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

NF-kappaB activity is regulated by its association with the inhibitory IkappaB proteins, among which IkappaBalpha and IkappaBbeta are the most abundant. IkappaB proteins are widely expressed in different cells and tissues and bind to similar combinations of NF-kappaB proteins. The degradation of IkappaB proteins allows nuclear translocation of NF-kappaB and hence plays a critical role in NF-kappaB activation. Previous studies have demonstrated that, although both IkappaB proteins are phosphorylated by the same IkappaB 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 IkappaBalpha and IkappaBbeta. We found that, whereas IKKalpha is a weak kinase for the N-terminal serines of both IkappaB isoforms, IKKbeta is an efficient kinase for those residues in IkappaBalpha. However, IKKbeta phosphorylates the N-terminal serines of IkappaBbeta far less efficiently, thereby providing an explanation for the slower rate of degradation observed for IkappaBbeta. 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 IkappaBalpha and IkappaBbeta to serve as substrates for IKKs and help provide a possible explanation for the differential degradation kinetics of IkappaBalpha and IkappaBbeta.