXR) function. Site-directed mutagenesis was performed to generate phosphorylation-deficient (hPXRT57A) and phosphomimetic (hPXRT57D) mutants. Gene reporter, Western blotting, immunocytochemistry, mammalian two-hybrid, and electrophoretic mobility shift assays were used to study cytochrome P450 3A4 (CYP3A4) promoter activation, protein levels, localization, cofactor interaction, and CYP3A4 promoter binding of the hPXR mutants, respectively. hPXRT57D, but not hPXRT57A, lost its transcriptional activity. Neither mutation altered hPXR's protein levels and interaction with steroid receptor coactivator-1. hPXR and hPXRT57A exhibited a homogenous nuclear distribution, whereas hPXRT57D exhibited a distinctive punctate nuclear localization pattern similar to that of hPXR mutants with impaired function that colocalize with silencing mediator of retinoid and thyroid receptors (SMRT), although silencing of SMRT did not rescue the altered function of hPXRT57D. However, hPXRT57D, but not hPXRT57A, impaired hPXR's ability to bind to the CYP3A4 promoter, consistent with the mutant's transactivation function. Furthermore, the 70-kDa form of ribosomal protein S6 kinase (p70 S6K) phosphorylated hPXR in vitro and inhibited its transcriptional activity, whereas hPXRT57A partially resisted the inhibitory effect of p70 S6K. Our studies identify a functionally significant phosphomimetic mutant (hPXRT57D) and show p70 S6K phosphorylation and regulation of hPXR transactivation to support the notion that phosphorylation plays important roles in regulating hPXR function.
Footnotes
-
This work was supported in part by the National Institutes of Health National Institute of General Medical Sciences [Grant GM60346] (to E.G.S.); the National Institutes of Health National Cancer Institute [Grant P30-CA21765]; and the American Lebanese Syrian Associated Charities and St. Jude Children's Research Hospital.
-
Parts of this work were previously presented as a poster as follows: Pondugula SR, Wu J, Brimer-Cline C, Schuetz EG, and Chen T (2008) Phosphomimetic mutation affects function, localization, and CYP3A4 promoter binding of pregnane X receptor. Nuclear Receptors: Bench to Bedside; 2008 Aug 27–31; Cold Spring Harbor, NY. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
-
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
-
doi:10.1124/dmd.108.024695.
-
ABBREVIATIONS: PXR, pregnane X receptor; NR, nuclear receptor; DBD, DNA binding domain; LBD, ligand binding domain; AF, activation function; PKA, protein kinase A; PKC, protein kinase C; CDK2, cyclin-dependent kinase 2; p70 S6K, protein S6 kinase; hPXR, human pregnane X receptor; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PCR, polymerase chain reaction; SRC, steroid receptor coactivator; DMSO, dimethyl sulfoxide; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol (systematic); SMRT, silencing mediator of retinoid and thyroid receptor; PBS, phosphate-buffered saline; RXR, retinoid X receptor; siRNA, small interfering RNA; RLU, relative luminescence unit; GST, glutathione S-transferase; RAR, retinoic acid receptor; PPAR, peroxisome proliferator-activated receptor; HNF, hepatic nuclear factor; GPS, Group-Based Phosphorylation Scoring Method; mTOR, mammalian target of rapamycin; PI3K, phosphatidylinositol 3-kinase; F-hPXR, fusion of a FLAG-tag to the N-terminal wild-type hPXR; Akt, protein kinase B; FKHR, forkhead in rhabdomyosarcoma; SR12813, 3,5-di-tert-butyl-4-hydroxystyrene-β,β-diphosphonic acid tetraethyl ester.
- Received September 18, 2008.
- Accepted January 22, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
DMD articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|