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-kappa B) transcription factor, a central player in immune response regulation, is based on phosphorylation of inhibitor of kappaB alpha (I kappa B alpha) by the Inhibitor of kappaB kinase (IKK) that triggers I kappa B alpha degradation. Although inhibitor of kappaB beta (I kappa B beta) is structurally similar to I kappa B alpha, its precise characteristics remain undefined. Herein, we report that the molecular interactivity of I kappa B beta with the kinase-active region of IKK subunit 2 (IKK2), as well as its phosphorylation status, differs markedly from those of I kappa B alpha. A mass spectrometry analysis revealed that I kappa B beta phosphorylation sites are distributed in its C-terminal region, whereas I kappa B alpha phosphorylation sites are located in the N-terminal region. Furthermore, IKK2 phosphorylation sites in I kappa B beta 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 I kappa B beta phosphorylation sites enhances its resistance to homeostatic proteasomal degradation. These findings contribute a novel concept in NF-kappa B/IKK signalling research.