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Quantitative and Dynamic Imaging of ATM Kinase Activity by Bioluminescence Imaging

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ATM Kinase

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1599))

Abstract

Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA damage response, including DNA double strand breaks (DSBs). ATM activation results in the initiation of a complex cascade of events facilitating DNA damage repair, cell cycle checkpoint control, and survival. Traditionally, protein kinases have been analyzed in vitro using biochemical methods (kinase assays using purified proteins or immunological assays) requiring a large number of cells and cell lysis. Genetically encoded biosensors based on optical molecular imaging such as fluorescence or bioluminescence have been developed to enable interrogation of kinase activities in live cells with a high signal to background. We have genetically engineered a hybrid protein whose bioluminescent activity is dependent on the ATM-mediated phosphorylation of a substrate. The engineered protein consists of the split luciferase-based protein complementation pair with a CHK2 (a substrate for ATM kinase activity) target sequence and a phospho-serine/threonine-binding domain, FHA2, derived from yeast Rad53. Phosphorylation of the serine residue within the target sequence by ATM would lead to its interaction with the phospho-serine-binding domain, thereby preventing complementation of the split luciferase pair and loss of reporter activity. Bioluminescence imaging of reporter-expressing cells in cultured plates or as mouse xenografts provides a quantitative surrogate for ATM kinase activity and therefore the cellular DNA damage response in a noninvasive, dynamic fashion.

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References

  1. Bhojani MS, Nyati S, Rao HR, Rehemtulla A (2010) Molecular imaging in lung cancer metastases. In: Lung cancer metastasis. Springer, New York, pp 267–287

    Google Scholar 

  2. Khan AP, Contessa JN, Nyati MK, Ross BD, Rehemtulla A (2011) Molecular imaging of epidermal growth factor receptor kinase activity. Anal Biochem 417(1):57–64. doi:10.1016/j.ab.2011.05.040

    Article  CAS  PubMed  Google Scholar 

  3. Khan AP, Schinske KA, Nyati S, Bhojani MS, Ross BD, Rehemtulla A (2010) High-throughput molecular imaging for the identification of FADD kinase inhibitors. J Biomol Screen 15(9):1063–1070

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Nyati S, Ranga R, Ross BD, Rehemtulla A, Bhojani MS (2010) Molecular imaging of glycogen synthase kinase-3β and casein kinase-1α kinases. Anal Biochem 405(2):246–254

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Nyati S, Ross BD, Rehemtulla A, Bhojani MS (2010) Novel molecular imaging platform for monitoring oncological kinases. Cancer Cell Int 10:23. doi:10.1186/1475-2867-10-23

    Article  PubMed  PubMed Central  Google Scholar 

  6. Nyati S, Schinske K, Ray D, Nyati M, Ross BD, Rehemtulla A (2011) Molecular imaging of TGF beta-Induced smad2/3 phosphorylation reveals a role for receptor tyrosine kinases in modulating TGF beta signaling. Clin Cancer Res 17(23):7424–7439. doi:10.1158/1078-0432.Ccr-11-1248

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Williams TM, Nyati S, Ross BD, Rehemtulla A (2013) Molecular imaging of the ATM kinase activity. Int J Radiat Oncol Biol Phys 86(5): 969–977. doi:10.1016/j.ijrobp.2013.04.028. S0360-3016(13)00457-4 [pii]

  8. Zhang L, Bhojani MS, Ross BD, Rehemtulla A (2008) Molecular imaging of protein kinases. Cell Cycle 7(3):314–317

    Article  CAS  PubMed  Google Scholar 

  9. Zhang L, Lee KC, Bhojani MS, Khan AP, Shilman A, Holland EC, Ross BD, Rehemtulla A (2007) Molecular imaging of Akt kinase activity. Nat Med 13(9):1114–1119

    Article  CAS  PubMed  Google Scholar 

  10. Zhang L, Virani S, Zhang Y, Bhojani MS, Burgess TL, Coxon A, Galban CJ, Ross BD, Rehemtulla A (2011) Molecular imaging of c-Met tyrosine kinase activity. Anal Biochem 412(1):1–8. doi:10.1016/j.ab.2011.01.028

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Johnson SA, You Z, Hunter T (2007) Monitoring ATM kinase activity in living cells. DNA Repair 6(9):1277–1284. doi:10.1016/j.dnarep.2007.02.025

    Article  CAS  PubMed  Google Scholar 

  12. Nyati S, Schinske-Sebolt K, Pitchiaya S, Chekhovskiy K, Chator A, Chaudhry N, Dosch J, Van Dort ME, Varambally S, Kumar-Sinha C, Nyati MK, Ray D, Walter NG, Yu H, Ross BD, Rehemtulla A (2015) The kinase activity of the Ser/Thr kinase BUB1 promotes TGF-beta signaling. Sci Signal 8(358):ra1. doi:10.1126/scisignal.2005379

    Article  PubMed  PubMed Central  Google Scholar 

  13. Schinske KA, Nyati S, Khan AP, Williams TM, Johnson TD, Ross BD, Tomás RP, Rehemtulla A (2011) A novel kinase inhibitor of FADD phosphorylation chemosensitizes through the inhibition of NF-κB. Mol Cancer Ther 10(10):1807–1817

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. McCaffrey A, Kay MA, Contag CH (2003) Advancing molecular therapies through in vivo bioluminescent imaging. Mol Imaging 2(2):75–86

    Article  CAS  PubMed  Google Scholar 

  15. Contag CH, Bachmann MH (2002) Advances in in vivo bioluminescence imaging of gene expression. Annu Rev Biomed Eng 4:235–260

    Article  CAS  PubMed  Google Scholar 

  16. Choy G, Choyke P, Libutti SK (2003) Current advances in molecular imaging: noninvasive in vivo bioluminescent and fluorescent optical imaging in cancer research. Mol Imaging 2(4):303–312

    Article  CAS  PubMed  Google Scholar 

  17. Greer LF 3rd, Szalay AA (2002) Imaging of light emission from the expression of luciferases in living cells and organisms: a review. Luminescence 17(1):43–74

    Article  CAS  PubMed  Google Scholar 

  18. Stacer AC, Nyati S, Moudgil P, Iyengar R, Luker KE, Rehemtulla A, Luker GD (2013) NanoLuc reporter for dual luciferase imaging in living animals. Mol Imaging 12(7):1–13

    PubMed  PubMed Central  Google Scholar 

  19. Luker KE, Smith MC, Luker GD, Gammon ST, Piwnica-Worms H, Piwnica-Worms D (2004) Kinetics of regulated protein-protein interactions revealed with firefly luciferase complementation imaging in cells and living animals. Proc Natl Acad Sci U S A 101(33):12288–12293

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Bakkenist CJ, Kastan MB (2003) DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 421(6922):499–506. doi:10.1038/nature01368

    Article  CAS  PubMed  Google Scholar 

  21. Bensimon A, Schmidt A, Ziv Y, Elkon R, Wang SY, Chen DJ, Aebersold R, Shiloh Y (2010) ATM-dependent and -independent dynamics of the nuclear phosphoproteome after DNA damage. Sci Signal 3(151):rs3. doi:10.1126/scisignal.2001034

  22. Bhatti S, Kozlov S, Farooqi AA, Naqi A, Lavin M, Khanna KK (2011) ATM protein kinase: the linchpin of cellular defenses to stress. Cell Mol Life Sci 68(18):2977–3006. doi:10.1007/s00018-011-0683-9

    Article  CAS  PubMed  Google Scholar 

  23. Choi S, Srivas R, Fu KY, Hood BL, Dost B, Gibson GA, Watkins SC, Van Houten B, Bandeira N, Conrads TP, Ideker T, Bakkenist CJ (2012) Quantitative proteomics reveal ATM kinase-dependent exchange in DNA damage response complexes. J Proteome Res 11(10):4983–4991. doi:10.1021/pr3005524

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Lavin MF, Kozlov S (2007) ATM activation and DNA damage response. Cell Cycle 6(8):931–942

    Article  CAS  PubMed  Google Scholar 

  25. Mu JJ, Wang Y, Luo H, Leng M, Zhang J, Yang T, Besusso D, Jung SY, Qin J (2007) A proteomic analysis of ataxia telangiectasia-mutated (ATM)/ATM-Rad3-related (ATR) substrates identifies the ubiquitin-proteasome system as a regulator for DNA damage checkpoints. J Biol Chem 282(24):17330–17334. doi:10.1074/jbc.C700079200

