Signal-induced degradation of IκBα requires sitespecific ubiquitination

The inhibitor protein IκBα controls the nuclear import of the transcription factor NF-κB. The inhibitory activity of IκBα is regulated from the cytoplasmic compartment by signal-induced proteolysis. Previous studies have shown that signal-dependent phosphorylation of serine residues 32 and 36 targets IκBα to the ubiquitin-proteasome pathway. Here we provide evidence that lysine residues 21 and 22 serve as the primary sites for signal-induced ubiquitination of IκBα. Conservative Lys → Arg substitutions at both Lys-21 and Lys-22 produce dominant-negative mutants of IκBα in vivo. These constitutive inhibitors are appropriately phosphorylated but fail to release NF-κB in response to multiple inducers, including viral proteins, cytokines, and agents that mimic antigenic stimulation through the T-cell receptor. Moreover, these Lys → Arg mutations prevent signaldependent degradation of IκBα in vivo and ubiquitin conjugation in vitro. We conclude that site-specific ubiquitination of phosphorylated IκBα at Lys-21 and/or Lys-22 is an obligatory step in the activation of NF-κB.