PURIFICATION, RECONSTITUTION, AND I-KAPPA-B ASSOCIATION OF THE C-REL P65 (RELA) COMPLEX, A STRONG ACTIVATOR OF TRANSCRIPTION

HeLa cells contain a DNA-binding activity which associates with a kappaB-like DNA element, termed Rel-related protein-binding element (RRBE), localized upstream of the human urokinase promoter. We have purified this activity from the HeLa cell cytosol and have shown that it represents a preformed heteromeric complex between p65 (RelA) and c-Rel. Coexpression of c-Rel and p65 (RelA) by in vitro translation formed a DNA-binding complex indistinguishable from purified cellular c-Rel-p65 (RelA) in mobility shift assays. The c-Rel-p65 (RelA) complex was also formed in COS7 cells upon coexpression of c-Rel and p65 (RelA) cDNAs. Cotransfection experiments with COS7 cells, using expression plasmids encoding p50, p65 (RelA), or c-Rel and reporter constructs containing a trimerized RRBE, revealed that c-Rel-p65 (RelA) is a potent activator of the RRBE, giving rise to transcriptional activity higher than that observed with NF-kappaB (p50-p65). In the cytosol, the c-Rel-p65 (RelA) complex existed in a latent, non-DNA-binding form but could be activated by detergent treatment, suggesting that it was associated with an IkappaB protein. Recombinant IkappaB-alpha inhibited the DNA-binding activity of c-Rel-p65 (RelA) via association with either c-Rel or p65 (RelA). Finally, NF-kappaB and c-Rel-p65 (RelA) complexes were found to be differentially expressed and regulated in different cells. The two complexes were present in equimolar amounts in HeLa cells and K562 cells. Stimulation with tetradecanoyl phorbol acetate (TPA) resulted in the nuclear translocation of both NF-kappaB and c-Rel-p65 (RelA) in HeLa cells and of NF-kappaB in HepG2 cells but had no effect on either complex in K562 cells. In addition, TPA stimulation of HepG2 cells induced the expression of a cytosolic latent c-Rel-p65 (RelA) complex which, however, was not translocated to the nucleus. In conclusion, our findings show that c-Rel-p65 (RelA) is an inducible and very potent transcriptional activator which is differentially activated in a cell-type-specific manner.