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Protein kinase C-mediated phosphorylation of orphan nuclear receptor TR2: effects on receptor stability and activity

Shaukat Ali Khan1, Sung Wook Park, Mostaqul Huq

  • 1Department of Pharmacology, University of Minnesota Medical School, Minneapolis, MN 55455, USA.

Proteomics
|September 1, 2005
PubMed

Insights

Orphan nuclear receptor TR2 phosphorylation by protein kinase C (PKC) enhances its stability and gene activation. Specifically, Ser-568 is crucial for TR2

Area of Science:

  • Molecular Biology
  • Cellular Signaling
  • Nuclear Receptor Research

Background:

  • Orphan nuclear receptors regulate gene expression.
  • Post-translational modifications, like phosphorylation, can modulate protein function.
  • The role of TR2 phosphorylation in its stability and activity was previously unclear.

Purpose of the Study:

  • To identify the enzymes responsible for TR2 phosphorylation.
  • To investigate the impact of TR2 phosphorylation on its protein stability.
  • To determine how TR2 phosphorylation affects its transcriptional activity on target genes.

Main Methods:

  • In vivo metabolic labeling to detect phosphorylation.
  • Kinase/phosphatase inhibitors to identify responsible enzymes.
  • In vitro phosphorylation assays with purified TR2 and protein kinase C (PKC).
  • LC-ESI-MS/MS to identify phosphorylation sites.
  • Site-directed mutagenesis to evaluate biological activity.

Main Results:

  • TR2 undergoes phosphorylation in vivo.
  • PKC-mediated phosphorylation enhances TR2's activation of the RARbeta gene.
  • PKC phosphorylation increases TR2 protein stability by preventing proteasomal degradation.
  • Ser-568 and Ser-461 were identified as PKC-mediated phosphorylation sites.
  • Mutation of Ser-568, but not Ser-461, significantly reduced TR2 stability and transcriptional activity.

Conclusions:

  • PKC-mediated phosphorylation is a key regulatory mechanism for TR2.
  • Phosphorylation at Ser-568 is critical for maintaining TR2 protein stability and transcriptional function.
  • TR2 phosphorylation by PKC enhances its role in gene regulation.

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