Reactive Oxygen Species (ROS)-Activated ATM-Dependent Phosphorylation of Cytoplasmic Substrates Identified by Large-Scale Phosphoproteomics Screen

Abstract

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

Rats with a missense mutation in Atm display neuroinflammation and neurodegeneration subsequent to accumulation of cytosolic DNA following unrepaired DNA damage

“…Whereas ATM activated by oxidative stress phosphorylated p53 and checkpoint kinase 2, it failed to phosphorylate histone H2AX variant or Kap1, both of which participate in the DDR at the level of chromatin. More recent data describe phosphorylation of ATM-dependent cytoplasmic substrates in response to oxidative stress [17]. The rescue of several aspects of the phenotype in A-T cells and Atm 2/2 mice by antioxidants provides further support for a role for ATM in protecting against oxidative stress [18][19][20].…”

Multifaceted roles of ATM in autophagy: From nonselective autophagy to selective autophagy

“…[2][3][4][5][6] ATM autophosphorylates Ser 1981, resulting in the dissociation of its inactivate dimer and the formation of an active monomer, 7 which coordinates the activation of cell cycle checkpoints and DDR and further maintains gene stability. 9,12,13 In the cytoplasm, however, ATM was shown to be located in peroxisomes, mitochondria, and endosomes, [14][15][16][17][18][19] and it participates in regulating oxidative stress, cell metabolism, and autophagy. The nuclear function of ATM is tightly associated with DNA damage, such as DDR and cell cycle checkpoints, through the ATM-Chk2 and ATM-ATR-Chk1 pathways, 11 as well as the MRN (Mre11-Rad590-Nb1) complex and MDC1.…”

“…The nuclear function of ATM is tightly associated with DNA damage, such as DDR and cell cycle checkpoints, through the ATM-Chk2 and ATM-ATR-Chk1 pathways, 11 as well as the MRN (Mre11-Rad590-Nb1) complex and MDC1. 9,12,13 In the cytoplasm, however, ATM was shown to be located in peroxisomes, mitochondria, and endosomes, [14][15][16][17][18][19] and it participates in regulating oxidative stress, cell metabolism, and autophagy. 20,21 PACS-2 is required for the IR-induced cytoplasmic localization of nuclear ATM.…”

Role of DNA Damage Response in Suppressing Malignant Progression of Chronic Myeloid Leukemia and Polycythemia Vera: Impact of Different Oncogenes

“…Cytoplasmic activation of ATM in PV likely reflects its response to ROS generated in PV bone marrow inflammatory microenvironment, as ATM phosphorylation by ROS requires its cytoplasmic localization [89]. In addition, the aforementioned extent of Chk2 activation in CML (high) and PV (low) likely reflects subcellular localization of P-ATM [90,91]. Our further (published in [82]) IHC staining of patients' bone marrow sections from PV revealed low nuclear staining for activated ATR (P-ATR at T1989), and barely detectable expression of a marker for oxidative DNA lesions 8-oxoguanine (8-oxoG), as well as very low staining for a marker of global nuclear DDR activation, Ser 139-phosphorylated histone H2AX (γH2AX) [82].…”