I-KAPPA-B/MAD-3 MASKS THE NUCLEAR-LOCALIZATION SIGNAL OF NF-KAPPA-B P65 AND REQUIRES THE TRANSACTIVATION DOMAIN TO INHIBIT NF-KAPPA-B P65 DNA-BINDING

The active nuclear form of the NF-kappaB transcription factor complex is composed of two DNA binding subunits, NF-kappaB p65 and NF-kappaB p50, both of which share extensive N-terminal sequence homology with the v-rel oncogene product. The NF-kappaB p65 subunit provides the transactivation activity in this complex and serves as an intracellular receptor for a cytoplasmic inhibitor of NF-kappaB, termed IkappaB. In contrast, NF-kappaB p50 alone fails to stimulate kappaB-directed transcription, and based on prior in vitro studies, is not directly regulated by IkappaB. To investigate the molecular basis for the critical regulatory interaction between NF-kappaB and IkappaB/MAD-3, a series of human NF-kappaB p65 mutants was identified that functionally segregated DNA binding, IkappaB-mediated inhibition, and IkappaB-induced nuclear exclusion of this transcription factor. Results from in vivo expression studies performed with these NF-kappaB p65 mutants revealed the following: 1) IkappaB/MAD-3 completely inhibits NF-kappaB p65-dependent transcriptional activation mediated through the human immunodeficiency virus type 1 kappaB enhancer in human T lymphocytes, 2) the binding of IkappaB/MAD3 to NF-kappaB p65 is sufficient to retarget NF-kappaB p65 from the nucleus to the cytoplasm, 3) selective deletion of the functional nuclear localization signal present in the Rel homology domain of NF-kappaB p65 disrupts its ability to engage IkappaB/MAD-3, and 4) the unique C-terminus of NF-kappaB p65 attenuates its own nuclear localization and contains sequences that are required for IkappaB-mediated inhibition of NF-kappaB p65 DNA binding activity. Together, these findings suggest that the nuclear localization signal and transactivation domain of NF-kappaB p65 constitute a bipartite system that is critically involved in the inhibitory function Of IkappaB/MAD-3. Unexpectedly, our in vivo studies also demonstrate that IkappaB/MAD-3 binds directly to NF-kappaB p50. This interaction is functional as it leads to retargeting of NF-kappaB 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-kappaB p65.