title: RBCK1, an E3 ubiquitin ligase, interacts with and ubiquinates the human pregnane X receptor.
abstract: The pregnane X receptor (PXR, NR1I2) plays a pivotal role in the disposition and detoxification of numerous foreign and endogenous chemicals by increasing transcription of numerous target genes, including phase I and II drug-metabolizing enzymes and transporters. In the present study, yeast two-hybrid screening identified an E3 ubiquitin ligase, RBCK1 (Ring-B-box-coiled-coil protein interacting with protein kinase C-1), as a human pregnane X receptor (hPXR)-interacting protein. Coimmunoprecipitation studies confirmed the interaction between RBCK1 and hPXR when both were ectopically expressed in AD-293 cells. Domain mapping studies showed that the interaction between RBCK1 and hPXR involves all RBCK1 domains. We further demonstrate that RBCK1 ubiquitinates hPXR, and this may target hPXR for degradation by the ubiquitin-proteasome pathway. Simultaneous ectopic overexpression of RBCK1 and PXR decreased PXR levels in AD-293 cells, and this decrease was inhibited by the proteasomal inhibitor MG-132 (carbobenzoxy-Leu-Leu-leucinal). Furthermore, overexpression of RBCK1 decreased endogenous levels of PXR in HepG2 cells. Of importance, ectopic overexpression and silencing of endogenous RBCK1 in primary human hepatocytes resulted in a decrease and increase, respectively, in endogenous PXR protein levels and in the induction of PXR target genes by rifampicin. These results suggest that RBCK1 is important for the ubiquitination of PXR and may play a role in its proteasomal degradation.
authors.affiliation: Laboratory of Toxicology & Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
authors.forename: Ritu
authors.initials: R
authors.lastname: Rana
authors.forename: Sherry
authors.initials: S
authors.lastname: Coulter
authors.forename: Harriet
authors.initials: H
authors.lastname: Kinyamu
authors.forename: Joyce A
authors.initials: JA
authors.lastname: Goldstein
mesh_terms: D005786:Gene Expression Regulation; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D047468:Immunoprecipitation; D007976:Leupeptins / Q000494:pharmacology; D000077297:Pregnane X Receptor; D061251:Primary Cell Culture; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors / Q000494:pharmacology; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D034622:RNA Interference; D011987:Receptors, Steroid / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D020798:Two-Hybrid System Techniques; D044767:Ubiquitin-Protein Ligases; D054875:Ubiquitination
chemical_list: D007976:Leupeptins; C000625852:NR1I2 protein, human; D000077297:Pregnane X Receptor; D061988:Proteasome Inhibitors; D011987:Receptors, Steroid; D014157:Transcription Factors; C504507:RBCK1 protein, human; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde