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.

Abstract

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.

Adenovirus‐mediated Gene Transduction of IkB or IkB Plus Bax Gene Drastically Enhances Tumor Necrosis Factor (TNF)‐induced Apoptosis in Human Gliomas

“…Nuclear importation of NF-κB is hindered because of the high-affinity association of its p65 subunit with a labile cytoplasmic inhibitor IκB. [22][23][24] However, cytoplasmic inhibitor IκB is rapidly degraded after TNF stimulation. 25) The IκBdN gene encodes an IκB protein that lacks the N-terminal 36 amino acids.…”

“…[18][19][20][21] The NF-κB p65 subunit serves as an intracellular receptor for the inhibitor kappa B alpha (IκBα), which prevents the nuclear import of NF-κB. [22][23][24] Various extracellular signals, such as TNF, rapidly uncouple the IκBα-dependent cytoplasmic retention of NF-κB. 25) The sensitivity to TNF-induced apoptosis was enhanced by stable expression of the N-and C-terminal phosphorylation mutant of IκBα in primary mouse and human fibroblast, human Jurkat lymphoma and T24 bladder carcinoma lines.…”

Nuclear localization signals and human disease

“…The roles that NF-jB plays in cell proliferation and preventing apoptosis, as well as the mechanisms that regulate these activities, are well defined. NF-jB is held in an inactive form in the cytoplasm by an inhibitor protein, inhibitor of jB (IjB), which binds to and masks the NLS of NF-jB preventing its nuclear import (93)(94)(95) (Fig. 2B).…”

Signal-induced site-specific phosphorylation targets I kappa B alpha to the ubiquitin-proteasome pathway.

“…Members of this inhibitor family share a structural domain comprised of five to six ankyrin-like repeats. The best-characterized IKB protein, IKBa, binds to the p50 {NF-KBlJ/ p65 {RelA} heterodimer of NF-KB and masks the nuclear localization signals of these proteins (Beg et al 1992; Ganchi et al 1992; Henkel et al 1992; Zabel et al 1993}. When cells are exposed to a variety of NF-KB inducers such as lipopolysaccharide {LPS}, phorbol esters, tumor necrosis factor-~ {TNRx}, and mterleukin-1 (IL-IJ, IKB~ is rapidly phosphorylated and degraded, and NF-KB translocates to the nucleus where it activates gene expression (Beg et al 1993~ Brown et al 1993~ Cordle et al 1993; Henkel et al 19931 Rice and Ernst 19931 Sun et al 1993Sun et al , 1994a). …”