The N-terminal domain of IκBα masks the nuclear localization signal(s) of p50 and c-Rel homodimers

Members of the Rel/NF-kappa B family of transcription factors are related to each other over a region of about 300 amino acids called the Rel Homology Domain (RHD), which governs DNA binding, dimerization, and binding to inhibitor. At the C-terminal end of the RHD, each protein has a nuclear localization signal (NLS), The crystal structures of the p50 and RelA family members show that the RHD consists of two regions: an N-terminal section which contains some of the DNA contacts and a C-terminal section which contains the remaining DNA contacts and controls dimerization, In unstimulated cells, the homo- or heterodimeric Rel/ NF-kappa B proteins are cytoplasmic by virtue of binding to an inhibitor protein (I kappa B) which somehow masks the NLS of each member of the dimer. The I kappa B proteins consist of an ankyrin-repeat-containing domain that is required for binding to dimers and N- and C-terminal domains that are dispensable for binding to most dimers. In this study, we examined the interaction between I kappa B alpha and Rel family homodimers by mutational analysis. We show that (i) the dimerization regions of p50, RelA, and c-Rel are sufficient for binding to I kappa B alpha, (ii) the NLSs of RelA and c-Rel are not required for binding to I kappa B alpha but do stabilize the interaction, (iii) the NLS of p50 is required for binding to I kappa B alpha, (iv) only certain residues within the p50 NLS are required for binding, and (v) in a p50-I kappa B alpha complex or a c-Rel-I kappa B alpha complex the N terminus of I kappa B alpha either directly or indirectly masks one or both of the dimer NLSs.