κB-Ras Binds to the Unique Insert within the Ankyrin Repeat Domain of IκBβ and Regulates Cytoplasmic Retention of IκBβ·NF-κB Complexes*

The IκBα and IκBβ proteins inhibit the transcriptional potential of active NF-κB dimers through stable complex formation. It has been shown that inactive IκBα·NF-κB complexes shuttle in and out of the nucleus, whereas IκBβ·NF-κB complexes are retained exclusively in the cytoplasm of resting cells. The biochemical mechanism underlying this functional difference and its consequences are unknown. Although the two IκB proteins are significantly homologous, IκBβ contains a unique 47-amino acid insertion of unknown function within its ankyrin repeat domain. In this study, we assess the role of the IκBβ insert in regulating cytoplasmic retention of IκBβ·NF-κB complexes. Deletion of the IκBβ insert renders IκBβ·NF-κB complexes capable of shuttling between the nucleus and cytoplasm, similar to IκBα·NF-κB complexes. A small Ras-like G-protein, κB-Ras, participates with the IκBβ insert to effectively mask the NF-κB nuclear localization potential. Similarly, a complex between NF-κB and a mutant IκBβ protein containing four serine to alanine mutations within its C-terminal proline, glutamic acid, serine, and threonine-rich sequence exhibits nucleocytoplasmic shuttling. This suggests a phosphorylation state-dependent role for the C-terminal proline, glutamic acid, serine, and threonine-rich sequence of IκBβ in proper localization of IκBβ·NF-κB complexes. These results are consistent with structural studies, which predicted that binary IκBβ·NF-κB complexes should be capable of nuclear translocation, and with previous observations that hypophosphorylated IκBβ·NF-κB complexes can reside in the nucleus.