[Cell Cycle 7:14, 2097-2099; 15 July 2008]; ©2008 Landes Bioscience
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A wide-ranging cellular response to UV damage of DNA Matthew P. Stokes* and Michael J. Comb Cell Signaling Technology; Danvers, Massachusetts USA
Key words: DNA damage, checkpoint, mass spectrometry, phosphorylation, ATM, ATR, UV, DDR, SILAC
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signaling pathways underlying the DNA damage response continues to be an active area of research. Until recently, known substrates of ATM/R were limited to a short list defined through individual discoveries. Recent research utilizing mass spectrometry has led to the identification of large numbers of novel ATM/R substrates induced by DNA damage.17-19 Our study made use of immunoaffinity purification of phosphopeptides containing the consensus ATM/R substrate motif (SQ/TQ) coupled with liquid chromatography-tandem mass spectrometry to identify substrates phosphorylated in response to UV damage of DNA. This work was a successful extension of our PhosphoScan® technology previously used to identify tyrosine phosphorylated peptides.20 The findings of our study agree with and extend those of a similar report on ATM/R substrates phosphorylated in response to double strand breaks caused by gamma irradiation (IR).18 These studies underscore the importance of the DNA damage response and the variety of cellular processes affected by damage.
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The kinases ATM and ATR are central to proper function of the DNA damage response. These kinases phosphorylate proteins to coordinate cell cycle progression and DNA damage repair/bypass. We have recently reported a large-scale identification of ATM/ATR substrates phosphorylated in response to UV damage of DNA. Overall 231 sites of phosphorylation were induced by UV damage of DNA or dependent on proper function of ATR. The study expanded the number of phosphorylation sites from protein classes known to be involved in the DNA damage response. Further, many sites were identified from protein types not thought to have a role in damage signaling. This observation suggests that the DNA damage response affects a much wider range of cellular processes than was previously appreciated. This study has also extended the successful use of the PhosphoScan® proteomic method from phospho-tyrosine to serine/threonine motifs, providing a general blueprint to use the method to study signaling pathways underlying a wide range of diseases.
Introduction
Results and Discussion
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To ensure survival, cells have evolved numerous mechanisms to respond to genomic insult, collectively termed the DNA damage response (DDR).1 Central to the function of the DDR are the PI3 kinase-like kinases ATM and ATR (ATM/R).2 ATM is primarily activated by double strand breaks, while ATR is activated in response to a variety of DNA lesions and replication perturbations.3-5 ATM/R in turn phosphorylate a number of substrates known to inhibit cell cycle progression and coordinate DNA lesion repair/bypass activities, including Chk1, Chk2 and p53.1,6-10 ATM/R phosphorylate serine or threonine residues in the context of glutamine at the +1 position (the SQ/TQ motif ).11 The importance of proper function of ATM and ATR is highlighted by debilitating diseases caused by mutations of these kinases as in ataxia telangiectasia (ATM mutation) and Seckel cell syndrome (ATR mutation).12,13 Other components of the DNA damage response are also involved in disease, such as FANC proteins in Fanconi’s Anemia, BRCA1 and 2 in breast and ovarian cancer, and the high frequency of p53 mutation in many cancer types.14-16 With such importance in disease, more completely identifying the *Correspondence to: Matthew P. Stokes; 3 Trask Lane; Danvers, Massachusetts 01923; Email:
[email protected] Submitted: 05/19/08; Accepted: 05/21/08 Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/6326 www.landesbioscience.com
We used 2 SQ/TQ-directed antibodies to immunoprecipitate phosphopeptides from UV damaged cells. This resulted in the identification of over 500 SQ/TQ sites, nearly 90% of which were novel human sites. Beyond simply generating lists of SQ/TQ sites in UV damaged cells, we determined which of these phosphorylation events were actually induced by UV damage of DNA. We compared phosphopeptide levels in undamaged and UV damaged cells using a label-free quantitative method measuring chromatographic peak apex intensities. This analysis resulted in identification of 216 SQ/TQ sites that were at least 2-fold more highly phosphorylated in UV damaged cells than undamaged cells. Stable isotope labeling of amino acids in cell culture (SILAC) quantitative analysis and Western blotting/Immunoprecipitation-Western blotting were used to confirm differential phosphorylation of a number of these sites. Additionally, we investigated changes in phosphopeptide levels upon UV damage between ATR-wild type and ATR-deficient cell lines since UV damage is dealt with primarily by the ATR branch of the DNA damage checkpoint. This study resulted in 39 sites more highly phosphorylated in UV damaged ATR-wild type cells than UV damaged ATR-deficient cells. Together, 231 SQ/TQ sites were either induced by UV damage of DNA, dependent on the presence of wild type ATR, or both. This represents a significant increase in the number of known DNA damage-inducible SQ/TQ sites from the 93 previously found (from the PhosphoSite® bioinformatics resource, www.phosphosite.org).
