A proteasome inhibitor prevents activation of NF‐kappa B and stabilizes a newly phosphorylated form of I kappa B‐alpha that is still bound to NF‐kappa B

• Published in: The EMBO journal Vol. 13; no. 22; pp. 5433 - 5441
• Main Authors: Traenckner, E.B., Wilk, S., Baeuerle, P.A.
• Format: Journal Article
• Language: English
• Published: England 15.11.1994
• Subjects: Amino Acid Sequence; Antioxidants - pharmacology; Cysteine Endopeptidases - physiology; Ethers, Cyclic - pharmacology; HeLa Cells; Humans; Models, Biological; Molecular Sequence Data; Multienzyme Complexes - physiology; NF-kappa B - metabolism; Okadaic Acid; Oligopeptides - pharmacology; Phosphorylation; Protease Inhibitors - pharmacology; Proteasome Endopeptidase Complex; Protein Binding; Protein Kinase Inhibitors; Protein Kinases - physiology; Protein Processing, Post-Translational; Proto-Oncogene Proteins - metabolism; Pyrrolidines - pharmacology; Thiocarbamates - pharmacology; Transcription Factor RelB; Transcription Factors; Tumor Necrosis Factor-alpha - pharmacology
• ISSN: 0261-4189; 1460-2075
• DOI: 10.1002/j.1460-2075.1994.tb06878.x

Activation of the inducible transcription factor NF‐kappa B involves removal of the inhibitory subunit I kappa B‐alpha from a latent cytoplasmic complex. It has been reported that I kappa B‐alpha is subject to both phosphorylation and proteolysis in the process of NF‐kappa B activation. In this study, we present evidence that the multicatalytic cytosolic protease (proteasome) is involved in the degradation of I kappa B‐alpha. Micromolar amounts of the peptide Cbz‐Ile‐Glu(O‐t‐Bu)‐Ala‐leucinal (PSI), a specific inhibitor of the chymotrypsin‐like activity of the proteasome, prevented activation of NF‐kappa B in response to tumor necrosis factor‐alpha (TNF) and okadaic acid (OA) through inhibition of I kappa B‐alpha degradation. The m‐calpain inhibitor Cbz‐Leu‐leucinal was ineffective. In the presence of PSI, a newly phosphorylated form of I kappa B‐alpha accumulated in TNF‐ and OA‐stimulated cells. However, the covalent modification of I kappa B‐alpha was not sufficient for activation of NF‐kappa B: no substantial NF‐kappa B DNA binding activity appeared in cells because the newly phosphorylated form of I kappa B‐alpha was still tightly bound to p65 NF‐kappa B. Pyrrolidinedithiocarbamate, an antioxidant inhibitor of NF‐kappa B activation which did not interfere with proteasome activities, prevented de novo phosphorylation of I kappa B‐alpha as well as its subsequent degradation. This suggests that phosphorylation of I kappa B‐alpha is equally necessary for the activation of NF‐kappa B.