Abstract
Nuclear translocation of the p50/p65 heterodimer is essential for NF-κB signaling. In unstimulated cells, p50/p65 is retained by the inhibitor IκBα in the cytoplasm that masks the p65-nuclear localization sequence (NLS). Upon activation, p50/p65 is translocated into the nucleus by the adapter importin α3 and the receptor importin β. Here, we describe a bipartite NLS in p50/p65, analogous to nucleoplasmin NLS but exposed in trans. Importin α3 accommodates the p50- and p65-NLSs at the major and minor NLS-binding pockets, respectively. The p50-NLS is the predominant binding determinant, while the p65-NLS induces a conformational change in the Armadillo 7 of importin α3 that stabilizes a helical conformation of the p65-NLS. Neither conformational change was observed for importin α1, which makes fewer bonds with the p50/p65 NLSs, explaining the preference for α3. We propose that importin α3 discriminates between the transcriptionally active p50/p65 heterodimer and p50/p50 and p65/65 homodimers, ensuring fidelity in NF-κB signaling.
Highlights
Nuclear translocation of the p50/p65 heterodimer is essential for NF-κB signaling
We found the p50/p65:ΔIBB-importin α3 complex has a radius of gyration (Rg) of 44.21 ± 0.28 Å (Fig. 7a, Supplementary Table 3), and a volume of correlation[61] mass of 124.4 ± 12.4 kDa, in close agreement with the theoretical MW ~ 129 kDa expected for a heterotrimer of p50/ p65:ΔIBB-importin α3 in a 1:1:1 stoichiometry (Supplementary Table 1)
The specificity of importin α3 for the canonical NF-κB p50/p65 heterodimer was first reported over fifteen years ago[19,20]
Summary
Nuclear translocation of the p50/p65 heterodimer is essential for NF-κB signaling. In unstimulated cells, p50/p65 is retained by the inhibitor IκBα in the cytoplasm that masks the p65-nuclear localization sequence (NLS). Importin α3 accommodates the p50- and p65-NLSs at the major and minor NLS-binding pockets, respectively. Cytoplasmic cargos targeted for active nuclear import expose a nuclear localization sequence (NLS) recognized by soluble transport receptors. Several pathways for nuclear import have been described, which share similar principles and rely on soluble transport factors of the importin β superfamily (or β-karyopherins)[4]. These macromolecules promote active, signal-mediated nuclear translocation of cargos by coordinating three activities: high-affinity binding to the NLS-. Importin β can import NLS-cargos directly by either binding to their exposed NLSs6 or through specialized adaptor proteins such as snurportin and importin α7. These NF-κB subunits exist as a variety of homodimers and heterodimers, of which p50/p65 represents the most abundant and a Helix 3
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