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.
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.(ABSTRACT TRUNCATED AT 250 WORDS)
Institute of Biochemistry, Albert-Ludwigs-University, Freiburg, Germany.
E B
EB
Traenckner
S
S
Wilk
P A
PA
Baeuerle
D000595:Amino Acid Sequence; D000975:Antioxidants / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000502:physiology*; D004988:Ethers, Cyclic / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000502:physiology*; D016328:NF-kappa B / Q000378:metabolism*; D019319:Okadaic Acid; D009842:Oligopeptides / Q000494:pharmacology; D010766:Phosphorylation; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000502:physiology; D011499:Protein Processing, Post-Translational; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D011759:Pyrrolidines / Q000494:pharmacology; D013859:Thiocarbamates / Q000494:pharmacology; D051841:Transcription Factor RelB; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D000975:Antioxidants; D004988:Ethers, Cyclic; D009097:Multienzyme Complexes; D016328:NF-kappa B; D009842:Oligopeptides; D011480:Protease Inhibitors; D047428:Protein Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D011759:Pyrrolidines; C497593:RELB protein, human; D013859:Thiocarbamates; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; C090535:benzyloxycarbonyl-isoleucyl-glutamyl(O-tert-butyl)-alanyl-leucinal; D051841:Transcription Factor RelB; D019319:Okadaic Acid; C020972:pyrrolidine dithiocarbamic acid; D011494:Protein Kinases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex