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).
Cressman
D E
DE
Department of Genetics, University of Pennsylvania, School of Medicine, Philadelphia 19104-6145.
Taub
R
R
D016475:3T3 Cells; D000818:Animals; D017487:Ankyrins / Q000737:chemistry; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism; D066298:In Vitro Techniques; D051379:Mice; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000037:antagonists & inhibitors*; D009687:Nuclear Proteins / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D051841:Transcription Factor RelB; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation
D017487:Ankyrins; D004268:DNA-Binding Proteins; D016328:NF-kappa B; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; C497594:Relb protein, mouse; D014157:Transcription Factors; D051841:Transcription Factor RelB