Polyubiquitylation of PARP-1 through ubiquitin K48 is modulated by activated DNA, NAD+, and dipeptides.
Poly(ADP-ribose) polymerase-1 (PARP-1) is the most abundant and the best-studied isoform of a family of enzymes that catalyze the polymerization of ADP-ribose from NAD(+) onto target proteins. PARP-1 is well known to involve in DNA repair, genomic stability maintenance, transcription regulation, apoptosis, and necrosis. Polyubiquitylation targets proteins towards degradation and regulates cell cycle progression, transcription, and apoptosis. Here we report polyubiquitylation of PARP-1 in mouse fibroblasts in the presence of proteasome inhibitor and in full-length recombinant PARP-1 in vitro under standard ubiquitylation assay conditions by immunoprecipitation and immunoblotting. Mutation of ubiquitin K48R but not ubiquitin K63R abolishes polyubiquitylation of PARP-1, indicating that K48 of ubiquitin was used in the formation of polyubiquitin chain and that ubiquitylated PARP-1 is likely destined for degradation. Full-length PARP-1 was ubiquitylated most likely at the N-terminal 24 kDa domain of PARP-1 as suggested by the inhibition of ubiquitylation by activated DNA and the absence of polyubiquitin in the C-terminal 89 kDa PARP-1 derived from caspase-catalyzed cleavage. NAD(+) inhibited ubiquitylation of PARP-1, while dipeptides ArgAla and LeuAla enhanced ubiquitylation of PARP-1. ATP inhibited the synthesis of poly(ADP-ribose) by PARP-1 and affinity purified polyubiquitylated PARP-1 was active in PAR synthesis. The results suggest polyubiquitylation of PARP-1 could regulate poly(ADP-ribosyl)ation of nuclear proteins by PARP-1 and consequently apoptosis and PARP-1 regulated cellular processes through ubiquitin-dependent degradation pathways.
Department of Chemistry, Georgetown University, Washington, District of Columbia 20057, USA.
Tao
T
Wang
Cynthia M
CM
Simbulan-Rosenthal
Mark E
ME
Smulson
P Boon
PB
Chock
David C H
DC
Yang
D000818:Animals; D002478:Cells, Cultured; D004247:DNA / Q000494:pharmacology; D004151:Dipeptides / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D051379:Mice; D020125:Mutation, Missense; D009243:NAD / Q000494:pharmacology; D009687:Nuclear Proteins / Q000378:metabolism; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
D004151:Dipeptides; D009687:Nuclear Proteins; D025801:Ubiquitin; D009243:NAD; D004247:DNA; C499321:Parp1 protein, mouse; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases