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Assaying Protein Kinase A Activity Using a FRET-Based Sensor Purified from Mammalian Cells

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cAMP Signaling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2483))

Abstract

Protein Kinase A (PKA) is the major intracellular receptor for cAMP. Research into this prototype kinase is supported by kinase assays that are typically performed in vitro using radio-labeled ATP. For in vivo studies, genetically encoded FRET-based sensors have become popular for monitoring PKA activity. Here, we show that it is also possible to apply such reporters in vitro. We describe how to express and purify milligram quantities of a FRET-based PKA activity reporter using cultured human embryonic kidney cells. We demonstrate how to utilize the purified reporter in a plate reader to determine the IC50 for the widely utilized PKA inhibitor H89 in the presence of a physiologically relevant concentration of ATP. The protocol takes advantage of the economical transfection reagent polyethylenimine and can be performed in a standard cell culture facility. Whereas assays based on radiolabelling are more sensitive, the approach presented here has several advantages: It enables continuous measurement of changes in substrate phosphorylation; a single preparation produces enough reporter for thousands of recordings; the reporter has a long shelf life; and it avoids the safety considerations that arise when working with radioactive material.

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References

  1. Bers DM, Xiang YK, Zaccolo M (2019) Whole-cell cAMP and PKA activity are epiphenomena, Nanodomain Signaling matters. Physiology (Bethesda) 34(4):240–249. https://doi.org/10.1152/physiol.00002.2019

    Article  CAS  Google Scholar 

  2. Nedvetsky PI, Tamma G, Beulshausen S, Valenti G, Rosenthal W, Klussmann E (2009) Regulation of aquaporin-2 trafficking. Handb Exp Pharmacol 190:133–157. https://doi.org/10.1007/978-3-540-79885-9_6

    Article  CAS  Google Scholar 

  3. Hell JW (2016) How Ca2+−permeable AMPA receptors, the kinase PKA, and the phosphatase PP2B are intertwined in synaptic LTP and LTD. Sci Signal 9(425):e2. https://doi.org/10.1126/scisignal.aaf7067

    Article  CAS  PubMed  Google Scholar 

  4. Walker C, Wang Y, Olivieri C, Karamafrooz A, Casby J, Bathon K, Calebiro D, Gao J, Bernlohr DA, Taylor SS, Veglia G (2019) Cushing's syndrome driver mutation disrupts protein kinase a allosteric network, altering both regulation and substrate specificity. Sci Adv 5(8):eaaw9298. https://doi.org/10.1126/sciadv.aaw9298

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Gold MG, Gonen T, Scott JD (2013) Local cAMP signaling in disease at a glance. J Cell Sci 126(Pt 20):4537–4543. https://doi.org/10.1242/jcs.133751

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Madhusudan TEA, Xuong NH, Adams JA, Ten Eyck LF, Taylor SS, Sowadski JM (1994) cAMP-dependent protein kinase: crystallographic insights into substrate recognition and phosphotransfer. Protein Sci 3(2):176–187. https://doi.org/10.1002/pro.5560030203

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Zhang P, Smith-Nguyen EV, Keshwani MM, Deal MS, Kornev AP, Taylor SS (2012) Structure and allostery of the PKA RIIbeta tetrameric holoenzyme. Science 335(6069):712–716. https://doi.org/10.1126/science.1213979

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Moore MJ, Adams JA, Taylor SS (2003) Structural basis for peptide binding in protein kinase a. role of glutamic acid 203 and tyrosine 204 in the peptide-positioning loop. J Biol Chem 278(12):10613–10618. https://doi.org/10.1074/jbc.M210807200

    Article  CAS  PubMed  Google Scholar 

  9. Knape MJ, Ahuja LG, Bertinetti D, Burghardt NC, Zimmermann B, Taylor SS, Herberg FW (2015) Divalent metal ions mg(2)(+) and ca(2)(+) have distinct effects on protein kinase a activity and regulation. ACS Chem Biol 10(10):2303–2315. https://doi.org/10.1021/acschembio.5b00271

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Gold MG, Reichow SL, O’Neill SE, Weisbrod CR, Langeberg LK, Bruce JE, Gonen T, Scott JD (2012) AKAP2 anchors PKA with aquaporin-0 to support ocular lens transparency. EMBO Mol Med 4(1):15–26. https://doi.org/10.1002/emmm.201100184

