I kappa B alpha can localize in the nucleus but shows no direct transactivation potential.

Although I kappa B is a cytoplasmic inhibitor of NF-kappa B and c-Rel that prevents nuclear translocation of NF-kappa B, some forms of I kappa B have been found in the nucleus. Given that some other proteins with ankyrin-type repeats are transcription factors, we wondered if a nuclear form of I kappa B alpha could itself be a transcriptional activator. We found that Gal4-I kappa B alpha fusion proteins strongly transactivate a Gal4 site-containing promoter in 3T3 fibroblasts. The I kappa B alpha domain responsible for this transactivation is not the acidic domain of I kappa B alpha, but the ankyrin repeat domain which is responsible for protein-protein interactions. To enhance our ability to detect cellular I kappa B alpha by immunofluorescence, we overexpressed the protein in transfected cells, and found that overexpressed I kappa B alpha is largely cytoplasmic in serum-deprived cells, but nuclear in serum-stimulated cells. However, in cell fractionation studies under all treatment conditions, I kappa B alpha appears mainly in cytoplasmic fractions, suggesting that it can rapidly move out of the nucleus through nuclear pores during extract preparation. Using double antibody immunoprecipitations, we found that I kappa B alpha in proliferating cells is strongly associated with RelA(p65). When I kappa B alpha is fused to the Gal4 DNA-binding domain, nuclear Gal4-I kappa B alpha is associated with RelA(p65). Thus, the activation domain of the associated RelA(p65) molecule could account for the ability of Gal4-I kappa B alpha to transactivate the Gal4 promoter. Unlike Bcl-3, an I kappa B which has been recently shown to directly transactivate through kappa B sites when associated with NFKB2 (p52), I kappa B alpha shows no ability to directly transactivate target promoters via its association with RelA(p65).