Evidence for regulation of NF-κB by poly(ADP-ribose) polymerase

The DNA-binding activity of NF-KB in nuclear extracts of poly(ADP-ribose) polymerase (PARP)-defective mutant L1210 cell clones was markedly increased and was inversely correlated with the PARP content in these cells. The DNA-binding activity of NF-kappa B in a clone with the lowest PARP content (Cl-3527, contained 6 % of PARP of wild type cells) was about 35-fold of that of the wild-type cells, whereas the change in the DNA-binding activity of AP-1 and SP-1 in the mutant was relatively small or not so significant. Transfection of a PARP-expressing plasmid to the mutant cells decreased the abnormally high levels of NF-kappa B complexes, especially p50/p65(Rel A) complex, to near the normal level. Moreover, poly(ADP-ribosyl)ation of nuclear extracts in vitro suppressed the ability of NF-kappa B to form a complex with its specific DNA probe by approx. 80 Ob. Further analysis with purified recombinant NF-KB proteins revealed that both rp50 and rMBP-p65 (Rel A) proteins, but not rGST-I kappa B, could be poly(ADP-ribosyl)ated in vitro and that the modification resulted in a marked decrease in the DNA-binding activity of rMBP-p65, whereas a slight activation was observed in rp50. Poly(ADP-ribosyl)ated p65/NF-kappa B was detected in the cytosol of wild type L1210 cells by immunoblotting with anti-poly(ADP-ribose) and anti-p65 antibodies. Taken together, these results strongly suggest that PARP is involved in the regulation of NF-kappa B through the protein modification.