Different mechanisms control signal-induced degradation and basal turnover of the NF-κB inhibitor IκBα in vivo

Abstract

The transcription factor NF-κB is sequestered in the cytoplasm by a family of IκB molecules. Upon cellular stimulation with diverse agents, one of these molecules, IκBα, is rapidly phosphorylated and subsequently degraded. This process triggers nuclear translocation of NF-κB and the successive activation of target genes. Independent of its rapid stimulation-induced breakdown, IκBα is inherently unstable and undergoes a continuous turnover. To compare the mechanisms and protein domains involved in inducible and basal degradation of IκBα in intact cells we employed a transfection strategy using tagged IκBα and ubiquitin molecules. We show that tumor necrosis factor alpha (TNFα) induced breakdown of IκBα but not its basal turnover coincides with ubiquitination in the aminoterminal signal response domain (SRD) of IκBα. Neither the SRD nor the carboxy-terminal PEST sequence is needed for basal turnover, which instead depends only on the core ankyrin repeat domain. Despite the differences in the requirements of protein domains and ubiquitin-conjugation for both degradation pathways, each one is mediated by the proteasome. This finding is important for understanding alternative modes of controlling NF-κB activity.