Nuclear receptor phosphorylation in xenobiotic signal transduction

Abstract

Nuclear pregnane X receptor (PXR, NR1I2) and constitutive active/androstane receptor (CAR, NR1I3) are nuclear receptors characterized in 1998 by their capability to respond to xenobiotics and activate cytochrome P450 (CYP) genes. An anti-epileptic drug, phenobarbital (PB), activates CAR and its target CYP2B genes, whereas PXR is activated by drugs such as rifampicin and statins for the CYP3A genes. Inevitably, both nuclear receptors have been investigated as ligand-activated nuclear receptors by identifying and characterizing xenobiotics and therapeutics that directly bind CAR and/or PXR to activate them. However, PB, which does not bind CAR directly, presented an alternative research avenue for an indirect ligand-mediated nuclear receptor activation mechanism: phosphorylation-mediated signal regulation. This review summarizes phosphorylation-based mechanisms utilized by xenobiotics to elicit cell signaling. First, the review presents how PB activates CAR (and other nuclear receptors) through a conserved phosphorylation motif located between two zinc fingers within its DNA-binding domain. PB-regulated phosphorylation at this motif enables nuclear receptors to form communication networks, integrating their functions. Next, the review discusses xenobiotic-induced PXR activation in the absence of the conserved DNA-binding domain phosphorylation motif. In this case, phosphorylation occurs at a motif located within the ligand-binding domain to transduce cell signaling that regulates hepatic energy metabolism. Finally, the review delves into the implications of xenobiotic-induced signaling through phosphorylation in disease development and progression.

Keywords: CAR; Nuclear receptors; PXR; cell signaling; drug metabolism; estrogen; gene regulation; induction; nuclear receptor; phenobarbital; phosphorylation; steroid hormone receptors.

Figure 1.

Xenobiotic-induced signals and diseases. Shown is a schematic representation of CAR/PXR-mediated signaling and diseases. Arrows, activation; stop bars, repression. Hatched arrows, pathways found in mice but not in humans. CAR- and PXR-mediated pathways are indicated in red and blue, respectively.

Figure 2.

Zinc finger of nuclear receptors. A, domain organization of nuclear receptor. B, two treble clef-fold zinc fingers of human estrogen receptor α (PDB code 1HCP) (31). Within the N-terminal treble clef zinc finger, one zinc knuckle (noncanonical turn with CXXC, which is enclosed by a broken red line), two β-strands (c and d; corresponding to those shown in Fig. S1), and one α-helix are depicted. Four cysteine residues functioning as ligands for a zinc ion (gray sphere) are also shown. This image was created on the RCSB PDB website using Mol* (32). C, amino acid sequence of two zinc fingers within the DBD of human CAR. Localizations of the well-conserved Thr³⁸ (in red) and extension of the P-box, D-box, and two α-helices are indicated.

Figure 3.

Phosphorylation motif conserved within the LBD. Nuclear receptor protein sequence alignment was performed with Clustal Omega (RRID:SCR_001591). The C-terminal portion of helix 8 and the N-terminal portion of helix 9 are indicated by green arrows on the top of this alignment as well as the corresponding sequences of PXR, which are highlighted in green. The conserved Ser/Thr is highlighted in orange. PXR and three steroid hormone receptors are boxed in blue.

Figure 4.

The phosphorylation-mediated CAR activation mechanism This schematically summarizes the work done over the last 20 years and presents the molecular mechanism by which phosphorylation regulates CAR activation. Prior to PB activation, CAR is phosphorylated as an inactive homodimer through Surface A in the cytoplasm. EGF stimulates ERK1/2 binding to the XRS of the LBD, stabilizing the homodimer. PB binds EGF receptor to attenuate the EGF signal, resulting in ERK1/2 dephosphorylation and dissociation from XRS to monomerize the CAR homodimer. The resulting phosphorylated monomer is dephosphorylated by PP2A for nuclear translocation and heterodimerization with RXRα.

Figure 5.

Phosphorylation, dimer interfaces, and diverse interaction. A, dynamic simulation of the helix. This is depicted from previous works (41) and modified by adding the side chain of phosphorylated Thr-38. The C-terminal portion of the helix is distorted by phosphorylation. B, two different dimer interfaces are pictured. C, modeling an ERα homodimer-CAR-RXRα tetramer. First, CAR homodimer (64) and CAR-RXRα heterodimer (65) are superimposed through their CAR monomers to model the CAR-CAR-RXRα trimer. One monomer of the ERα homodimer (PDB code 1ERE) is superimposed with the first CAR monomer of the CAR-CAR-RXRα trimer. By removing the CAR monomer, an ERα homodimer-CAR-RXRα tetramer is modeled (66).

Figure 6.

Nuclear receptor integration via conserved phosphorylation. Shown is a schematic representation of the PB-induced process utilized to integrate CAR, ERα, and RORα on the Sult1e1 promoter. ERα homodimerizes and CAR heterodimerizes with RXRα through Surface B, allowing CAR to interact with ERα through Surface A. Having two Surfaces A, the phosphorylated ERα homodimer can interact with RORα, sandwiched between RORα and the CAR-RXRα heterodimer, and forms a hexamer. This interaction with phosphorylated ERα regulates RORα phosphorylation, converting RORα from a transcriptional repressor to an activator.

Figure 7.

Function of conserved phosphorylation motif in the LBD. A, PXR phosphorylated at Ser-350 is a low-glucose response signal. This model is depicted from previous work (55) and modified. CDK2 regulates phosphorylation of PXR at Ser-350 by VRK1 in response to low glucose. Phosphorylated PXR binds PP2Cα that dephosphorylates SGK2, regulating genes. Similarly, ligand-activated PXR heterodimerizes with RXR to regulate dephosphorylation of SGK2. B, intramolecular interactions of Ser-673 in GR molecule. Left, cartoon diagram of the crystal structure (PDB code 1M2Z) of hGR (pink) with bound dexamethasone (cyan). The C-terminal region is colored gray, and the Ser-673 side-chain hydroxyl group is shown in red. Right, Ser-673 forming a side-chain hydrogen bond with Leu-670 and a backbone hydrogen bond with Leu-772.