In Vivoandin VitroRecruitment of an IκBα-Ubiquitin Ligase to IκBα Phosphorylated by IKK, Leading to Ubiquitination

Abstract

Activation of the transcriptional factor NF-κB is triggered by signal-dependent degradation of its inhibitor protein IκB through the ubiquitin (Ub)–proteasome pathway. We found here that a phosphorylated IκBα immunoprecipitated (IP-pIκBα) from the crude extract of HeLa cells which had been treated with tumor necrosis factor-α (TNFα) caused a dramatic ubiquitination of itself, termed autoubiquitination, when incubated with ATP, Ub, and E1-activating and E2-conjugating enzymes. IP-pIκBα also catalyzed ubiquitination of anin vitrosynthesized35S-IκBα previously phosphorylated by IκB-kinase (IKK) which is referred to as transubiquitination. No appreciable activity of auto- and transubiquitination was observed in an unphosphorylated IP-IκBα. Moreover, the putative IκBα-Ub ligase (IκBα-E3) present in HeLa cell cytosol associatedin vitrowith an IKK-phosphorylated recombinant IκBα, a process independent of NF-κB binding to IκBα or TNFα stimulation. Replacement of the two Ser residues at positions 32 and 36 corresponding to IKK phosphorylation sites by Ala resulted in almost complete prevention of binding of an IκBα-E3 to IκBα. These results indicate that phosphorylation of IκBα is necessary and sufficient for recruitment of this IκBα-E3 to associate with IκBα.