Suppression of p65 phosphorylation coincides with inhibition of IkappaBalpha polyubiquitination and degradation.
Transcription factor nuclear factor-kappaB (NF-kappaB) is held in the cytoplasm in an inactive state by IkappaB inhibitors. Oncogenic activation of NF-kappaB is achieved by stimulus-induced ubiquitination and subsequent proteasome-mediated degradation of IkappaBalpha. Once released from the inhibitor, NF-kappaB/p65 enters the nucleus. A pre-requisite for cytokine-induced IkappaBalpha ubiquitination and degradation is the phosphorylation of IkappaBalpha at S32/S36. Phosphorylation of IkappaBalpha alone, however, is not sufficient to trigger its degradation, suggesting other events must be required for regulating IkappaBalpha degradation. In this study, we tested the hypothesis that phosphorylation of p65 at 536 is required for TNF-alpha induced IkappaBalpha proteolysis that in turn controls p65 nuclear translocation. We observed that, without affecting IkappaBalpha phosphorylation, MEK1 inhibitor U0126 treatment inhibited not only p65-S536 phosphorylation but also TNF-alpha-induced polyubiquitination of IkappaBalpha thereby inhibiting IkappaBalpha degradation. With p65 S536 phosphorylation mutants and mimics, we further observed that the structural mutation of p65 serine 536 to alanine inhibited the recruitment of ubiquitin to the p65-containing complex. As a consequence of suppressing polyubiquitination of the p65-containing complex, degradation of p65 phosphorylation mutant-bound IkappaBalpha was also inhibited. Accordingly, the nuclear translocation of phosphorylation-impaired p65 was significantly reduced. These findings suggest that p65 phosphorylation plays a key role in stimulus-induced IkappaBalpha ubiquitination.
Hu
Jing
J
Gene Regulation Section, Laboratory of Cancer Prevention, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, Maryland 21702, USA.
Haseebuddin
Mohammed
M
Young
Matthew
M
Colburn
Nancy H
NH
D000818:Animals; D002070:Butadienes / Q000494:pharmacology; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004817:Epidermis / Q000187:drug effects / Q000378:metabolism; D020672:I-kappa B Proteins / Q000378:metabolism*; D015151:Immunoblotting; D047468:Immunoprecipitation; D048369:MAP Kinase Kinase 1 / Q000037:antagonists & inhibitors; D051379:Mice; D009154:Mutation / Q000235:genetics; D000072000:NF-KappaB Inhibitor alpha; D009570:Nitriles / Q000494:pharmacology; D010766:Phosphorylation; D021381:Protein Transport; D051996:Transcription Factor RelA / Q000235:genetics / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014452:Ubiquitins / Q000378:metabolism*
D002070:Butadienes; D004791:Enzyme Inhibitors; D020672:I-kappa B Proteins; C000606205:Nfkbia protein, mouse; D009570:Nitriles; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; C113580:U 0126; D014452:Ubiquitins; D000072000:NF-KappaB Inhibitor alpha; D048369:MAP Kinase Kinase 1; C482127:Map2k1 protein, mouse