Common structural constituents confer I kappa B activity to NF-kappa B p105 and I kappa B/MAD-3.

The vertebrate NF-kappa B/c-rel inhibitors MAD-3/I kappa B alpha, I kappa B gamma/pdI and bcl-3 all share a conserved ankyrin repeat domain (ARD) consisting of six complete repeats, a short acidic motif and/or an incomplete seventh repeat. We present here a detailed analysis of the domain in p105/pdI and MAD-3/I kappa B involved in inhibition of DNA binding and in protein interaction with rel factors. We demonstrate that in both cases an acidic region and six ankyrin-like repeats are sufficient and required for protein interaction with the rel factors. However, for p105/pdI to achieve the high affinity needed to suppress DNA binding, an incomplete seventh repeat is required in addition. Both pdI and MAD-3 associate with rel proteins by forming heterotrimeric complexes, as shown by native gel analysis and by cross-linking. Furthermore, we demonstrate that deletion of only three amino acids in the first repeat converts the subunit specificity of the p105 ARD into that of MAD-3/I kappa B. We conclude that functionally the ARD in these molecules has a modular structure, with different subregions determining the specificity for the NF-kappa B subunits p50 and p65.     

NF-kappa B2 p100 is a pro-apoptotic protein with anti-oncogenic function

Nuclear factor-kappa B (NF-kappa B) promotes cell survival by upregulating expression of anti-apoptotic genes, a process that is antagonized by inhibitors of kappa B (I kappa B) factors. The only NF-kappa B family member known to be mutated in human cancer is NF-kappa B2 p100 (ref. 2), a factor with I kappa B activity. Here, we report the isolation from irradiated mouse tumour cells of a complex that induces caspase-8 activity in cell-free assays and identify p100 as an essential component of this complex. Expression of p100 profoundly sensitizes cells to death-receptor-mediated apoptosis through a pathway that is independent of I kappa B-like activity. The carboxyl terminus of p100 contains a death domain that is absent from all known tumour-derived mutants. This death domain mediates recruitment of p100 into death machinery complexes after ligand stimulation and is essential for p100's pro-apoptotic activity. p100 also sensitizes NIH3T3 cells to apoptosis triggered by oncogenic Ras, resulting in a marked inhibition of transformation that is rescued by suppression of endogenous caspase-8. These observations thus identify an I kappa B-independent apoptotic activity of NF-kappa B2 p100 and help explain its unique tumour suppressor role.     

Related subunits of NF-kappa B map to two distinct loci associated with translocations in leukemia, NFKB1 and NFKB2.

The chromosomal locations of the human genes NFKB1 and NFKB2, which encode two alternative DNA binding subunits of the NF-kappa B complex, p105 and p49/p100, respectively, have been determined. p105 was assigned to 4q21.1-q24 and p49/p100 to chromosome 10 by Southern blot analysis of panels of human/Chinese hamster cell hybrids. The locations were confirmed by fluorescence in situ hybridization and mapped with greater resolution to 4q23 and 10q24, respectively. These results demonstrate that these members of the NF-kappa B family are unlinked. Interestingly, p49/p100 as well as p105 maps to regions associated with certain types of acute lymphoblastic leukemia.     

Interaction of the C-terminal region of p105 with the nuclear localisation signal of p50 is required for inhibition of NF-kappa B DNA binding activity.

DNA binding of the homodimeric p50 subunit of NF-kappa B was inhibited by a bacterially expressed protein containing the ankyrin repeats present in the C-terminus of the p105 precursor but not by the I kappa B protein MAD-3. However p50 was retained on protein affinity matrices containing either the C-terminal ankyrin repeats of p105 or MAD-3. To investigate the interaction between p50 and proteins containing ankyrin repeats we have used a number of approaches to probe the accessibility of the p50 nuclear localisation signal in the protein complex. A monoclonal antibody recognising a linear epitope either very close to, or including, the nuclear localisation signal of the p50 protein could immunoprecipitate p50 homodimers but was unable to precipitate the protein when it was bound to the C-terminal region of p105. A close association between the nuclear localisation signal of p50 and the C-terminal region of p105 was also suggested by protease accessibility experiments. While the nuclear localisation signal of free p50 is extremely susceptible to cleavage with trypsin the same site is masked in the presence of the C-terminal ankyrin repeats of p105 and, to a lesser extent MAD-3. Removal of the nuclear localisation signal by trypsin digestion generates a protein that is fully competent for DNA binding but is refractile to inhibition by the C-terminal ankyrin repeats of p105. Addition of DNA destabilises complexes between p50 and ankyrin repeat containing proteins, increasing the susceptibility of the nuclear localisation signal to trypsin cleavage. The data suggest that there is a rapid exchange of p50 between complexes containing DNA or I kappa B proteins via a metastable complex containing DNA, p50 and I kappa B.     

Tumor necrosis factor and interleukin-1 lead to phosphorylation and loss of I kappa B alpha: a mechanism for NF-kappa B activation.

Nuclear factor kappa B (NF-kappa B) is a critical regulator of several genes which are involved in immune and inflammation responses. NF-kappa B, consisting of a 50-kDa protein (p50) and a 65-kDa protein (p65), is bound to a cytoplasmic retention protein called I kappa B. Stimulation of cells with a variety of inducers, including cytokines such as tumor necrosis factor and interleukin-1, leads to the activation and the translocation of p50/65 NF-kappa B into the nucleus. However, the in vivo mechanism of the activation process remains unknown. Here, we provide the first evidence that the in vivo mechanism of NF-kappa B activation is through the phosphorylation and subsequent loss of its inhibitor, I kappa B alpha. We also show that both I kappa B alpha loss and NF-kappa B activation are inhibited in the presence of antioxidants, demonstrating that the loss of I kappa B alpha is a prerequisite for NF-kappa B activation. Finally, we demonstrate that I kappa B alpha is rapidly resynthesized after loss, indicating that an autoregulatory mechanism is involved in the regulation of NF-kappa B function. We propose a mechanism for the activation of NF-kappa B through the modification and loss of I kappa B alpha, thereby establishing its role as a mediator of NF-kappa B activation.