ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage
25 May 2007-Science (American Association for the Advancement of Science)-Vol. 316, Iss: 5828, pp 1160-1166
TL;DR: A large-scale proteomic analysis of proteins phosphorylated in response to DNA damage on consensus sites recognized by ATM and ATR is performed and more than 900 regulated phosphorylation sites encompassing over 700 proteins are identified.
Abstract: Cellular responses to DNA damage are mediated by a number of protein kinases, including ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related). The outlines of the signal transduction portion of this pathway are known, but little is known about the physiological scope of the DNA damage response (DDR). We performed a large-scale proteomic analysis of proteins phosphorylated in response to DNA damage on consensus sites recognized by ATM and ATR and identified more than 900 regulated phosphorylation sites encompassing over 700 proteins. Functional analysis of a subset of this data set indicated that this list is highly enriched for proteins involved in the DDR. This set of proteins is highly interconnected, and we identified a large number of protein modules and networks not previously linked to the DDR. This database paints a much broader landscape for the DDR than was previously appreciated and opens new avenues of investigation into the responses to DNA damage in mammals.
Citations
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TL;DR: The authors' improving understanding of DNA-damage responses is providing new avenues for disease management, and these responses are biologically significant because they prevent diverse human diseases.
Abstract: The prime objective for every life form is to deliver its genetic material, intact and unchanged, to the next generation. This must be achieved despite constant assaults by endogenous and environmental agents on the DNA. To counter this threat, life has evolved several systems to detect DNA damage, signal its presence and mediate its repair. Such responses, which have an impact on a wide range of cellular events, are biologically significant because they prevent diverse human diseases. Our improving understanding of DNA-damage responses is providing new avenues for disease management.
4,871 citations
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TL;DR: This review will focus on how the DDR controls DNA repair and the phenotypic consequences of defects in these critical regulatory functions in mammals.
3,678 citations
Cites background from "ATM and ATR substrate analysis reve..."
...A large number of DDR-deficient mice and various human DDR syndromes, such as A-T, BS, and FA, display aberrant meiotic progression and infertility (Table 2) (Biton et al., 2008; Bohr, 2008; Matzuk and Lamb, 2008)....
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TL;DR: Evidence that cancer and diseases of aging are two sides of the DNAdamage problem is presented, followed by an account of the derailment of genome guardian mechanisms in cancer and of how this cancerspecific phenomenon can be exploited for treatment.
Abstract: NA damage has emerged as a major culprit in cancer and many diseases related to aging. The stability of the genome is supported by an intricate machinery of repair, damage tolerance, and checkpoint pathways that counteracts DNA damage. In addition, DNA damage and other stresses can trigger a highly conserved, anticancer, antiaging survival response that suppresses metabolism and growth and boosts defenses that maintain the integrity of the cell. Induction of the survival response may allow interventions that improve health and extend the life span. Recently, the first candidate for such interventions, rapamycin (also known as sirolimus), has been identified. 1 Compromised repair systems in tumors also offer opportunities for intervention, making it possible to attack malignant cells in which maintenance of the genome has been weakened. Time-dependent accumulation of damage in cells and organs is associated with gradual functional decline and aging. 2 The molecular basis of this phenomenon is unclear, 3-5 whereas in cancer, DNA alterations are the major culprit. In this review, I present evidence that cancer and diseases of aging are two sides of the DNAdamage problem. An examination of the importance of DNA damage and the systems of genome maintenance in relation to aging is followed by an account of the derailment of genome guardian mechanisms in cancer and of how this cancerspecific phenomenon can be exploited for treatment.
1,917 citations
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TL;DR: New insights are provided into the mechanisms that control ATR activation, which have helped to explain the overlapping but non-redundant activities of ATR and ATM in DNA-damage signalling, and have clarified the crucial functions of AtR in maintaining genome integrity.
Abstract: Genome maintenance is a constant concern for cells, and a coordinated response to DNA damage is required to maintain cellular viability and prevent disease. The ataxia-telangiectasia mutated (ATM) and ATM and RAD3-related (ATR) protein kinases act as master regulators of the DNA-damage response by signalling to control cell-cycle transitions, DNA replication, DNA repair and apoptosis. Recent studies have provided new insights into the mechanisms that control ATR activation, have helped to explain the overlapping but non-redundant activities of ATR and ATM in DNA-damage signalling, and have clarified the crucial functions of ATR in maintaining genome integrity.
1,687 citations
Cites background from "ATM and ATR substrate analysis reve..."
...The list of ATR substrates is rapidly expanding owing to large-scale proteomic profiling methodologie...
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TL;DR: This work has witnessed an explosion in understanding of DNA damage sensing, signaling, and the complex interplay between protein phosphorylation and the ubiquitin pathway employed by the DDR network to execute the response to DNA damage.