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A wide-ranging cellular response to UV damage of DNA
Table 1 Summary data table of protein classes containing damage-inducible protein phosphorylation sites identified in our study (either more highly phosphorylated upon UV damage or ATR-dependent phosphorylation in response to UV damage) Protein class
Number of sites
Adaptor/scaffold
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Adhesion or extracellular matrix
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Apoptosis
4
Cell cycle regulation
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Chromatin, DNA-binding, -repair or -replication
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Inhibitor
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Phosphatase
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Protease
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Protein kinase, atypical
1
Protein kinase, Ser/Thr
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Receptor, channel, transporter or cell surface
7 15
Transcriptional regulator
36
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RNA processing
Translational regulator Ubiquitin conjugating system
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This increase is represented graphically using a proportional Venn diagram in Figure 1A. There were only 9 sites common to the two groups, meaning 222 of the regulated sites found are novel. Overall, 22 protein classes were represented in this dataset, listed in Table 1. There were increases in the number of sites in some expected protein classes, such as cell cycle proteins, chromatin/DNA binding, DNA repair, DNA replication proteins, and transcriptional regulators (Fig. 1B–D). There were also a number of protein classes not previously appreciated to be involved in ATM/R-mediated DNA damage signaling. These are highlighted in red in Table 1, and include such classes as cytoskeletal and nuclear envelope proteins. The large number of phosphorylation sites and diversity of protein classes phosphorylated in response to UV damage of DNA suggests that damage-dependent ATM/R signaling affects many more cellular processes than previously appreciated, a finding that is in agreement with the recent study by Matsuoka et al.18 What is the implication of ATM/R affecting so many more cellular pathways than previously known? It may be that some pathways are affected differentially by varying levels of damage. As currently understood, very low levels of DNA damage are unable to activate checkpoint signaling. Once the level of damage crosses some threshold the checkpoint is activated, inhibiting cell cycle machinery and activating repair and replication factors to remove or bypass the lesions. If the amount of damage is too great for the cell to effectively deal with, apoptotic mechanisms are activated and the cell essentially gives up the fight against the huge amount of genetic insult that has been incurred. The finding that so many cellular pathways are affected by damage suggests the possibility that somewhere between activation of ATM/ATR and programmed cell death other thresholds may exist for modulation of specific cellular processes. Increasing levels of DNA damage might differentially activate various branches of the DNA damage response. It will be of interest to determine if varying levels of DNA damage result in phosphorylation of all
G protein or regulator
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Figure 1. Proportional Venn diagrams showing the number of previously known DNA damage-inducible human SQ/TQ sites (blue circles) and novel substrates identified in our study (red circles). The number of sites for each protein class are shown, along with number of sites in common between the two datasets (yellow). (A) All sites identified in the study versus all sites previously known. (B–D) Phosphorylation sites identified in the study versus known sites for Cell Cycle (B), Chromatin, DNA-Binding, -Repair or -Replication Proteins (C), and Transcriptional Regulators (D).
Enzyme, misc.
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Cytoskeletal
Vesicle Unknown function TOTAL
2 10 2 38 231
The number of phosphorylation sites for each protein class is shown. Protein classes not previously known to be phosphorylated in response to UV damage of DNA are shown in red.
substrates identified, or if subsets are phosphorylated at different levels of damage. Successful extension of PhosphoScan® to different phosphorylation motifs and other post-translational modifications will facilitate the study of signaling in disease mechanisms (a list of all motif antibodies now available for PhosphoScan® analysis can be found at http://www.cellsignal.com/technologies/phosphoscan/motif_abs. html). Studies using our phosphotyrosine antibody have uncovered novel signaling pathways that drive cancer cell survival.21,22 Aberrant phosphorylation of serine and threonine in cancer can now also be studied using our library of motif antibodies with the following general strategy: First, cancerous cells can be compared to non-cancerous control cells, or cancerous cells can be compared before and after some inhibitor treatment using Western blotting, immunofluorescence, immunohistochemistry, etc., with the suite of motif antibodies available. This will allow selection of motif antibodies that show differences between cancerous and non-cancerous tissue or minus/plus treatment. The antibodies that give interesting results in the screen can then be used in PhosphoScan® to identify the changes in phosphorylation, and thus provide information as to the mechanisms underlying carcinogenesis and cancer progression.
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A wide-ranging cellular response to UV damage of DNA
Beyond cancer biology other disease mechanisms can be explored using this methodology. SQ/TQ antibodies for example can be used to study signaling in ATM/ATR driven diseases such as Seckel cell syndrome and ataxia telangiectasia. Ubiquitin-branch antibodies that recognize sites of ubiquitination can be used to study the potential role of ubiquitination pathways in neurodegenerative diseases.23 The role of phosphorylation in diabetes, heart disease, or any disease in which post-translational modifications are thought to play a role can be studied. The potential for discovery using immunoaffinity purification coupled with mass spectrometry is ever expanding and will continue to be an important method in the study of cancer biology and beyond.
