IκB/MAD-3 masks the nuclear localization signal of NF-κB p65 and requires the transactivation domain to inhibit NF-κB p65 DNA binding

The active nuclear form of the NF-κB transcription factor complex is composed of two DNA binding subunits, NF-κB p65 and NF-κB p50, both of which share extensive N-terminal sequence homology with the v-rel oncogene product. The NF-κB p65 subunit provides the transactivation activity in this complex and serves as an intracellular receptor for a cytoplasmic inhibitor of NF-κB, termed IκB. In contrast, NF-κB p50 alone fails to stimulate κB-directed transcription, and based on prior in vitro studies, is not directly regulated by IκB. To investigate the molecular basis for the critical regulatory interaction between NF-κB and IκB/MAD-3, a series of human NF-κB p65 mutants was identified that functionally segregated DNA binding, IκB-mediated inhibition, and IκB-induced nuclear exclusion of this transcription factor. Results from in vivo expression studies performed with these NF-κB p65 mutants revealed the following: 1) IκB/MAD-3 completely inhibits NF-κB p65-dependent transcriptional activation mediated through the human immunodeficiency virus type 1 κB enhancer in human T lymphocytes, 2) the binding of IκB/MAD-3 to NF-κB p65 is sufficient to retarget NF-κB p65 from the nucleus to the cytoplasm, 3) selective deletion of the functional nuclear localization signal present in the Rel homology domain of NF-κB p65 disrupts its ability to engage IκB/MAD-3, and 4) the unique C-terminus of NF-κB p65 attenuates its own nuclear localization and contains sequences that are required for IκB-mediated inhibition of NF-κB p65 DNA binding activity. Together, these findings suggest that the nuclear localization signal and transactivation domain of NF-κB p65 constitute a bipartite system that is critically involved in the inhibitory function of IκB/MAD-3. Unexpectedly, our in vivo studies also demonstrate that IκB/MAD-3 binds directly to NF-κB p50. This interaction is functional as it leads to retargeting of NF-κB p50 from the nucleus to the cytoplasm. However, no loss of DNA binding activity is observed, presumably reflecting the unique C-terminal domain that is distinct from that present in NF-κB p65.