Differential phosphorylation of the signal-responsive domain of IκBα and IκBβ by IκB kinases

Abstract

NF-κB activity is regulated by its association with the inhibitory IκB proteins, among which IκBα and IκBβ are the most abundant. IκB proteins are widely expressed in different cells and tissues and bind to similar combinations of NF-κB proteins. The degradation of IκB proteins allows nuclear translocation of NF-κB and hence plays a critical role in NF-κB activation. Previous studies have demonstrated that, although both IκB proteins are phosphorylated by the same IκB kinase (IKK) complex, and their ubiquitination and degradation following phosphorylation are carried out by the same ubiquitination/degradation machinery, their kinetics of degradation are quite different. To better understand the underlying mechanism of the differences in degradation kinetics, we have carried out a systematic, comparative analysis of the ability of the IKK catalytic subunits to phosphorylate IκBα and IκBβ. We found that, whereas IKKα is a weak kinase for the N-terminal serines of both IκB isoforms, IKKβ is an efficient kinase for those residues in IκBα. However, IKKβ phosphorylates the N-terminal serines of IκBβ far less efficiently, thereby providing an explanation for the slower rate of degradation observed for IκBβ. Mutational analysis indicated that the regions around the two N-terminal serines collectively influence the relative phosphorylation efficiency, and no individual residue is critical. These findings provide the first systematic analysis of the ability of IκBα and IκBβ to serve as substrates for IKKs and help provide a possible explanation for the differential degradation kinetics of IκBα and IκBβ.