Suppression of p65 phosphorylation coincides with inhibition of I kappa B alpha polyubiquitination and degradation

Transcription factor nuclear factor-kappa B (NF-kappa B) is held in the cytoplasm in an inactive state by I kappa B inhibitors Oncogenic activation of NF-kappa B is achieved by stimulus-induced ubiquitination and subsequent proteasome-mediated degradation of I kappa B alpha. Once released from the inhibitor, NF-kappa B/p65 enters the nucleus. A pre-requisite for cytokine-induced I kappa B alpha ubiquitination and degradation is the phosphorylation Of I kappa B alpha at S32/S36. Phosphorylation Of I kappa B alpha alone, however, is not sufficient to trigger its degradation, suggesting other events must be required for regulating I kappa B alpha degradation. In this study, we tested the hypothesis that phosphorylation of p65 at 536 is required for TNF-alpha. induced I kappa B alpha proteolysis that in turn controls p65 nuclear translocation. We observed that, without affecting I kappa B alpha phosphorylation, MEK1 inhibitor U0126 treatment inhibited not only p65-S536 phosphorylation but also TNF-alpha-induced polyubiquitination Of I kappa B alpha thereby inhibiting I kappa B alpha 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 I kappa B alpha 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 I kappa B alpha ubiquitination. Published 2005 Wiley-Liss, Inc.(dagger)

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