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 RBCK1ubiquitinates 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.

Threonine-408 Regulates the Stability of Human Pregnane X Receptor through Its Phosphorylation and the CHIP/Chaperone-Autophagy Pathway

“…Since hPXR stability was previously shown to be regulated via phosphorylation-facilitated ubiquitination through dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 2 (DYRK2) and ubiquitin protein ligase E3 component n-recognition 5 (UBR5) as well as via ubiquitination by the Ring-B-box-coiled-coil protein interacting with PKC-1 (RBCK1) and its proteasomal degradation (Rana et al, 2013), we herein examined the effects of proteasome inhibitors on the stability of the T408D mutant. Calpain inhibitor I at 10 mM and lactacystin at 5 mM, which did not significantly suppress cell growth, enhanced the basic levels of CYP3A4 mRNA and also the CYP3A4 mRNA levels induced by rifampicin, respectively, in HepG2 cells exogenously expressing the hPXR WT (Supplemental Fig.…”

“…Since the knockdown of E3 ligases RBCK1 and UBR5 was previously reported to result in the accumulation of cellular hPXR and concomitant increases in the induction of hPXR target genes by rifampicin (Rana et al, 2013;Ong et al, 2014), we evaluated the effects of calpain inhibitor 1 and geldanamycin on the expression of CHIP, RBCK1, and UBR5 mRNAs. The inhibition of proteasome and Hsp90 did not markedly affect the expression of CHIP, RBCK1, or UBR5 mRNAs; the calpain inhibitor at 10 mM slightly increased CHIP and UBR5 mRNA levels but did not alter those of RBCK1 mRNA (Supplemental Fig.…”

“…Since the stability of hPXR is known to be regulated through a proteasome degradation pathway (Rana et al, 2013;Ong et al, 2014) and the hPXR T408D mutant accumulates in the cytoplasm in the presence of proteasome inhibitor MG132 (Sugatani et al, 2012), the phosphorylation of hPXR at threonine-408 appears to be involved in the regulation of its stability. However, the molecular mechanisms regulating the stability and function of the T408D mutant have not been elucidated in detail (Sugatani et al, 2012).…”

SUMOylation and Ubiquitylation Circuitry Controls Pregnane X Receptor Biology in Hepatocytes

“…We and others have also previously shown PXR to be a target for the ubiquitin-signaling pathway (Masuyama et al, 2002;Staudinger et al, 2011;Rana et al, 2013), and it is well known that ubiquitylation is an integral part of canonical NR-mediated gene expression (Dennis and O'Malley, 2005). Several studies have shown that phosphorylation controls PXR biologic function as well (Lichti-Kaiser et al, 2009a,b;Pondugula et al, 2009;Sugatani et al, 2012Sugatani et al, , 2014Smutny et al, 2013;Elias et al, 2014).…”

“…Ubiquitylation of PXR has previously been demonstrated by our group and others (Masuyama et al, 2002;Staudinger et al, 2011;Rana et al, 2013), and pharmacological inhibition of the 26S proteasome in cells inhibits PXR function (Staudinger et al, 2011). However, the precise molecular nature of the ubiquitin chain formation, specific lysine residue on PXR that is the target of ubiquitin, and biologic significance of the ubiquitin-SUMO interaction at the level of PXR are not currently well defined.…”