title: A phosphomimetic mutation at threonine-57 abolishes transactivation activity and alters nuclear localization pattern of human pregnane x receptor.
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(57) affects human PXR (hPXR) function. Site-directed mutagenesis was performed to generate phosphorylation-deficient (hPXR(T57A)) and phosphomimetic (hPXR(T57D)) 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. hPXR(T57D), but not hPXR(T57A), lost its transcriptional activity. Neither mutation altered hPXR's protein levels and interaction with steroid receptor coactivator-1. hPXR and hPXR(T57A) exhibited a homogenous nuclear distribution, whereas hPXR(T57D) 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 hPXR(T57D). However, hPXR(T57D), but not hPXR(T57A), 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 hPXR(T57A) partially resisted the inhibitory effect of p70 S6K. Our studies identify a functionally significant phosphomimetic mutant (hPXR(T57D)) and show p70 S6K phosphorylation and regulation of hPXR transactivation to support the notion that phosphorylation plays important roles in regulating hPXR function.
authors.affiliation: Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Mail Stop 1000, Memphis, TN 38105, USA.
authors.forename: Satyanarayana R
authors.initials: SR
authors.lastname: Pondugula
authors.forename: Cynthia
authors.initials: C
authors.lastname: Brimer-Cline
authors.forename: Jing
authors.initials: J
authors.lastname: Wu
authors.forename: Erin G
authors.initials: EG
authors.lastname: Schuetz
authors.forename: Rakesh K
authors.initials: RK
authors.lastname: Tyagi
authors.forename: Taosheng
authors.initials: T
authors.lastname: Chen
mesh_terms: D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism*; D051544:Cytochrome P-450 CYP3A / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D007150:Immunohistochemistry; D018716:Molecular Mimicry; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D000077297:Pregnane X Receptor; D011987:Receptors, Steroid / Q000378:metabolism*; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism; D013912:Threonine / Q000235:genetics*; D015533:Transcriptional Activation
chemical_list: D000077297:Pregnane X Receptor; D011987:Receptors, Steroid; D013912:Threonine; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D038762:Ribosomal Protein S6 Kinases, 70-kDa