The eukaryotic transcription factor nuclear factor-kappa B (NF-kappa B) participates in many parts of the genetic program mediating T lymphocyte activation and growth. Nuclear expression of NF-kappa B occurs after its induced dissociation from its cytoplasmic inhibitor I kappa B alpha. Phorbol ester and tumor necrosis factor-alpha induction of nuclear NF-kappa B is associated with both the degradation of performed I kappa B alpha and the activation of I kappa B alpha gene expression. Transfection studies indicate that the I kappa B alpha gene is specifically induced by the 65-kilodalton transactivating subunit of NF-kappa B. Association of the newly synthesized I kappa B alpha with p65 restores intracellular inhibition of NF-kappa B DNA binding activity and prolongs the survival of this labile inhibitor. Together, these results show that NF-kappa B controls the expression of I kappa B alpha by means of an inducible autoregulatory pathway.

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

ReIA (p65) functions as the critical transactivating component of the heterodimeric p50-p65 NF-icB complex and contains a hh-affinity binding site for its cytoplasmic inhibitor, IkBa. After cellular activation, IKcBa is rapidly degraded in concert with the induced nuclear translocation of NF-ic. The present study demonstrates that tumor necrosis factor a-induced degradation of IicBa in human T cells is preceded by its rapid phosphorylation in vivo. However, these effects on IicBa result in nuclear mobilization of only a fraction of the entire cytoplasmic pool of RelA. Subsequent studies have revealed that (W) cytoplasmic ReIA is stably associated not only with IdcBa but also with other ankyrin motifrich proteins including the products of the NF-KB2 (plO0) and NF-ucBl (p105) genes; (it) in contrast to RelA-IhBa, ReIAp1OO cytoplasmic complexes are not dissociated following tumor necrosis factor a activation; (ii) p1O0 functions as a potent inhibitor of RelA-mediated transcription in vivo; (iv) the interaction of RelA and p1O0 involves the conserved Rel homology domain of both proteins but not the nuclear localization signal ofReIA, which is required for IucBa binding; (v) p1O0 inhibition of RelA function requires the C-terminal ankyrin motif domain, which mediates cytoplasmic retention of RelA; and (vW) as observed with IkcBa, nuclear RelA stimulates p1O0 mRNA and protein expression. These findings thus reveal the presence of a second inducible autoregulated inhibitory pathway that helps ensure the rapid but transient action of nuclear NF-icB.