A Phosphomimetic Mutation at Threonine-57 Abolishes Transactivation Activity and Alters Nuclear Localization Pattern of Human Pregnane X Receptor

Abstract

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 Thr ) and show p70 S6K phosphorylation and regulation of hPXR transactivation to support the notion that phosphorylation plays important roles in regulating hPXR function.

Protein Phosphatase 2Cβl Regulates Human Pregnane X Receptor-Mediated CYP3A4 Gene Expression in HepG2 Liver Carcinoma Cells

“…The CMV-Renilla luciferase plasmid was purchased from Promega (Madison, WI). Transient transfections were performed using FuGENE 6 (Roche Diagnostics, Indianapolis, IN) as described previously (Lin et al, 2008;Pondugula et al, 2009b). In brief, transfections were performed in 6-well culture plates with 2 g of total plasmid DNA per well.…”

“…HepG2 human liver carcinoma cell line was propagated as described previously (Lin et al, 2008;Pondugula et al, 2009b) in growth media containing Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT), 100 U/ml penicillin (Invitrogen), 100 g/ml streptomycin (Invitrogen), 2 mM L-glutamine (Invitrogen), and 1 mM sodium pyruvate (Invitrogen). The pcDNA3-hPXR, FLAGpcDNA3-hPXR, and pGL3-CYP3A4-luc (CYP3A4 luciferase reporter) plasmids were generated as described previously (Lin et al, 2008).…”

“…The mammalian two-hybrid assays were performed as described previously to investigate the effect of PP2C␤l on the interaction between hPXR and steroid receptor coactivator-1 (SRC-1) (Pondugula et al, 2009b). In brief, pBIND-SRC-1 (Gal4-SRC-1) (500 ng) and a luciferase reporter pG5-luc (1000 ng), together with either pACT (empty vector) (200 ng), pACT-hPXR (VP16-hPXR) (200 ng), or pcDNA3-PP2C␤l (600 ng), as indicated, were cotransfected into HepG2/Control shRNA or HepG2/PP2C␤l shRNA cells for 24 h. pcDNA3 was added to ensure that the total plasmid DNA in each transfection reaction was 2.5 g. The cells were then treated with DMSO or 5 M rifampicin for 24 h before luciferase assays.…”

“…Recent studies demonstrated a role for phosphorylation-dependent signaling events in regulating PXR-mediated CYP gene expression (Pondugula et al, 2009a). Kinases such as protein kinase A (Ding and Staudinger, 2005a;Lichti-Kaiser et al, 2009), protein kinase C (Ding and Staudinger, 2005b), cyclin-dependent kinase (CDK)2 (Lin et al, 2008), and p70 ribosomal S6 kinase (Pondugula et al, 2009b) phosphorylate PXR and regulate PXR-mediated CYP gene expression. Furthermore, CDK1, casein kinase II, and glycogen synthase kinase 3 also phosphorylate PXR (Lichti-Kaiser et al, 2009), although the functional significance of these phosphorylations is unknown.…”

Pregnane X Receptor in Drug Development

“…A systematic approach to mutating serine/threonine (S/T) residues to aspartic acid (D) revealed that Ser8Asp, Thr57Asp, Ser208Asp, and Thr408Asp resulted in a decrease in PXR transactivation (Lichti-Kaiser et al 2009a). Notably, we determined the phosphomimetic mutant of PXR, Thr57Asp, also exhibited an altered pattern of subcellular localization (Pondugula et al 2009a). Collectively, phosphorylation of PXR confers a negative regulatory effect, possibly through altering its pattern of subcellular localization or affecting its interaction with corepressors or coactivators.…”