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

EMBO J. 1994 Nov 15;13(22):5433-41.

Abstract

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)

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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

Substances

  • Antioxidants
  • Ethers, Cyclic
  • Multienzyme Complexes
  • NF-kappa B
  • Oligopeptides
  • Protease Inhibitors
  • Protein Kinase Inhibitors
  • Proto-Oncogene Proteins
  • Pyrrolidines
  • RELB protein, human
  • Thiocarbamates
  • Transcription Factors
  • Tumor Necrosis Factor-alpha
  • benzyloxycarbonyl-isoleucyl-glutamyl(O-tert-butyl)-alanyl-leucinal
  • Transcription Factor RelB
  • Okadaic Acid
  • pyrrolidine dithiocarbamic acid
  • Protein Kinases
  • Cysteine Endopeptidases
  • Proteasome Endopeptidase Complex