Systems-wide Analysis of Serine ADP-Ribosylation Reveals Widespread Occurrence and Site-Specific Overlap with Phosphorylation

Abstract

ADP-ribosylation (ADPr) is a reversible posttranslational modification involved in a range of cellular processes. Here, we report system-wide identification of serine ADPr in human cells upon oxidative stress. High-resolution mass spectrometry and unrestricted data processing confirm that serine residues are the major target of ADPr in HeLa cells. Proteome-wide analysis identifies 3,090 serine ADPr sites, with 97% of acceptor sites modulating more than 2-fold upon oxidative stress, while treatment with the poly (ADP-ribose) polymerase (PARP) inhibitor olaparib abrogates this induction. Serine ADPr predominantly targets nuclear proteins, while structural-predictive analyses reveal that serine ADPr preferentially targets disordered protein regions. The identified ADP-ribosylated serines significantly overlap with known phosphorylated serines, and large-scale phosphoproteomics analysis provides evidence for site-specific crosstalk between serine ADPr and phosphorylation. Collectively, we demonstrate that serine ADPr is a widespread modification and a major nuclear signaling response to oxidative stress, with a regulatory scope comparable to other extensive posttranslational modifications. By combining unbiased proteomics analyses with unrestricted data processing, Larsen et al. demonstrate that serine residues are the primary target of system-wide ADP-ribosylation in response to oxidative stress. Moreover, the authors show that a significant degree of serine residues are co-targeted by ADP-ribosylation and phosphorylation in the DNA damage response.