IκBβ, but Not IκBα, Functions as a Classical Cytoplasmic Inhibitor of NF-κB Dimers by Masking Both NF-κB Nuclear Localization Sequences in Resting Cells

Abstract

NF-κB dimers, inhibitor IκB proteins, and NF-κB· IκB complexes exhibit distinct patterns in partitioning between nuclear and cytoplasmic cellular compartments. IκB-dependent modulation of NF-κB subcellular localization represents one of the more poorly understood processes in the NF-κB signaling pathway. In this study, we have combined in vitro biochemical and cell-based methods to elucidate differences in NF-κB regulation exhibited by the inhibitors IκBβ and IκBα. We show that although both IκBα and IκBβ bind to NF-κB with similar global architecture and stability, significant differences exist that contribute to their unique functional roles. IκBβ derives its high affinity toward NF-κB dimers by binding to both NF-κB subunit nuclear localization signals. In contrast, IκBα contacts only one NF-κB NLS and employs its carboxyl-terminal proline, glutamic acid, serine, and threonine-rich region for high affinity NF-κB binding. We show that the presence of one free NLS in the NF-κB·IκBα complex renders it a dynamic nucleocytoplasmic complex, whereas NF-κB· IκBβ complexes are localized to the cytoplasm of resting cells.