Reactive oxygen species (ROS)-activated ATM-dependent phosphorylation of cytoplasmic substrates identified by large-scale phosphoproteomics screen

Kozlov, Sergei V., Waardenberg, Ashley J., Engholm-Keller, Kasper, Arthur, Jonathan W., Graham, Mark E., and Lavin, Martin (2015) Reactive oxygen species (ROS)-activated ATM-dependent phosphorylation of cytoplasmic substrates identified by large-scale phosphoproteomics screen. Molecular and Cellular Proteomics, 15 (3). pp. 1032-1047.

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Ataxia-telangiectasia, mutated (ATM) protein plays a central role in phosphorylating a network of proteins in response to DNA damage. These proteins function in signaling pathways designed to maintain the stability of the genome and minimize the risk of disease by controlling cell cycle checkpoints, initiating DNA repair, and regulating gene expression. ATM kinase can be activated by a variety of stimuli, including oxidative stress. Here, we confirmed activation of cytoplasmic ATM by autophosphorylation at multiple sites. Then we employed a global quantitative phosphoproteomics approach to identify cytoplasmic proteins altered in their phosphorylation state in control and ataxia-telangiectasia (A-T) cells in response to oxidative damage. We demonstrated that ATM was activated by oxidative damage in the cytoplasm as well as in the nucleus and identified a total of 9,833 phosphorylation sites, including 6,686 high-confidence sites mapping to 2,536 unique proteins. A total of 62 differentially phosphorylated peptides were identified; of these, 43 were phosphorylated in control but not in A-T cells, and 19 varied in their level of phosphorylation. Motif enrichment analysis of phosphopeptides revealed that consensus ATM serine glutamine sites were overrepresented. When considering phosphorylation events, only observed in control cells (not observed in A-T cells), with predicted ATM sites phosphoSerine/phosphoThreonine glutamine, we narrowed this list to 11 candidate ATM-dependent cytoplasmic proteins. Two of these 11 were previously described as ATM substrates (HMGA1 and UIMCI/RAP80), another five were identified in a whole cell extract phosphoproteomic screens, and the remaining four proteins had not been identified previously in DNA damage response screens. We validated the phosphorylation of three of these proteins (oxidative stress responsive 1 (OSR1), HDGF, and ccdc82) as ATM dependent after H2O2 exposure, and another protein (S100A11) demonstrated ATM-dependence for translocation from the cytoplasm to the nucleus. These data provide new insights into the activation of ATM by oxidative stress through identification of novel substrates for ATM in the cytoplasm.

Item ID: 55657
Item Type: Article (Research - C1)
ISSN: 1535-9484
Copyright Information: Copyright © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
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Funders: National Health and Medical Research Council (NHMRC), Lundbeck Foundation (LF), Danish Council for Independent Research (DCIR), European Union FP7
Projects and Grants: MOBILEX Postdoctoral Fellowship DFF 1325-00154, European Union FP7 Marie Curie Actions - COFUND program
Date Deposited: 25 Sep 2018 02:26
FoR Codes: 06 BIOLOGICAL SCIENCES > 0601 Biochemistry and Cell Biology > 060109 Proteomics and Intermolecular Interactions (excl Medical Proteomics) @ 50%
11 MEDICAL AND HEALTH SCIENCES > 1101 Medical Biochemistry and Metabolomics > 110106 Medical Biochemistry: Proteins and Peptides (incl Medical Proteomics) @ 50%
SEO Codes: 97 EXPANDING KNOWLEDGE > 970106 Expanding Knowledge in the Biological Sciences @ 100%
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