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

The DNA-binding activity of NF-KB in nuclear extracts of poly(ADP-ribose) p olymerase (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 o f 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. Trans fection of a PARP-expressing plasmid to the mutant cells decreased the abno rmally high levels of NF-kappa B complexes, especially p50/p65(Rel A) compl ex, to near the normal level. Moreover, poly(ADP-ribosyl)ation of nuclear e xtracts in vitro suppressed the ability of NF-kappa B to form a complex wit h its specific DNA probe by approx. 80 Ob. Further analysis with purified r ecombinant NF-KB proteins revealed that both rp50 and rMBP-p65 (Rel A) prot eins, but not rGST-I kappa B, could be poly(ADP-ribosyl)ated in vitro and t hat the modification resulted in a marked decrease in the DNA-binding activ ity 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 t he regulation of NF-kappa B through the protein modification.