The enzymatic and DNA binding activity of PARP-1 are not required for NF-kappa B coactivator function.
Poly(ADP-ribose) polymerase 1 (PARP-1)-deficient mice are protected against septic shock, diabetes type I, stroke, and inflammation. We report that primary cells from PARP-1(-/-) animals are impaired in kappa B-dependent transcriptional activation induced by different stimuli involved in inflammatory and genotoxic stress signaling. PARP-1 was also required for p65-mediated transcriptional activation. PARP-1 enzymatic inhibitors did not inhibit the transcriptional activation of a kappa B-dependent reporter gene in wild type cells. Remarkably, neither the enzymatic activity nor the DNA binding activity of PARP-1 was required for kappa B-dependent transcriptional activation in PARP-1(-/-) cells complemented with different PARP-1 mutants. However, PARP-1 interacted in vitro directly with both subunits of NF-kappa B (p50 and p65), and mapping of the interaction domains revealed that both subunits bind to different PARP-1 domains. Furthermore, a PARP-1 mutant lacking the enzymatic and DNA binding activity interacted comparably to the wild type PARP-1 with p65 or p50. Finally, we showed that PARP-1 is activating the natural inducible nitric-oxide synthase and P-selectin promoter in a kappa B-dependent manner upon stimulation of the cells with inflammatory stimuli or cotransfection of p65. Our results provide evidence that neither the DNA binding nor the enzymatic activity of PARP-1 but its direct protein-protein interaction with both subunits of NF-kappa B is required for its coactivator function, thus expanding the role of PARP-1 as an essential and novel classical transcriptional coactivator for kappa B-dependent gene expression in vivo.
Hassa
P O
PO
Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
Covic
M
M
Hasan
S
S
Imhof
R
R
Hottiger
M O
MO
D001402:B-Lymphocytes / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D017931:DNA Primers; D004789:Enzyme Activation; D006801:Humans; D018836:Inflammation Mediators / Q000494:pharmacology; D009153:Mutagens / Q000494:pharmacology; D016328:NF-kappa B / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D019007:P-Selectin / Q000235:genetics; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D013601:T-Lymphocytes / Q000378:metabolism; D014158:Transcription, Genetic
D017931:DNA Primers; D018836:Inflammation Mediators; D009153:Mutagens; D016328:NF-kappa B; D019007:P-Selectin; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D011065:Poly(ADP-ribose) Polymerases