Phosphorylation of Farnesoid X Receptor at Serine 154 Links Ligand Activation With Degradation

Abstract

Comparison of 11 human nuclear receptor amino acid sequences revealed a conserved phosphorylation motif within their DNA-binding domains as an intramolecular signal that regulates proteolytic degradation. Nuclear receptors use this signal to either degrade or proscribe degradation through either the proteasome or nonproteasome pathways. A phosphomimetic farnesoid X receptor (FXR) S154D mutant neither bound to nor trans-activated an FXR-response element-driven reporter gene and was rapidly degraded in COS-1 cells. Ectopically expressed FXR had increased Ser154 phosphorylation in COS-1 cells after ligand treatment, and knock-down of the nuclear vaccinia-related kinase 1 (VRK1) greatly reduced this phosphorylation. FXR was phosphorylated at Ser154 in the nucleus of centrilobular hepatocytes only in ligand-treated mice. Thus, FXR Ser154 phosphorylation is a rheostat for activation and subsequent degradation that controls receptor levels and activity.

Phenobarbital Meets Phosphorylation of Nuclear Receptors

“…This conserved phosphorylation motif has only sporadically been the subject of investigations with a limited number of nuclear receptors, such as the vitamin D receptor (VDR), hepatocyte-enriched nuclear factor (HNF) 4a, and FXR (Hsieh et al, 1991;Sun et al, 2007;Gineste et al, 2008). Our current work systematically examined 11 different human nuclear receptors by expressing them in COS-1 cells (Hashiguchi et al, 2016). Among these 11 nuclear receptors, 8 (FXR, CAR, VDR, thyroid hormone receptora, Rev-erb a, RAR-related receptor a, HNF4a, and peroxisome proliferator-activated receptor a) degraded through ubiquitination and proteasomes in COS-1 cells upon a phosphomimetic mutation at their conserved motifs.…”

“…In addition, the fact that phosphorylation of this motif was not confirmed in endogenous nuclear receptors in tissues in vivo may have perpetuated this ignorance. In addition to CAR, three more nuclear receptors [estrogen receptor (ER) a at serine 216, farnesoid X receptor (FXR) at serine 154, and retinoid X receptor (RXR) at threonine 162] are phosphorylated in mice in vivo (Shindo et al, 2013 unpublished;Hashiguchi et al, 2016;unpublished observations). Further investigations may establish this phosphorylation in numerous nuclear receptors, shedding light on the conserved motif of the DBD and providing us with an opportunity to integrate nuclear receptors.…”

“…Only four members of the subfamily 3C and NR1I2 (PXR) do not conserve this motif. Some data are from a previous publication (Hashiguchi et al, 2016). These conserved phosphorylation residues are in red and boxed.…”

“…In addition to CAR, two nuclear receptors (FXR and ERa) are now confirmed to be phosphorylated at their conserved motifs in vivo in mice. FXR becomes phosphorylated at serine 154 in the nucleus of the liver of ligand-treated mice, in which nuclear kinase vaccinia-related kinase 1 appears to phosphorylate serine 154 (Hashiguchi et al, 2016). As suggested by the FXR S154D mutant, phosphorylated FXR does not bind or activate FXRE and it is degraded by proteasomes either in the nucleus or cytoplasm.…”

Phosphorylated Nuclear Receptor CAR Forms a Homodimer To Repress Its Constitutive Activity for Ligand Activation

“…These results shed light on how a phosphorylation-based system provides the basis for an intermolecular switch as the general mechanism that enables CAR to confer responsiveness by controlling constitutive activity. Moreover, since Thr 38 is conserved as a phosphorylation motif in a majority of nuclear receptors (10), phosphorylation and homodimerization may be common mechanisms for many constitutively activated nuclear receptors to regulate their activities.…”