Identification of the ADP-ribosylation sites in the PARP-1 automodification domain: analysis and implications.
Poly(ADP-ribose) polymerase-1 (PARP-1) is a multimodular (domains A, B, C, D, E, and F) nuclear protein that participates in many fundamental cellular activities. Stimulated by binding to nicked DNA, PARP-1 catalyzes poly(ADP-ribosyl)ation of the acceptor proteins and itself using NAD(+) as a substrate. Early studies suggested that domain D is likely an interface for protein-protein interaction between PARP-1 and its targets and is also the primary region for automodification. However, determination of the modification sites has been complicated by the heterogeneous nature of the poly(ADP-ribose) polymer. Here we report a strategy to identify the modification sites on domain D using the PARP-1 E988Q mutant, which only catalyzes mono(ADP-ribosyl)ation. Trypsin digestion of the modified domain D followed by LC-MS/MS analysis led to the identification of three ADP-ribosylation sites in domain D (D387, E488, and E491). Our data also show, in contrast to early reports, that automodification of PARP-1 is not limited to domain D but occurs beyond this region. In addition, domain D is not essential for PARP-1 activity since PARP-1 mutant having domain D deleted is still catalytically active. Two synthetic peptides with amino acid sequences derived from the ADP-ribosylation sites of domain D were also demonstrated to act as PARP-1 substrates. The methodology and the results reported herein will facilitate future studies of PARP-1 catalysis.
Tao
Zhihua
Z
Institute for Cellular and Molecular Biology, University of Texas, Austin, Texas 78712, USA.
Gao
Peng
P
Liu
Hung-wen
HW
D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain; D006801:Humans; D008958:Models, Molecular; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary
D000244:Adenosine Diphosphate; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases