Mechanism of direct degradation of IkappaBalpha by 20S proteasome

Abstract

IkappaBalpha regulates activation of the transcription factor NF-kappaB. NF-kappaB is activated in response to several stimuli, i.e. proinflamatory cytokines, infections, and physical stress. This signal dependent pathway involves IkappaBalpha phosphorylation, ubiquitylation, and degradation by 26S proteasome. A signal independent (basal) turnover of IkappaBalpha has also been described. Here, we show that IkappaBalpha can be directly degraded by 20S proteasomes. Deletion constructs of IkappaBalpha allow us to the determine that N-terminal (DeltaN 1-70) and C-terminal regions (DeltaC 280-327, removing the PEST region) of IkappaBalpha are not required for IkappaBalpha degradation, while a further C-terminal deletion including part of the arm repeats (DeltaC2 245-327) almost completely suppress the degradation by 20S proteasome. Binding and competition experiments demonstrate that the degradation of IkappaBalpha involves specific interactions with alpha2(C3) subunit of the proteasome. Finally, p65/relA (not itself a substrate for 20S proteasome) inhibits the degradation of IkappaBalpha by the proteasome. These results recapitulate in vitro the main characteristics of signal independent (basal) turnover of IkappaBalpha demonstrated in intact cells.