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-->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-->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.
D001120:Arginine / Q000737:chemistry; D001483:Base Sequence; D002451:Cell Compartmentation; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D006801:Humans; D008213:Lymphocyte Activation; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D020569:Proto-Oncogene Proteins c-rel; D013329:Structure-Activity Relationship; D051841:Transcription Factor RelB; D014157:Transcription Factors; D014452:Ubiquitins / Q000378:metabolism*
D017931:DNA Primers; D009097:Multienzyme Complexes; D016328:NF-kappa B; D011518:Proto-Oncogene Proteins; D020569:Proto-Oncogene Proteins c-rel; C497593:RELB protein, human; D014157:Transcription Factors; D014452:Ubiquitins; D051841:Transcription Factor RelB; D010768:Phosphoserine; D001120:Arginine; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D008239:Lysine