At low clinically relevant radiation doses, the underlying mechanism of NF-κB activation by ionizing radiation (IR) is not understood. Herein we demonstrate that IR (<5 Gy) -induced activation of NF-κB proceeds in the absence of IKK-dependent phosphorylation and degradation of its cytoplasmic inhibitor, IκBα. Our previous studies demonstrated that a constitutive, Ca2+ dependent nitric oxide synthase (nNOS) is activated within minutes of exposing epithelial cells to low doses of IR and one consequence was the Tyr nitration of IκBα. Nitrated IκBα did not bind the p65/p50 heterodimer of NF-κB and nuclear import of the dimer p65/p50 complex was inhibited by NOS inhibitors. To further explore the mechanism all 8 Tyrs of IκBα were individually mutated to Phe. CHO-cells were transfected with wt and mutant IκBα and either irradiated (5 Gy) or cell lysates were treated with peroxynitrite (200μM). Nitrated proteins were purified by affinity purification with anti-nitroTyr and blotted for IκBα or p50/p65. Be either nitration method only 181Tyr and 305Tyr were nitrated. In subsequent studies cells were co-transfected with the NF-κB reporter gene and either myc-tagged wt or IκBα Tyr mutants. As expected, over-expression of wild type and IκBα mutants inhibited basal NF-κB reporter activity (≈90%). Radiation-induced activation was still observed, however, and equally stimulated in cells expressing wt or Tyr305Phe mutants. In contrast, radiation-stimulated NF-κB reporter activity was completely blocked in cells expressing the Tyr181Phe mutant, demonstrating an important role for 181Tyr in ionizing radiation-induced activation of NF-κB. Crystallographic studies show that IκBα orients itself such that fingers 3/4, 4/5, and 5/6 contact the p50 subunit. 181Tyr and 182Asn extending from finger 3/4 have multiple contacts with p50. 181Tyr, in particular, plays an important role in these interactions since it forms hydrogen bonds with p50 252Lys and 258Arg, π-stacks with 351Tyr, and makes multiple van der Waals contacts with 261Ala, 327Pro, and 349Leu. Our experimental results with exogenous ONOO− as well as site-directed mutagenesis studies, confirm the role of 181Tyr in IκBα/NF-κB complex formation. The above experimentation indicates a new mechanism for NF-κB activation. Because Tyr-nitration is not commonly studied, it may be an undiscovered but important component for NF-κB activation by stimuli other than radiation. Tyr-nitration and disruption of the interactions between 181Tyr of IκBα and 351Tyr of p50 may be representative of a post-translational modification and structural motif critical for the stability of other protein complexes involving the stacking interactions of aromatic amino acids.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]