SIGNAL-INDUCED DEGRADATION OF I-KAPPA-B-ALPHA REQUIRES SITE-SPECIFIC UBIQUITINATION

The inhibitor protein I kappa B alpha controls the nuclear import of t he transcription factor NF-kappa B. The inhibitory activity of I kappa B alpha is regulated from the cytoplasmic compartment by signal-induc ed proteolysis. Previous studies have shown that signal-dependent phos phorylation of serine residues 32 and 36 targets I kappa B alpha to th e ubiquitin-proteasome pathway. Here we provide evidence that lysine r esidues 21 and 22 serve as the primary sites for signal-induced ubiqui tination ofI kappa B alpha. Conservative Lys --> Arg substitutions at both Lys-21 and Lys-22 produce dominant-negative mutants of I kappa B alpha in vivo. These constitutive inhibitors are appropriately phospho rylated but fail to release NF-kappa B in response to multiple inducer s, including viral proteins, cytokines, and agents that mimic antigeni c stimulation through the T-cell receptor. Moreover, these Lys --> Arg mutations prevent signal-dependent degradation of I kappa B alpha in vivo and ubiquitin conjugation in vitro. We conclude that site-specifi c ubiquitination of phosphorylated I kappa B alpha at Lys-21 and/or Ly s-22 is an obligatory step in the activation of NF-kappa B.