title: Identification and characterization of phosphorylation sites within the pregnane X receptor protein.
abstract: Pregnane X receptor (PXR) is a xenobiotic sensor regulating the expression of genes involved in xenobiotic detoxification and elimination. Phosphorylation plays an important role in modulating PXR activity and several phosphorylation sites have been predicted and characterized in in vitro experiments. Although PXR has been shown to be a phosphoprotein in vivo, the exact residues that are phosphorylated remain elusive. Using mass spectrometry, we identified for the first time S114, T133/135, S167, and S200 residues that are phosphorylated in PXR following an in vitro kinase assay using cyclin-dependent kinase 2. We further found that the phosphorylation at S114, T133, and T135 occurred in PXR isolated from cells. We tested the phosphodeficient and phosphomimetic mutants corresponding to all the sites identified and determined that phosphorylation at S114 attenuates the transcriptional activity of PXR, consistent with the observation that the S114D mutant displayed reduced association with the PXR-targeted DNA response element. Phosphomimetic mutations at either T133 or T135 did not show a significant change in transcriptional activity however, the dual phosphomimetic mutant T133D/T135D displayed reduced transcriptional activity. Subcellular localization studies showed a varied distribution of the mutants suggesting that the regulation of PXR is much more complex than what we can observe by just overexpressing the mutants. Thus, our results provide the first direct evidence that PXR is phosphorylated at specific residues and suggest that further investigation is warranted to fully understand the regulation of PXR by phosphorylation.
authors.affiliation: Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.
authors.forename: Ayesha
authors.initials: A
authors.lastname: Elias
authors.affiliation: St. Jude Proteomics Facility, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.
authors.forename: Anthony A
authors.initials: AA
authors.lastname: High
authors.affiliation: St. Jude Proteomics Facility, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.
authors.forename: Ashutosh
authors.initials: A
authors.lastname: Mishra
authors.affiliation: Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.
authors.forename: Su Sien
authors.initials: SS
authors.lastname: Ong
authors.affiliation: Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Wu
authors.affiliation: St. Jude Proteomics Facility, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA; Departments of Structural Biology and Developmental Neurobiology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.
authors.forename: Junmin
authors.initials: J
authors.lastname: Peng
authors.affiliation: Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA. Electronic address: taosheng.chen@stjude.org.
authors.forename: Taosheng
authors.initials: T
authors.lastname: Chen
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D051357:Cyclin-Dependent Kinase 2; D057809:HEK293 Cells; D056945:Hep G2 Cells; D006801:Humans; D007313:Insecta; D010766:Phosphorylation / Q000502:physiology; D000077297:Pregnane X Receptor; D017510:Protein Folding / Q000187:drug effects; D011987:Receptors, Steroid / Q000737:chemistry* / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics
chemical_list: D000077297:Pregnane X Receptor; D011987:Receptors, Steroid; D051357:Cyclin-Dependent Kinase 2
keywords: Drug metabolism; PXR; Phosphomimetic Mutations.; Phosphorylation