NF-κB Is Transported into the Nucleus by Importin α3 and Importin α4*

NF-κB transcription factors are retained in the cytoplasm in an inactive form until they are activated and rapidly imported into the nucleus. We identified importin α3 and importin α4 as the main importin α isoforms mediating TNF-α-stimulated NF-κB p50/p65 heterodimer translocation into the nucleus. Importin α3 and α4 are close relatives in the human importin α family. We show that importin α3 isoform also mediates nuclear import of NF-κB p50 homodimer in nonstimulated cells. Importin α3 is shown to directly bind to previously characterized nuclear localization signals (NLSs) of NF-κB p50 and p65 proteins. Importin α molecules are known to have armadillo repeats that constitute the N-terminal and C-terminal NLS binding sites. We demonstrate by site-directed mutagenesis that NF-κB p50 binds to the N-terminal and p65 to the C-terminal NLS binding site of importin α3. In vitro competition experiments and analysis of cellular NF-κB suggest that NF-κB binds to importin α only when it is free of IκBα. The present study demonstrates that the nuclear import of NF-κB is a highly regulated process mediated by a subset of importin α molecules.