Tyrosine Nitration of IκBα: A Novel Mechanism for NF-κB Activation†,‡

The Rel/NF-κB family of transcription factors mediate cellular responses to oxidative and other stresses (1). NF-κB transcription factors are formed by the homo-or heterodimerization of proteins of the Rel family including p50, p52, p65 (RelA), c-Rel and RelB. The most abundant and best-understood dimer is p65/p50. This dimer exists in the cytoplasm complexed with an inhibitor protein, IκBα, that masks the NF-κB nuclear localization sequence (2).Stimulation of cells with cytokines such as tumor necrosis factor (TNFα) results in the phosphorylation, ubiquitination and degradation of IκBα, permitting the unmasked p65/p50 heterodimer to translocate into the nucleus (2). Ionizing radiation (IR) also activates NF-κB by mechanisms not fully understood. At high IR doses (>10Gy) activation appears similar to that observed for TNFα. DNA damage-inducible kinases, ATM and DNA-PK, activate IκBα kinases (e.g., IKKβ) stimulating the phosphorylation, ubiquitination and proteasome degradation of IκBα (1, 3). IKKβ activity and IκBα degradation, however, do not appear necessary for NF-κB activation at lower, therapeutic doses of IR, e.g. (4, 5). Indeed, IR inhibits proteasomal activities at doses as low as 0.2 Gy with maximal inhibition at 2 Gy (4, 6). IKKβ has other functions including phosphorylation of S536 in the transactivation domain of the p65 subunit (7–10). Serine-536 phosphorylation is independent of IκBα phosphorylation and important for enhancing p65 transactivation potential and modulating gene target selection. Recent studies demonstrate that NO• and a metabolic derivative, peroxynitrite (ONOO−) modulate NF-κB activity (11–15). For example, addition of a ONOO− donor to cultured cells stimulates NF-κB reporter activity without IκBα degradation (11). These findings and the evidence that a Ca2+ dependent constitutive NO• synthase activity in epithelial cells is transiently stimulated by low IR doses (16–19) prompted the following investigation of NO• signaling in IR-induced activation of NF-κB.