title: Hydrogen peroxide activates IkappaB kinases through phosphorylation of serine residues in the activation loops.
abstract: The cellular redox state regulates nuclear factor-kappaB (NF-kappaB) signaling systems. We investigated the effects of H2O2 on inhibitor of NF-kappaB (IkappaB) kinases (IKKalpha and IKKbeta), which phosphorylate IkappaB leading to its degradation and NF-kappaB 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-kappaB activation. Studies using IKK mutants and an antibody against phosphorylated IKK proteins revealed that phosphorylation of serine residues, Ser180 of IKKalpha and Ser181 of IKKbeta, in the activation loops was essential for the H2O2-mediated activation of IKK. H2O2-induced activation of IKKalpha and IKKbeta was reduced by IKKbeta and IKKalpha kinase-negative mutants, respectively, indicating that IKKalpha and IKKbeta were stimulated by H2O2 in an interdependent manner. These results suggest that oxidative radical stress has stimulatory effects on NF-kappaB through the activation of IKK, which is mediated by the phosphorylation of serine residues in the activation loops.
authors.affiliation: Department of Life Science, Faculty of Science, Himeji Institute of Technology, Akoh-gun, Hyogo, Japan. h_kamata@sci.himeji-tech.ac.jp
authors.forename: Hideaki
authors.initials: H
authors.lastname: Kamata
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Manabe
authors.forename: Shin ichi
authors.initials: Si
authors.lastname: Oka
authors.forename: Keiko
authors.initials: K
authors.lastname: Kamata
authors.forename: Hajime
authors.initials: H
authors.lastname: Hirata
mesh_terms: D024202:Electrophoretic Mobility Shift Assay; D004789:Enzyme Activation / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D051550:I-kappa B Kinase; D007527:Isoenzymes / Q000187:drug effects / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000187:drug effects* / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology
chemical_list: D007527:Isoenzymes; D016328:NF-kappa B; D012694:Serine; D006861:Hydrogen Peroxide; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human