Signal-dependent and -independent degradation of free and NF-κb-bound IκBα

Abstract

A family of inhibitory IκB molecules regulates the activation of the transcription factor NF-κB. One member of the IκB family, IκBα, plays a major role in the rapid signal-induced activation of NF-κB. IκBα itself is transcriptionally regulated by NF-κB allowing for a tight autoregulatory loop that is beth sensitive to and rapidly influenced by NF-κB activating stimuli. For this pathway to remain primed both for rapid activation of NF- κB in the presence of signal and then to suppress NF-κB activation once that signal is removed, IκBα must be exquisitely regulated. The regulation of IκBα is mainly accomplished through phosphorylation, ubiquitination, and subsequent degradation. The mechanism(s) that regulate IκBα degradation needs to be able to target IκBα for degradation in both its NF-κB bound and free states in the cell. In this study, we utilize a full-length IκBα mutant that is unable to associate to Re1A/p65. We show that the signal- induced IκB kinase (IKK) phosphorylation sites on IκBα can only significantly influence the regulation of signal-dependent but not signal- independent turnover of IκBα. We also demonstrate that the constitutive carboxyl-terminal casein kinase II phosphorylation sites are necessary for the proper regulation of both signal-dependent and -independent turnover of IκBα. These findings further elucidate how the phosphorylation of IκBa influences the complex regulatory mechanisms involved in maintaining a sensitive NF-κB pathway.