Role of I kappa B alpha ubiquitination in signal-induced activation of NF-kappa B in vivo

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