Cyclic AMP-dependent Protein Kinase Signaling Modulates Pregnane x Receptor Activity in a Species-specific Manner

Abstract

Pregnane x receptor is a ligand-activated transcription factor that regulates drug-inducible expression of several key cytochrome P450 enzymes and drug transporter proteins in liver and intestine in a species-specific manner. Activation of this receptor modulates several key biochemical pathways, including gluconeogenesis, ␤-oxidation of fatty acids, fatty acid uptake, cholesterol homeostasis, and lipogenesis. It is of current interest to determine whether the interaction between pregnane x receptor and these key biochemical pathways is evolutionarily conserved. We show here that activation of the cyclic AMP-dependent protein kinase signaling pathway synergizes with pregnane x receptor-mediated gene activation in mouse hepatocytes. Conversely, cyclic AMP-dependent protein kinase signaling has a repressive effect upon pregnane x receptor-mediated gene activation in rat and human hepatocytes. We show that the human pregnane x receptor protein can serve as an effective substrate for catalytically active cyclic AMP-dependent protein kinase in vitro. Metabolic labeling of the protein in vivo indicates that human pregnane x receptor exists as a phosphoprotein and that activation of cyclic AMP-dependent protein kinase signaling modulates the phosphorylation status of pregnane x receptor. Activation of cyclic AMP-dependent protein kinase signaling also modulates the interactions of pregnane x receptor with protein cofactors. Our results define the species-specific impact of cyclic AMP-dependent protein kinase signaling on pregnane x receptor and provide a molecular explanation of cyclic AMP-dependent protein kinase-mediated repression of human pregnane x receptor activity. Taken together, our results identify a novel mode of regulation of pregnane x receptor activity and highlight prominent functional differences in the process across species.

Threonine-290 Regulates Nuclear Translocation of the Human Pregnane X Receptor through Its Phosphorylation/Dephosphorylation by Ca2+/Calmodulin-Dependent Protein Kinase II and Protein Phosphatase 1

“…PKA and PKC are known to contribute to the cell signaling involved in ligand-inducible PXR activation; PKC and PKA cell signaling represses the transcriptional activity of PXR by increasing the strength of the interaction between PXR and the nuclear receptor corepressor protein NCoR, and the human PXR protein can serve as an effective substrate for catalytically active PKA (Ding and Staudinger, 2005a,b;Lichti-Kaiser et al, 2009b). We demonstrated here for the first time that CaMKII directly phosphorylated the threonine-290 of hPXR in vitro, while PKA and PKC, two activitydependent kinases with similar consensus sequences to CaMKII, could also phosphorylate serines/threonines, except for the threonine-290 of hPXR (Fig.…”

Pregnane X Receptor in Drug Development

“…The negative regulation of PXR by the cell-cycle-regulated Cdk2 suggests that PXR is subject to cell cycle regulation. PXR was also reported to be a substrate in in vitro kinase assays for a panel of kinases; protein kinase C (PKC) (Ding and Staudinger 2005b), 70 kDa ribosomal S6 kinase (p70S6K), glycogen synthase kinase 3 (GSK3), and casein kinase II (CK2) (Lichti-Kaiser et al 2009b), further suggesting that PXR may be modulated by a wide range of protein kinases. However, to this end, in vivo phosphorylation of PXR remains undetectable.…”

“…Treatment of primary rat and human hepatocytes with protein kinase A (PKA) activator leads to the attenuation of Cyp3A1 (Cyp3A1 is the rat ortholog of human CYP3A4) and CYP3A4 mRNA levels, respectively. This can be attributed, in part, to the phosphorylation of PXR by PKA (Ding & Staudinger 2005a;Lichti-Kaiser et al 2009b). However, a similar treatment of mouse hepatocytes resulted in an increase in Cyp3a11 mRNA levels, suggesting species specificity within PXR (Lichti-Kaiser et al 2009b).…”

“…This can be attributed, in part, to the phosphorylation of PXR by PKA (Ding & Staudinger 2005a;Lichti-Kaiser et al 2009b). However, a similar treatment of mouse hepatocytes resulted in an increase in Cyp3a11 mRNA levels, suggesting species specificity within PXR (Lichti-Kaiser et al 2009b). Work from our laboratory demonstrated that the treatment of HepG2 cells with flavonoids leads to an increase in CYP expression through modulating the activity of cyclin-dependent kinase 5 (Cdk5).…”

Protein Phosphatase 2Cβl Regulates Human Pregnane X Receptor-Mediated CYP3A4 Gene Expression in HepG2 Liver Carcinoma Cells

“…Kinases such as protein kinase A (Ding and Staudinger, 2005a;Lichti-Kaiser et al, 2009), protein kinase C (Ding and Staudinger, 2005b), cyclin-dependent kinase (CDK)2 (Lin et al, 2008), and p70 ribosomal S6 kinase (Pondugula et al, 2009b) phosphorylate PXR and regulate PXR-mediated CYP gene expression. Furthermore, CDK1, casein kinase II, and glycogen synthase kinase 3 also phosphorylate PXR (Lichti-Kaiser et al, 2009), although the functional significance of these phosphorylations is unknown.…”