Mechanism of direct degradation of IκBα by 20S proteasome

I kappa B alpha regulates activation of the transcription factor NF-kappa B. NF-kappa B is activated in response to several stimuli, i.e. proinflamatory cytokines, infections, and physical stress. This signal dependent pathway involves I kappa B alpha phosphorylation, ubiquitylation, and degradation by 26S proteasome. A signal independent (basal) turnover of I kappa B alpha has also been described. Here, we show that I kappa B alpha can be directly degraded by 20S proteasomes. Deletion constructs of I kappa B alpha allow us to the determine that N-terminal (Delta N 1-70) and C-terminal regions (Delta C 280-327, removing the PEST region) of I kappa B alpha are not required for I kappa B alpha degradation, while a further C-terminal deletion including part of the arm repeats(Delta C2 245-327) almost completely suppress the degradation by 20S proteasome. Binding and competition experiments demonstrate that the degradation of I kappa B alpha involves specific interactions with alpha 2(C3) subunit of the proteasome. Finally, p65/re1A (not itself a substrate for 20S proteasome) inhibits the degradation of I kappa B alpha by the proteasome. These results recapitulate in vitro the main characteristics of signal independent (basal) turnover of I kappa B alpha demonstrated in intact cells. (c) 2005 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.