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Tyrosine nitration on p65: a novel mechanism to rapidly inactivate nuclear factor-kappaB

Sung Wook Park1, M D Mostaqul Huq, Xinli Hu

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

Molecular & Cellular Proteomics : MCP
|January 20, 2005
PubMed

Insights

Nitric oxide (NO) inactivates Nuclear Factor-kappaB (NF-kappaB) by nitrating specific tyrosine residues on its p65 subunit. This tyrosine nitration causes p65 to dissociate from p50, leading to NF-kappaB pathway suppression.

Area of Science:

  • Biochemistry
  • Cellular signaling
  • Molecular biology

Background:

  • Nitric oxide (NO) is a crucial signaling molecule involved in various cellular processes.
  • NO mediates post-translational protein modifications, including cysteinyl-nitrosylation and tyrosine (Tyr) nitration.
  • Nuclear Factor-kappaB (NF-kappaB) is a key transcription factor regulating immune responses and inflammation.

Purpose of the Study:

  • To investigate the mechanism by which NO modulates NF-kappaB activity.
  • To identify the specific protein modifications and residues involved in NO-mediated NF-kappaB regulation.

Main Methods:

  • Utilized sodium nitroprusside (a NO donor) and deferoxamine (a peroxynitrite scavenger).
  • Employed Western blot with a nitrotyrosine-specific antibody to detect protein nitration.
  • Applied liquid chromatography-coupled nanoelectrospray mass spectrometry for precise residue identification.
  • Conducted mutation studies to validate the functional significance of identified residues.

Main Results:

  • Sodium nitroprusside rapidly suppressed NF-kappaB activity, an effect reversible by deferoxamine.
  • Western blot confirmed predominant nitration of the p65 subunit of NF-kappaB on Tyr residues.
  • Mass spectrometry identified specific nitration sites at Tyr-66 and Tyr-152 on p65.
  • Tyr-66 and Tyr-152 nitration induced p65 dissociation from p50, association with IkappaBalpha, and cytoplasmic sequestration, leading to NF-kappaB inactivation.

Conclusions:

  • NO rapidly inactivates NF-kappaB activity through a novel Tyr nitration-mediated pathway.
  • Specific Tyr nitration of p65 at residues 66 and 152 is critical for NO-induced NF-kappaB suppression.
  • This mechanism highlights a direct regulatory role of NO in controlling NF-kappaB signaling.

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