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Updated: Mar 15, 2026

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists
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Phosphorylation of Farnesoid X Receptor at Serine 154 Links Ligand Activation With Degradation

Takuyu Hashiguchi1, Shingo Arakawa1, Shogo Takahashi1

  • 1Pharmacogenetics Section (T.H., S.A., T.S., M.N.), Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709; and Laboratory of Metabolism (S.T., F.J.G.), Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.

Molecular Endocrinology (Baltimore, Md.)
|August 30, 2016
PubMed

Insights

Nuclear receptors utilize a conserved phosphorylation motif to regulate protein degradation. Farnesoid X receptor (FXR) phosphorylation at Ser154 controls its activity and degradation via proteasome or nonproteasome pathways.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Cell Biology

Background:

  • Nuclear receptors are crucial transcriptional regulators.
  • Their activity is tightly controlled by post-translational modifications, including phosphorylation.
  • Proteolytic degradation is a key mechanism for regulating nuclear receptor levels.

Purpose of the Study:

  • To investigate the role of a conserved phosphorylation motif in nuclear receptor degradation.
  • To elucidate the regulatory mechanism of farnesoid X receptor (FXR) stability and activity.

Main Methods:

  • Comparative analysis of 11 human nuclear receptor amino acid sequences.
  • Site-directed mutagenesis to create a phosphomimetic FXR S154D mutant.
  • Reporter gene assays in COS-1 cells to assess FXR binding and trans-activation.
  • Western blotting to detect FXR phosphorylation and degradation.
  • VRK1 knockdown experiments using siRNA.
  • In vivo phosphorylation studies in ligand-treated mice.

Main Results:

  • A conserved phosphorylation motif in the DNA-binding domain of nuclear receptors was identified as an intramolecular degradation signal.
  • The phosphomimetic FXR S154D mutant exhibited impaired DNA binding and trans-activation, along with rapid degradation.
  • Ligand treatment increased Ser154 phosphorylation of ectopically expressed FXR in COS-1 cells.
  • Knockdown of nuclear vaccinia-related kinase 1 (VRK1) significantly reduced FXR Ser154 phosphorylation.
  • FXR Ser154 phosphorylation was observed in the nucleus of mouse hepatocytes specifically after ligand treatment.

Conclusions:

  • FXR Ser154 phosphorylation acts as a rheostat, modulating receptor activation and subsequent degradation.
  • This phosphorylation event controls FXR levels and activity through proteasome and nonproteasome degradation pathways.
  • The findings reveal a novel regulatory mechanism for nuclear receptor homeostasis and function.

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