Tyrosine nitration on p65 -: A novel mechanism to rapidly inactivate nuclear factor-κB

NO is an important factor that induces post-translational modifications of proteins by cellular reduction and oxidation mechanism: cysteinyl-nitrosylation or Tyr nitration. Nuclear factor (NF)-kappa B activity can be rapidly suppressed by sodium nitroprusside, a NO donor. This effect was effectively reversed by peroxynitrite scavenger deferoxamine, suggesting a Tyr nitration-mediated mechanism. Western blot with nitrotyrosine- specific antibody demonstrated that the p65 subunit of NF-kappa B was predominantly nitrated on Tyr residues. Tyr nitration of p65 induced its dissociation from p50, its association with I kappa B alpha, and subsequent sequestration of p65 in the cytoplasm by I kappa B alpha-mediated export. Liquid chromatography-coupled nanoelectrospray mass spectrometry revealed specific nitration on Tyr-66 and Tyr-152 residues of p65. Mutation studies confirmed that both Tyr-66 and Tyr-152 residues were important for the direct effects of NO on p65, which resulted in more p65 export and inactivation of NF-kappa B activity. This study identified a novel and efficient pathway where NO rapidly inactivated NF-kappa B activity by inducing Tyr nitration on p65.