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 r emoval of the inhibitory subunit I kappa B-alpha from a latent cytopla smic complex, It has been reported that I kappa B-alpha is subject to both phosphorylation and proteolysis in the process of NF-kappa B acti vation. In this study, we present evidence that the multicatalytic cyt osolic protease (proteasome) is involved in the degradation of I kappa B-alpha. Micromolar amounts of the peptide Cbz-Ile-Glu(O-t-Bu)-Ala-le ucinal (PSI), a specific inhibitor of the chymotrypsin-like activity o f the proteasome, prevented activation of NF-kappa B in response to tu mor necrosis factor-alpha (TNF) and okadaic acid (OA) through inhibiti on of I kappa B-alpha degradation. The m-calpain inhibitor Cbz-Leu-leu cinal was ineffective. In the presence of PSI, a newly phosphorylated form of I kappa B-alpha accumulated in TNF- and OA-stimulated cells. H owever, the covalent modification of I kappa B-alpha was not sufficien t 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. Pyrrolidinedi thiocarbamate, an antioxidant inhibitor of NF-kappa B activation which did not interfere with proteasome activities, prevented de novo phosp horylation of I kappa B-alpha as well as its subsequent degradation. T his suggests that phosphorylation of I kappa B-alpha is equally necess ary for the activation of NF-kappa B. In contrast to cell-free experim ents, in intact cells the kinase reaction did not release I kappa B-al pha from NF-kappa B, but appeared to tag the inhibitor for subsequent and rapid degradation by a chymotrypsin-like subunit of the proteasome .