The cellular redox state regulates nuclear factor-U UB (NF-U UB) signaling systems. We investigated the effects of H 2 O 2 on inhibitor of NF-U UB (IU UB) kinases (IKKK K and IKKL L), which phosphorylate IU UB leading to its degradation and NF-U UB 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 H 2 O 2 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. H 2 O 2 also markedly activated IKK in cells that were pretreated with TNF or phorbol myristate acetate. Electrophoretic mobility shift assay revealed that H 2 O 2 enhanced TNFinduced NF-U UB activation. Studies using IKK mutants and an antibody against phosphorylated IKK proteins revealed that phosphorylation of serine residues, Ser180 of IKKK K and Ser181 of IKKL L, in the activation loops was essential for the H 2 O 2 -mediated activation of IKK. H 2 O 2 -induced activation of IKKK K and IKKL L was reduced by IKKL L and IKKK K kinase-negative mutants, respectively, indicating that IKKK K and IKKL L were stimulated by H 2 O 2 in an interdependent manner. These results suggest that oxidative radical stress has stimulatory effects on NF-U UB through the activation of IKK, which is mediated by the phosphorylation of serine residues in the activation loops. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

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