1,639 citations
References
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TL;DR: The inability to repair DNA damage properly in mammals leads to various disorders and enhanced rates of tumour development, and this work has shown that direct activation of DNA repair networks is needed to correct this problem.
Abstract: The inability to repair DNA damage properly in mammals leads to various disorders and enhanced rates of tumour development. Organisms respond to chromosomal insults by activating a complex damage response pathway. This pathway regulates known responses such as cell-cycle arrest and apoptosis (programmed cell death), and has recently been shown to control additional processes including direct activation of DNA repair networks.
3,230 citations
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TL;DR: All life on earth must cope with constant exposure to DNA-damaging agents such as the Sun's radiation, and how cells respond to DNA damage are critical determinants of whether that individual will develop cancer.
Abstract: All life on earth must cope with constant exposure to DNA-damaging agents such as the Sun's radiation. Highly conserved DNA-repair and cell-cycle checkpoint pathways allow cells to deal with both endogenous and exogenous sources of DNA damage. How much an individual is exposed to these agents and how their cells respond to DNA damage are critical determinants of whether that individual will develop cancer. These cellular responses are also important for determining toxicities and responses to current cancer therapies, most of which target the DNA.
2,725 citations
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TL;DR: Chk2 directly phosphorylated p53 on serine 20, which is known to interfere with Mdm2 binding, and provides a mechanism for increased stability of p53 by prevention of ubiquitination in response to DNA damage.
Abstract: Chk2 is a protein kinase that is activated in response to DNA damage and may regulate cell cycle arrest. We generated Chk2-deficient mouse cells by gene targeting. Chk2-/- embryonic stem cells failed to maintain gamma-irradiation-induced arrest in the G2 phase of the cell cycle. Chk2-/- thymocytes were resistant to DNA damage-induced apoptosis. Chk2-/- cells were defective for p53 stabilization and for induction of p53-dependent transcripts such as p21 in response to gamma irradiation. Reintroduction of the Chk2 gene restored p53-dependent transcription in response to gamma irradiation. Chk2 directly phosphorylated p53 on serine 20, which is known to interfere with Mdm2 binding. This provides a mechanism for increased stability of p53 by prevention of ubiquitination in response to DNA damage.
1,305 citations
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TL;DR: Applying this approach to several cell systems, including cancer cell lines, shows it can be used to identify activated protein kinases and their phosphorylated substrates without prior knowledge of the signaling networks that are activated, a first step in profiling normal and oncogenic signaling networks.
Abstract: Tyrosine kinases play a prominent role in human cancer, yet the oncogenic signaling pathways driving cell proliferation and survival have been difficult to identify, in part because of the complexity of the pathways and in part because of low cellular levels of tyrosine phosphorylation. In general, global phosphoproteomic approaches reveal small numbers of peptides containing phosphotyrosine. We have developed a strategy that emphasizes the phosphotyrosine component of the phosphoproteome and identifies large numbers of tyrosine phosphorylation sites. Peptides containing phosphotyrosine are isolated directly from protease-digested cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. Applying this approach to several cell systems, including cancer cell lines, shows it can be used to identify activated protein kinases and their phosphorylated substrates without prior knowledge of the signaling networks that are activated, a first step in profiling normal and oncogenic signaling networks.
1,144 citations
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TL;DR: The in vitro substrates recognized by most yeast protein kinases are described, with the use of proteome chip technology, and these results will provide insights into the mechanisms and roles of protein phosphorylation in many eukaryotes.
Abstract: Protein phosphorylation is estimated to affect 30% of the proteome and is a major regulatory mechanism that controls many basic cellular processes. Until recently, our biochemical understanding of protein phosphorylation on a global scale has been extremely limited; only one half of the yeast kinases have known in vivo substrates and the phosphorylating kinase is known for less than 160 phosphoproteins. Here we describe, with the use of proteome chip technology, the in vitro substrates recognized by most yeast protein kinases: we identified over 4,000 phosphorylation events involving 1,325 different proteins. These substrates represent a broad spectrum of different biochemical functions and cellular roles. Distinct sets of substrates were recognized by each protein kinase, including closely related kinases of the protein kinase A family and four cyclin-dependent kinases that vary only in their cyclin subunits. Although many substrates reside in the same cellular compartment or belong to the same functional category as their phosphorylating kinase, many others do not, indicating possible new roles for several kinases. Furthermore, integration of the phosphorylation results with protein-protein interaction and transcription factor binding data revealed novel regulatory modules. Our phosphorylation results have been assembled into a first-generation phosphorylation map for yeast. Because many yeast proteins and pathways are conserved, these results will provide insights into the mechanisms and roles of protein phosphorylation in many eukaryotes.
923 citations