A proteasome inhibitor prevents activation of NF-κB and stabilizes a newly phosphorylated form of IκB-α that is still bound to NF-κB

Activation of the inducible transcription factor NF-κB involves removal of the inhibitory subunit IκB-α from a latent cytoplasmic complex. It has been reported that IκB-α is subject to both phosphorylation and proteolysis in the process of NF-κB activation. In this study, we present evidence that the multicatalytic cytosolic protease (proteasome) is involved in the degradation of IκB-α. 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-κB in response to tumor necrosis factor-α (TNF) and okadaic acid (OA) through inhibition of IκB-α degradation. The m-calpain inhibitor Cbz-Leu-leucinal was ineffective. In the presence of PSI, a newly phosphorylated form of IκB-α accumulated in TNF- and OA-stimulated cells. However, the covalent modification of IκB-α was not sufficient for activation of NF-κB: no substantial NF-κB DNA binding activity appeared in cells because the newly phosphorylated form of IκB-α was still tightly bound to p65 NF-κB. Pyrrolidinedithiocarbamate, an antioxidant inhibitor of NF-κB activation which did not interfere with proteasome activities, prevented de novo phosphorylation of IκB-α as well as its subsequent degradation. This suggests that phosphorylation of IκB-α is equally necessary for the activation of NF-κB. In contrast to cell-free experiments, in intact cells the kinase reaction did not release IκB-α from NF-κB, but appeared to tag the inhibitor for subsequent and rapid degradation by a chymotrypsin-like subunit of the proteasome.