A novel complex between the p65 subunit of NF-kappa B and c-Rel binds to a DNA element involved in the phorbol ester induction of the human urokinase gene.

The NF-kappa B subunits, p50 and p65, have extensive sequence homology with the c-rel proto-oncogene and the Drosophila morphogen dorsal. It has recently been shown that in vitro translated c-Rel can bind to DNA and form a complex with p50. However, the conditions for DNA binding of c-Rel in vivo and its DNA sequence specificity have not been established. Here we report the identification a novel heterodimeric complex that binds to a kappa B-like, phorbol ester (TPA) responsive DNA sequence, 5'-GGGAAAGTAC-3', in the 5' flanking region of the human urokinase (uPA) gene. This sequence was shown to bind two protein complexes, LC and UC. LC was indistinguishable from NF-kappa B as it reacted with antibodies recognizing the p50 subunit of NF-kappa B, and was shown by UV crosslinking to contain the p50 and p65 subunits of NF-kappa B. UC, on the other hand, strongly reacted with anti-v-Rel, but not with the anti-p50 antibodies, and was shown by crosslinking to contain 75 kDa and 85 kDa protein-DNA adducts. The 75 kDa and the 85 kDa adducts could be immunoprecipitated only by anti-p65 and anti-c-Rel antibodies, respectively, showing that c-Rel formed a heterodimer with p65. Both protein complexes were present in inactive forms in HeLa cell cytosol, and their nuclear translocation was induced by TPA. DNA binding of UC and LC could, furthermore, be inhibited by I kappa B-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)     

PEST-dependent cytoplasmic retention of v-Rel by I(kappa)B-alpha: evidence that I(kappa)B-alpha regulates cellular localization of c-Rel and v-Rel by distinct mechanisms.

Association of c-Rel with the inhibitor of kappaB-alpha (IkappaB-alpha) protein regulates both cellular localization and DNA binding. The ability of v-Rel, the oncogenic viral counterpart of avian c-Rel, to evade regulation by p40, the avian IkappaB-alpha protein, contributes to v-Rel-mediated oncogenesis. The yeast two-hybrid system was utilized to dissect Rel:IkappaB-alpha interactions in vivo. We find that distinct domains in c-Rel and v-Rel are required for association with p40. Furthermore, while the ankyrin repeat domain of p40 is sufficient for association with c-Rel, both the ankyrin repeat domain and the PEST domain are required for association with v-Rel. Two amino acid differences between c-Rel and v-Rel that are principally responsible for PEST-dependent association of v-Rel with p40 were identified. These same amino acids were principally responsible for PEST-dependent cytoplasmic retention of v-Rel by p40. The presence of mutations in c-Rel that were sufficient to confer PEST-dependent association of the mutant c-Rel protein with p40 did not increase the weak oncogenicity of c-Rel. However, the introduction of these two c-Rel-derived amino acids into v-Rel markedly reduced the oncogenicity of v-Rel. Deletion of the NLS of either c-Rel or v-Rel did not abolish association with p40, but did confer PEST-dependent association of c-Rel with p40. Surprisingly, deletion of the nuclear localization signal in v-Rel did not affect oncogenicity by v-Rel. Analysis of several mutant c-Rel and v-Rel proteins demonstrated that association of Rel proteins with p40 is necessary but not sufficient for cytoplasmic retention. These results are not consistent with the hypothesis that p40 regulates cellular localization of v-Rel and c-Rel by the same mechanism. Rather, these results support the hypothesis that p40 regulates cellular localization of v-Rel and c-Rel by distinct mechanisms.     

Constitutive and inducible kappa B binding activities in the cytosol of v-Rel-transformed lymphoid cells.

Constitutive and inducible kapp B binding activities associated with v-Rel and c-Rel in the cytosol of v-Rel-transformed cells have been identified. These activities were resolved by electrophoretic mobility shift assay and chromatographic techniques into a high-molecular-weight protein-DNA complex designated complex I containing v- and c-Rel and lower-molecular-weight complexes II, III and IV which contained only v-Rel and which were stimulated by nucleotides, low pH, and detergent. These experiments suggest that interaction of v-Rel and c-Rel decreases the DNA-binding activity of each.     

Lipopolysaccharide induction of tissue factor gene expression in monocytic cells is mediated by binding of c-Rel/p65 heterodimers to a kappa B-like site.

Exposure of monocytic cells to bacterial lipopolysaccharide (LPS) activates the NF-kappa B/Rel family of proteins and leads to the rapid induction of inflammatory gene products, including tissue factor (TF). TF is the primary cellular initiator of the coagulation protease cascades. Here we report the characterization of a nuclear complex from human monocytic cells that bound to a kappa B-like site, 5'-CGGAGTTTCC-3', in the 5'-flanking region of the human TF gene. This nuclear complex was activated by LPS with kinetics that preceded induction of the TF gene. In vitro binding studies demonstrated that the TF site bound translated c-Rel and p65 homodimers but not p50/p65 heterodimers or p50 homodimers. Base-pair substitutions in the TF site indicated that the presence of a cytosine at position 1 precluded binding of NF-kappa B. In fact, under low-ionic-strength conditions, the TF complex did not migrate with translated p50/p65 dimers but instead comigrated with c-Rel/p65 dimers. Antibodies against the NF-kappa B and Rel proteins and UV cross-linking studies revealed the presence of c-Rel and p65 and the absence of p50 in the TF complex and further showed that c-Rel/p65 heterodimers selectively bound to the TF kappa B-like site. Functional studies indicated that the TF site conferred LPS inducibility on a heterologous promoter and was transactivated by c-Rel or p65. Taken together, our results demonstrated that binding of c-Rel/p65 heterodimers to a novel kappa B-like site mediated LPS induction of TF gene expression in monocytic cells.