The NF‐κB regulator IκBβ exhibits different molecular interactivity and phosphorylation status from IκBα in an IKK2‐catalysed reaction

Activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB) transcription factor, a central player in immune response regulation, is based on phosphorylation of inhibitor of kappaB alpha (IkappaBalpha) by the Inhibitor of kappaB kinase (IKK) that triggers IkappaBalpha degradation. Although inhibitor of kappaB beta (IkappaBbeta) is structurally similar to IkappaBalpha, its precise characteristics remain undefined. Herein, we report that the molecular interactivity of IkappaBbeta with the kinase-active region of IKK subunit 2 (IKK2), as well as its phosphorylation status, differs markedly from those of IkappaBalpha. A mass spectrometry analysis revealed that IkappaBbeta phosphorylation sites are distributed in its C-terminal region, whereas IkappaBalpha phosphorylation sites are located in the N-terminal region. Furthermore, IKK2 phosphorylation sites in IkappaBbeta are found in a region distinct from typical degradation signals, such as phosphodegron and proline/glutamic acid/serine/threonine-rich sequence (PEST) motifs. Mutation of the IkappaBbeta phosphorylation sites enhances its resistance to homeostatic proteasomal degradation. These findings contribute a novel concept in NF-kappaB/IKK signalling research.