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References 1. Zhou BB, Elledge SJ. The DNA damage response: putting checkpoints in perspective. Nature 2000; 408:433-9. 2. Abraham RT. PI3-kinase related kinases: ‘big’ players in stress-induced signaling pathways. DNA Repair (Amst) 2004; 3:883-7. 3. Tibbetts RS, Brumbaugh KM, Williams JM, Sarkaria JN, Cliby WA, Shieh SY, Taya Y, Prives C, Abraham RT. A role for ATR in the DNA damage-induced phosphorylation of p53. Genes Dev 1999; 13:152-7. 4. Kastan MB, Lim DS. The many substrates and functions of ATM. Nat Rev Mol Cell Biol 2000; 1:179-86. 5. Cliby WA, Roberts CJ, Cimprich KA, Stringer CM, Lamb JR, Schreiber SL, Friend SH. Overexpression of a kinase-inactive ATR protein causes sensitivity to DNA-damaging agents and defects in cell cycle checkpoints. Embo J 1998; 17:159-69. 6. Zhao H, Piwnica-Worms H. ATR-mediated checkpoint pathways regulate phosphorylation and activation of human Chk1. Mol Cell Biol 2001; 21:4129-39. 7. Sanchez Y, Wong C, Thoma RS, Richman R, Wu Z, Piwnica-Worms H, Elledge SJ. Conservation of the Chk1 checkpoint pathway in mammals: linkage of DNA damage to Cdk regulation through Cdc25. Science 1997; 277:1497-501. 8. Matsuoka S, Rotman G, Ogawa A, Shiloh Y, Tamai K, Elledge SJ. Ataxia telangiectasiamutated phosphorylates Chk2 in vivo and in vitro. Proc Natl Acad Sci USA 2000; 97:10389-94. 9. Mailand N, Falck J, Lukas C, Syljuasen RG, Welcker M, Bartek J, Lukas J. Rapid destruction of human Cdc25A in response to DNA damage. Science 2000; 288:1425-9. 10. Traven A, Heierhorst J. SQ/TQ cluster domains: concentrated ATM/ATR kinase phosphorylation site regions in DNA-damage-response proteins. Bioessays 2005; 27:397-407. 11. Kim ST, Lim DS, Canman CE, Kastan MB. Substrate specificities and identification of putative substrates of ATM kinase family members. J Biol Chem 1999; 274:37538-43. 12. Lavin MF, Shiloh Y. The genetic defect in ataxia-telangiectasia. Annu Rev Immunol 1997; 15:177-202. 13. O’Driscoll M, Ruiz-Perez VL, Woods CG, Jeggo PA, Goodship JA. A splicing mutation affecting expression of ataxia-telangiectasia and Rad3-related protein (ATR) results in Seckel syndrome. Nat Genet 2003; 33:497-501. 14. Ahmad SI, Hanaoka F, Kirk SH. Molecular biology of Fanconi anaemia—an old problem, a new insight. Bioessays 2002; 24:439-48. 15. Scully R. Role of BRCA gene dysfunction in breast and ovarian cancer predisposition. Breast Cancer Res 2000; 2:324-30. 16. Levine AJ. p53, the cellular gatekeeper for growth and division. Cell 1997; 88:323-31. 17. Stokes MP, Rush J, Macneill J, Ren JM, Sprott K, Nardone J, Yang V, Beausoleil SA, Gygi SP, Livingstone M, Zhang H, Polakiewicz RD, Comb MJ. Profiling of UV-induced ATM/ATR signaling pathways. Proc Natl Acad Sci USA 2007; 104:19855-60. 18. Matsuoka S, Ballif BA, Smogorzewska A, McDonald ER, 3rd, Hurov KE, Luo J, Bakalarski CE, Zhao Z, Solimini N, Lerenthal Y, Shiloh Y, Gygi SP, Elledge SJ. ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage. Science 2007; 316:1160-6. 19. Smolka MB, Albuquerque CP, Chen SH, Zhou H. Proteome-wide identification of in vivo targets of DNA damage checkpoint kinases. Proc Natl Acad Sci USA 2007; 104:10364-9. 20. Rush J, Moritz A, Lee KA, Guo A, Goss VL, Spek EJ, Zhang H, Zha XM, Polakiewicz RD, Comb MJ. Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. Nat Biotechnol 2005; 23:94-101. 21. Guo A, Villen J, Kornhauser J, Lee KA, Stokes MP, Rikova K, Possemato A, Nardone J, Innocenti G, Wetzel R, Wang Y, MacNeill J, Mitchell J, Gygi SP, Rush J, Polakiewicz RD, Comb MJ. Signaling networks assembled by oncogenic EGFR and c-Met. Proc Natl Acad Sci USA 2008; 105:692-7. 22. Rikova K, Guo A, Zeng Q, Possemato A, Yu J, Haack H, Nardone J, Lee K, Reeves C, Li Y, Hu Y, Tan Z, Stokes M, Sullivan L, Mitchell J, Wetzel R, Macneill J, Ren JM, Yuan J, Bakalarski CE, Villen J, Kornhauser JM, Smith B, Li D, Zhou X, Gygi SP, Gu TL, Polakiewicz RD, Rush J, Comb MJ. Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. Cell 2007; 131:1190-203. 23. Ciechanover A, Schwartz AL. The ubiquitin system: pathogenesis of human diseases and drug targeting. Biochim Biophys Acta 2004; 1695:3-17.
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