Signal-induced degradation of I-kappa-B-alpha requires site-specific ubiquitination

The inhibitor protein I[kappa]B[alpha] controls the nuclear import of the transcription factor NF-[kappa]B. The inhibitory activity of I[kappa]B[alpha] 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 I[kappa]B[alpha] 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 I[kappa]B[alpha]. Conservative Lys [right arrow]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 phosphorylated but fail to release NF-[kappa]B in response to multiple inducers, including viral proteins, cytokines, and agents that mimic antigenic stimulation through the T-cell receptor. Moreover, these Lys [right arrow]Arg mutations prevent signal-dependent degradation of I[kappa]B[alpha] in vivo and ubiquitin conjugation in vitro. We conclude that site-specific ubiquitination of phosphorylated I[kappa]B[alpha] at Lys-21 and/or Lys-22 is an obligatory step in the activation of NF-[kappa]B.