Hydrogen peroxide activates IκB kinases through phosphorylation of serine residues in the activation loops

The cellular redox state regulates nuclear factor-κB (NF-κB) signaling systems. We investigated the effects of H₂O₂ on inhibitor of NF-κB (IκB) kinases (IKKα and IKKβ), which phosphorylate IκB leading to its degradation and NF-κB activation. Tumor necrosis factor (TNF) stimulation increased IKK activity within 10 min, and then IKK activity decreased gradually within 30 min in HeLa cells. Stimulation of the cells with H₂O₂ induced a slight activation of IKK within 30 min. Furthermore, co-stimulation with TNF suppressed the downregulation of IKK and sustained the activation for more than 30 min. H₂O₂ also markedly activated IKK in cells that were pretreated with TNF or phorbol myristate acetate. Electrophoretic mobility shift assay revealed that H₂O₂ enhanced TNF-induced NF-κB activation. Studies using IKK mutants and an antibody against phosphorylated IKK proteins revealed that phosphorylation of serine residues, Ser180 of IKKα and Ser181 of IKKβ, in the activation loops was essential for the H₂O₂-mediated activation of IKK. H₂O₂-induced activation of IKKα and IKKβ was reduced by IKKβ and IKKα kinase-negative mutants, respectively, indicating that IKKα and IKKβ were stimulated by H₂O₂ in an interdependent manner. These results suggest that oxidative radical stress has stimulatory effects on NF-κB through the activation of IKK, which is mediated by the phosphorylation of serine residues in the activation loops.