Crucial role of the amino-terminal tyrosine residue 42 and the carboxyl-terminal PEST domain of I kappa B alpha in NF-kappa B activation by an oxidative stress

Activation of transcription factor NF-kappa B involves the signal-dependent degradation of basally phosphorylated inhibitors such as I kappa B alpha. In response to proinflammatory cytokines or mitogens, the transduction mach inery has recently been characterized, but the activation mechanism upon ox idative stress remains unknown. In the present work, we provide several lin es of evidence that NF-kappa B activation in a T lymphocytic cell line (EL4 ) by hydrogen peroxide (H2O2) did not involve phosphorylation of the serine residues 32 and 36 in the amino-terminal part of I kappa B alpha. Indeed, mutation of Ser(32) and Ser(36) blocked IL-1 beta- or PMA-induced NF-kappa B activation, but had no effect on its activation by H2O2, Although I kappa B alpha was phosphorylated upon exposure to H2O2, tyrosine residue 42 and the C-terminal PEST (proline-glutamic acid-serine-threonine) domain played an important role. Indeed, mutation of tyrosine 42 or serine/threonine resi dues of the PEST domain abolished NF-kappa B activation by H2O2, while it h ad no effect on activation by IL-1 beta or PMA-ionomycin, This H2O2-inducib le phosphorylation was not dependent on I kappa B kinase activation, but co uld involve casein kinase II, because an inhibitor of this enzyme (5,6 dich loro-1-beta-D-ribofuranosyl-benzimidazole) blocks NF-kappa B activation. H2 O2-induced I kappa B alpha phosphorylation was followed by its degradation by calpain proteases or through the proteasome, Taken together, our finding s suggest that NF-kappa B activation by H2O2 involves a new mechanism that is totally distinct from those triggered by proinflammatory cytokines or mi togens.