NF-κB is transported into the nucleus by importin α3 and importin α4

NF-kappa B transcription factors are retained in the cytoplasm in an inactive form until they are activated and rapidly imported into the nucleus. We identified importin alpha 3 and importin alpha 4 as the main importin alpha isoforms mediating TNF-alpha-stimulated NF-kappa B p50/p65 heterodimer translocation into the nucleus. Importin alpha 3 and alpha 4 are close relatives in the human importin alpha family. We show that importin alpha 3 isoform also mediates nuclear import of NF-kappa B p50 homodimer in nonstimulated cells. Importin alpha 3 is shown to directly bind to previously characterized nuclear localization signals (NLSs) of NF-kappa B p50 and p65 proteins. Importin alpha 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-kappa B p50 binds to the N-terminal and p65 to the C-terminal NLS binding site of importin alpha 3. In vitro competition experiments and analysis of cellular NF-kappa B suggest that NF-kappa B binds to importin alpha only when it is free of I kappa B alpha. The present study demonstrates that the nuclear import of NF-kappa B is a highly regulated process mediated by a subset of importin alpha molecules.