    Article  CAS  PubMed  Google Scholar 

  26. Kim ST, Lim DS, Canman CE, Kastan MB (1999) Substrate specificities and identification of putative substrates of ATM kinase family members. J Biol Chem 274(53):37538–37543

    Article  CAS  PubMed  Google Scholar 

  27. Sancar A, Lindsey-Boltz LA, Kang TH, Reardon JT, Lee JH, Ozturk N (2010) Circadian clock control of the cellular response to DNA damage. FEBS Lett 584(12):2618–2625. doi:10.1016/j.febslet.2010.03.017

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Stracker TH, Roig I, Knobel PA, Marjanovic M (2013) The ATM signaling network in development and disease. Front Genet 4:37. doi:10.3389/fgene.2013.00037

    Article  PubMed  PubMed Central  Google Scholar 

  29. Weber AM, Ryan AJ (2015) ATM and ATR as therapeutic targets in cancer. Pharmacol Ther 149:124–138. doi:10.1016/j.pharmthera.2014.12.001

    Article  CAS  PubMed  Google Scholar 

  30. Shiotani B, Zou L (2009) Single-stranded DNA orchestrates an ATM-to-ATR switch at DNA breaks. Mol Cell 33(5):547–558. doi:10.1016/j.molcel.2009.01.024

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Kozlov S, Gueven N, Keating K, Ramsay J, Lavin MF (2003) ATP activates ataxia-telangiectasia mutated (ATM) in vitro. Importance of autophosphorylation. J Biol Chem 278(11):9309–9317

    Article  CAS  PubMed  Google Scholar 

  32. Williams TM, Nyati S, Ross BD, Rehemtulla A (2013) Molecular imaging of the ATM kinase activity. Int J Radiat Oncol Biol Phys 86(5):969–977. doi:10.1016/j.ijrobp.2013.04.028

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Durocher D, Jackson SP (2002) The FHA domain. FEBS Lett 513(1):58–66

    Article  CAS  PubMed  Google Scholar 

  34. Filippakopoulos P, Muller S, Knapp S (2009) SH2 domains: modulators of nonreceptor tyrosine kinase activity. Curr Opin Struct Biol 19(6):643–649. doi:10.1016/j.sbi.2009.10.001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Schlessinger J (1994) SH2/SH3 signaling proteins. Curr Opin Genet Dev 4(1):25–30

    Article  CAS  PubMed  Google Scholar 

  36. Frosina G (2009) DNA repair and resistance of gliomas to chemotherapy and radiotherapy. Mol Cancer Res 7(7):989–999. doi:10.1158/1541-7786.MCR-09-0030

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

We would like to acknowledge the members of the Center for Molecular Imaging at the University of Michigan for their valuable input and support for the studies. This work was supported by the National Institutes of Health grant R01CA193690 (AR), P01CA087634 (BDR, AR and SN), as well as a P30CA046592 award to the University of Michigan Cancer Center.

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Authors and Affiliations

  1. Center for Molecular Imaging, University of Michigan, Ann Arbor, MI, 48109, USA

    Shyam Nyati, Brian Dale Ross & Alnawaz Rehemtulla

  2. Department of Radiation Oncology, University of Michigan, 109 Zina Pitcher place, AAT-BSRB, Level A, Room # 628, Ann Arbor, MI, 48109 2200, USA

    Shyam Nyati

  3. Department of Radiation Oncology, University of Michigan, Ann Arbor, MI, 48109, USA

    Grant Young & Alnawaz Rehemtulla

  4. Department of Radiology, University of Michigan, Ann Arbor, MI, 48109, USA

    Brian Dale Ross

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  1. Shyam Nyati
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  2. Grant Young
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  4. Alnawaz Rehemtulla
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Corresponding author

Correspondence to Shyam Nyati .

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Editors and Affiliations

  1. University of Queensland Centre for Clinical Research (UQCCR), University of Queensland, Herston, Queensland, Australia

    Sergei V. Kozlov

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Nyati, S., Young, G., Ross, B.D., Rehemtulla, A. (2017). Quantitative and Dynamic Imaging of ATM Kinase Activity by Bioluminescence Imaging. In: Kozlov, S. (eds) ATM Kinase. Methods in Molecular Biology, vol 1599. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6955-5_8

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  • DOI: https://doi.org/10.1007/978-1-4939-6955-5_8

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6953-1

  • Online ISBN: 978-1-4939-6955-5

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