Role of IκBα ubiquitination in signal-induced activation of NF-κB in vivo

Abstract

In unstimulated cells, the transcription factor NF-κB is held in the cytoplasm in an inactive state by the inhibitor protein IκBα. Stimulation of cells results in rapid phosphorylation and degradation of IκBα, thus releasing NF-κB, which translocates to the nucleus and activates transcription of responsive genes. Here we demonstrate that in cells where proteasomal degradation is inhibited, signal induction by tumor necrosis factor α results in the rapid accumulation of higher molecular weight forms of IκBα that dissociate from NF-κB and are consistent with ubiquitin conjugation. Removal of the high molecular weight forms of IκBα by a recombinant ubiquitin carboxyl-terminal hydrolase and reactivity of the immunopurified material with a monoclonal antibody specific for ubiquitin indicated that IκBα was conjugated to multiple copies of ubiquitin. Western blot analysis of immunopurified IκBα from cells expressing epitope-tagged versions of IκBα and ubiquitin revealed the presence of multiple copies of covalently bound tagged ubiquitin. An S32A/S36A mutant of IκBα that is neither phosphorylated nor degraded in response to signal induction fails to undergo inducible ubiquitination in vivo. Thus signal-induced activation of NF-κB involves phosphorylation-dependent ubiquitination of IκBα, which targets the protein for rapid degradation by the proteasome and releases NF- κB for translocation to the nucleus.