The human pregnane X receptor (hPXR), a major chemical toxin sensor, is a ligand-induced transcription factor activated by various xenobiotics and toxins, resulting in the transcriptional upregulation of detoxifying enzymes. To date, little is known about the upstream regulation of hPXR. Using mass spectrometry analysis and kinome-wide siRNA screen, we report that the E3 ligase UBR5 and DYRK2 regulates hPXR stability. UBR5 knockdown resulted in accumulation of cellular hPXR and a concomitant increase in hPXR activity, whereas the rescue of UBR5 knockdown decreased the cellular hPXR level and activity. Importantly, UBR5 exerted its effect in concert with the serine/threonine kinase DYRK2, as the knockdown of DYRK2 phenocopied UBR5 knockdown. hPXR was a substrate for DYRK2, and DYRK2-dependent phosphorylation on hPXR facilitated its subsequent ubiquitination by UBR5. This is the first report of the posttranslational regulation of hPXR via phosphorylation-facilitated ubiquitination by DYRK2 and UBR5. Our results reveal the role of the ubiquitin-proteasomal pathway in modulating hPXR activity and indicate that pharmacological inhibitors to the ubiquitin-proteasomal pathway that regulates hPXR stability may negatively affect treatment outcome from unintended hPXR-mediated drug-drug interactions.
The human pregnane X receptor (PXR) regulates genes involved in drug metabolism and disposition. PXR associates with multiple corepressors that attenuate and coactivators that enhance its activity. PXR plays a vital role in the drug metabolism pathway, and a comprehensive examination of PXR-associated proteins will provide greater insight into the regulation of the receptor and possible therapeutic implications. We performed a mass spectrometric screen to identify PXR-associated proteins. Here we report that the tumor suppressor protein p53 can associate with PXR and downregulate its activity. A loss-offunction p53 mutant (R175H) interacts with PXR but does not repress its activity. Mutant p53 can relieve the suppressive effect of wild-type p53 by competing with its interaction with PXR, suggesting that protein-protein interaction is required but not sufficient for p53 to repress PXR activity. Interestingly, a PXR variant with a naturally occurring deletion of a conserved, unique sequence in the ligand binding domain (PXRΔ174-210) did not interact with p53, indicating that the PXR-p53 interaction is specific. Using a chromatin immunoprecipitation assay, we showed that p53 inhibits the binding of PXR to the CYP3A4 promoter. The loss of p53 function in tumor cells leads to aberrant cell proliferation, apoptosis, carcinogenesis, and altered sensitivity to chemotherapeutic drugs, whereas PXR contributes to chemoresistance in many cancer cells. Our findings show for the first time that wild-type p53 can negatively regulate PXR by physically associating with it. Thus, PXR and p53 appear to play important yet opposing roles in the sensitivity of tumor cells to chemotherapy.
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.
The human pregnane X receptor (hPXR), a member of the nuclear receptor superfamily, senses xenobiotics and controls the transcription of genes encoding drug-metabolizing enzymes and transporters. The regulation of hPXR’s transcriptional activation of its target genes is important for xenobiotic detoxification and endobiotic metabolism, and hPXR dysregulation can cause various adverse drug effects. Studies have implicated the putative phosphorylation site serine 350 (Ser350) in regulating hPXR transcriptional activity, but the mechanism of regulation remains elusive. Here we investigated the transactivation of hPXR target genes in vitro and in vivo by hPXR with a phosphomimetic mutation at Ser350 (hPXRS350D). The S350D phosphomimetic mutation reduced the endogenous expression of cytochrome P450 3A4 (an hPXR target gene) in HepG2 and LS180 cells. Biochemical assays and structural modeling revealed that Ser350 of hPXR is crucial for formation of the hPXR–retinoid X receptor alpha (RXRα) heterodimer. The S350D mutation abrogated heterodimerization in a ligand-independent manner, impairing hPXR-mediated transactivation. Further, in a novel humanized transgenic mouse model expressing the hPXRS350D transgene, we demonstrated that the S350D mutation alone is sufficient to impair hPXR transcriptional activity in mouse liver. This transgenic mouse model provides a unique tool to investigate the regulation and function of hPXR, including its non-genomic function, in vivo. Our finding that phosphorylation regulates hPXR activity has implications for development of novel hPXR antagonists and for safety evaluation during drug development.
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