The role of the kappa enhancer and its binding factor NF-kappa B in the developmental regulation of kappa gene transcription

We report here on a comparison of plasmacytoma cell lines that differ markedly in their ability to express kappa immunoglobulin genes introduced by transfection, but nevertheless express their endogenous kappa genes at comparable levels. The cell line that fails to express exogenous kappa genes is nonpermissive for kappa enhancer function, apparently because it lacks a specific kappa enhancer-binding nuclear factor (NF-kappa B). We show that this same nuclear factor is also lacking in pre-B cells and that treatment of these cells with bacterial lipopolysaccharide induces the appearance of NF-kappa B in nuclear extracts and concomitantly activates the kappa enhancer. These findings indicate that factor NF-kappa B controls kappa enhancer activity, and that this activity is only transiently required during B cell maturation.     

Mechanism of I kappa B alpha binding to NF-kappa B dimers

X-ray crystal structures of the NF-kappa B.I kappa B alpha complex revealed an extensive and complex protein-protein interface involving independent structural elements present in both I kappa B alpha and NF-kappa B. In this study, we employ a gel electrophoretic mobility shift assay to assess and quantitate the relative contributions of the observed interactions toward overall complex binding affinity. I kappa B alpha preferentially binds to the p50/p65 heterodimer and p65 homodimer, with binding to p50 homodimer being significantly weaker. Our results indicate that the nuclear localization sequence and the region C-terminal to it of the NF-kappa B p65 subunit is a major contributor to NF-kappa B. I kappa B alpha complex formation. Additionally, there are no contacts between the corresponding nuclear localization signal tetrapeptide of p50 and I kappa B alpha. A second set of interactions involving the acidic C-terminal/PEST-like region of I kappa B alpha and the NF-kappa B p65 subunit N-terminal domain also contributes binding energy toward formation of the complex. This interaction is highly dynamic and nonspecific in nature, as shown by oxidative cysteine cross-linking. Phosphorylation of the C-terminal/PEST-like region by casein kinase II further enhances binding.     

Mechanism of kappa B DNA binding by Rel/NF-kappa B dimers

The DNA binding of three different NF-kappaB dimers, the p50 and p65 homodimers and the p50/p65 heterodimer, has been examined using a combination of gel mobility shift and fluorescence anisotropy assays. The NF-kappaB p50/p65 heterodimer is shown here to bind the kappaB DNA target site of the immunoglobulin kappa enhancer (Ig-kappaB) with an affinity of approximately 10 nm. The p50 and p65 homodimers bind to the same site with roughly 5- and 15-fold lower affinity, respectively. The nature of the binding isotherms indicates a cooperative mode of binding for all three dimers to the DNA targets. We have further characterized the role of pH, salt, and temperature on the formation of the p50/p65 heterodimer-Ig-kappaB complex. The heterodimer binds to the Ig-kappaB DNA target in a pH-dependent manner, with the highest affinity between pH 7.0 and 7.5. A strong salt-dependent interaction between Ig-kappaB and the p50/p65 heterodimer is observed, with optimum binding occurring at monovalent salt concentrations below 75 mm, with binding becoming virtually nonspecific at a salt concentration of 200 mm. Binding of the heterodimer to DNA was unchanged across a temperature range between 4 degrees C and 42 degrees C. The sensitivity to ionic environment and insensitivity to temperature indicate that NF-kappaB p50/p65 heterodimers form complexes with specific DNA in an entropically driven manner.     

The Drosophila RBP-J kappa gene encodes the binding protein for the immunoglobulin J kappa recombination signal sequence.

We previously isolated a cDNA encoding the 60-kDa murine protein (RBP-J kappa protein) that specifically binds to the immunoglobulin J kappa recombination signal sequence. The RBP-J kappa gene is highly conserved in a wide variety of organisms including man, Xenopus, Drosophila, and yeast. We have isolated and characterized the Drosophila homologue of the RBP-J kappa gene. The Drosophila RBP-J kappa gene was mapped to the polytene region 35BC of chromosome 2. The nucleotide sequence of this gene indicates that it is not one of the known genes located in the 35 BC region. The nucleotide and amino acid sequences of the Drosophila and mouse RBP-J kappa genes are 60 and 75% homologous, respectively. The central 248-residue regions of RBP-J kappa proteins of the two species are 93% homologous and include the 40-residue integrase motif. The Drosophila RBP-J kappa protein expressed in COS cells bound to the J kappa recognition sequence with the same specificity as the murine counterpart. These results suggest that Drosophila may have a site-specific recombination system which utilizes the immunoglobulin recombination signal sequence. Implications for evolution of immunoglobulin gene rearrangement were also discussed.     

DNA binding and I kappa B inhibition of the cloned p65 subunit of NF-kappa B, a rel-related polypeptide

The sequence and biochemical properties of the product of the cloned cDNA for the p65 subunit of nuclear factor kappa B (NF-kappa B) have been determined. The cDNA has an open reading frame of 549 amino acids capable of encoding a 60 kd protein. NF-kappa B p65 contains an amino-terminal region of 320 amino acids with extensive similarity to the oncogene c-rel and lesser similarity to NF-kappa B p50. In vitro translated p65 forms a DNA-binding complex with NF-kappa B p50, and the binding of this complex can be specifically inhibited by purified I kappa B. Progressive carboxy-terminal deletions of p65 show that, contrary to previous assumptions, p65 does include a DNA-binding domain that in vivo might become activated only through hetero-oligomerization with p50. DNA binding by truncated p65 is inhibited by I kappa B, thus mapping the I kappa B interaction domain to the rel-homologous region and suggesting that I kappa B exerts its inhibitory effect upon NF-kappa B primarily through interaction with 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.     

Epstein-Barr virus encodes a novel homolog of the bcl-2 oncogene that inhibits apoptosis and associates with Bax and Bak

The sequenced gammaherpesviruses each contain a single viral bcl-2 homolog (v-bcl-2) which may encode a protein that functions in preventing the apoptotic death of virus-infected cells. Epstein-Barr virus (EBV), a gammaherpesvirus associated with several lymphoid and epithelial malignancies, encodes the v-Bcl-2 homolog BHRF1. In this report the previously uncharacterized BALF1 open reading frame in EBV is identified as having significant sequence similarity to other v-bcl-2 homologs and cellular bcl-2. Transfection of cells with a BALF1 cDNA conferred apoptosis resistance. Furthermore, a recombinant green fluorescent protein-BALF1 fusion protein suppressed apoptosis and associated with Bax and Bak. These results indicate that EBV encodes a second functional v-bcl-2.