The sequence-specific DNA binding of NF-kappa B is reversibly regulated by the automodification reaction of poly (ADP-ribose) polymerase 1.
Recent studies suggest that the synthesis of protein-bound ADP-ribose polymers catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1) regulates eucaryotic gene expression, including the NF-kappaB-dependent pathway. Here, we report the molecular mechanism by which PARP-1 activates the sequence-specific binding of NF-kappaB to its oligodeoxynucleotide. We co-incubated pure recombinant human PARP-1 and the p50 subunit of NF-kappaB (NF-kappaB-p50) in the presence or absence of betaNAD+ in vitro. Electrophoretic mobility shift assays showed that, when PARP-1 was present, NF-kappaB-p50 DNA binding was dependent on the presence of betaNAD+. DNA binding by NF-kappaB-p50 was not efficient in the absence of betaNAD+. In fact, the binding was not efficient in the presence of 3-aminobenzamide (3-AB) either. Thus, we conclude that NF-kappaB-p50 DNA binding is protein-poly(ADP-ribosyl)ation dependent. Co-immunoprecipitation and immunoblot analysis revealed that PARP-1 physically interacts with NF-kappaB-p50 with high specificity in the absence of betaNAD+. Because NF-kB-p50 was not an efficient covalent target for poly(ADP-ribosyl)ation, our results are consistent with the conclusion that the auto-poly(ADP-ribosyl)ation reaction catalyzed by PARP-1 facilitates the binding of NF-kappaB-p50 to its DNA by inhibiting the specific protein.protein interactions between NF-kappaB-p50 and PARP-1. We also report the activation of NF-kappaB DNA binding by the automodification reaction of PARP-1 in cultured HeLa cells following exposure to H(2)O(2). In these experiments, preincubation of HeLa cells with 3-AB, prior to oxidative damage, strongly inhibited NF-kappaB activation in vivo as well.
Chang
W J
WJ
Department of Molecular Biology and Immunology, University of North Texas Health Science Center at Fort Worth, Fort Worth, TX 76107, USA.
Alvarez-Gonzalez
R
R
D001549:Benzamides / Q000494:pharmacology; D001667:Binding, Competitive; D002384:Catalysis; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D015151:Immunoblotting; D016328:NF-kappa B / Q000378:metabolism*; D052002:NF-kappa B p50 Subunit; D010100:Oxygen / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D001549:Benzamides; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D011994:Recombinant Proteins; C025160:3-aminobenzamide; D004247:DNA; D006861:Hydrogen Peroxide; D011065:Poly(ADP-ribose) Polymerases; D010100:Oxygen