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Chijiwa T, Mishima A, Hagiwara M, Sano M, Hayashi K, Inoue T, Naito K, Toshioka T, Hidaka H (1990) Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. J Biol Chem 265(9):5267–5272

    Article  CAS  Google Scholar 

  12. Ma H, Deacon S, Horiuchi K (2008) The challenge of selecting protein kinase assays for lead discovery optimization. Expert Opin Drug Discov 3(6):607–621. https://doi.org/10.1517/17460441.3.6.607

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Zhang P, Knape MJ, Ahuja LG, Keshwani MM, King CC, Sastri M, Herberg FW, Taylor SS (2015) Single turnover autophosphorylation cycle of the PKA RIIbeta holoenzyme. PLoS Biol 13(7):e1002192. https://doi.org/10.1371/journal.pbio.1002192

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Hastie CJ, McLauchlan HJ, Cohen P (2006) Assay of protein kinases using radiolabeled ATP: a protocol. Nat Protoc 1(2):968–971. https://doi.org/10.1038/nprot.2006.149

    Article  CAS  PubMed  Google Scholar 

  15. Davies SP, Reddy H, Caivano M, Cohen P (2000) Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351(Pt 1):95–105. https://doi.org/10.1042/0264-6021:3510095

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Grigoriu S, Bond R, Cossio P, Chen JA, Ly N, Hummer G, Page R, Cyert MS, Peti W (2013) The molecular mechanism of substrate engagement and immunosuppressant inhibition of calcineurin. PLoS Biol 11(2):e1001492. https://doi.org/10.1371/journal.pbio.1001492

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Patel N, Stengel F, Aebersold R, Gold MG (2017) Molecular basis of AKAP79 regulation by calmodulin. Nat Commun 8(1):1681. https://doi.org/10.1038/s41467-017-01715-w

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Luzi NM, Lyons CE, Peterson DL, Ellis KC (2017) Characterization of PKACalpha enzyme kinetics and inhibition in an HPLC assay with a chromophoric substrate. Anal Biochem 532:45–52. https://doi.org/10.1016/j.ab.2017.06.001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Aricescu AR, Lu W, Jones EY (2006) A time- and cost-efficient system for high-level protein production in mammalian cells. Acta Crystallogr D Biol Crystallogr 62(Pt 10):1243–1250. https://doi.org/10.1107/S0907444906029799

    Article  CAS  PubMed  Google Scholar 

  20. Greenwald EC, Mehta S, Zhang J (2018) Genetically encoded fluorescent biosensors illuminate the spatiotemporal regulation of Signaling networks. Chem Rev 118(24):11707–11794. https://doi.org/10.1021/acs.chemrev.8b00333

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Patel N, Gold MG (2015) The genetically encoded tool set for investigating cAMP: more than the sum of its parts. Front Pharmacol 6:164. https://doi.org/10.3389/fphar.2015.00164

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Walker-Gray R, Stengel F, Gold MG (2017) Mechanisms for restraining cAMP-dependent protein kinase revealed by subunit quantitation and cross-linking approaches. Proc Natl Acad Sci U S A 114(39):10414–10419. https://doi.org/10.1073/pnas.1701782114

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Gribble FM, Loussouarn G, Tucker SJ, Zhao C, Nichols CG, Ashcroft FM (2000) A novel method for measurement of submembrane ATP concentration. J Biol Chem 275(39):30046–30049. https://doi.org/10.1074/jbc.M001010200

    Article  CAS  PubMed  Google Scholar 

  24. Zhang JF, Liu B, Hong I, Mo A, Roth RH, Tenner B, Lin W, Zhang JZ, Molina RS, Drobizhev M, Hughes TE, Tian L, Huganir RL, Mehta S, Zhang J (2020) An ultrasensitive biosensor for high-resolution kinase activity imaging in awake mice. Nat Chem Biol 17(1):39–46. https://doi.org/10.1038/s41589-020-00660-y

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Ma L, Jongbloets BC, Xiong WH, Melander JB, Qin M, Lameyer TJ, Harrison MF, Zemelman BV, Mao T, Zhong H (2018) A highly sensitive A-kinase activity reporter for imaging Neuromodulatory events in awake mice. Neuron 99(4):665–679. e665. https://doi.org/10.1016/j.neuron.2018.07.020

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Murray AJ (2008) Pharmacological PKA inhibition: all may not be what it seems. Sci Signal 1(22):re4. https://doi.org/10.1126/scisignal.122re4

    Article  PubMed  Google Scholar 

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Acknowledgements

This work was supported by a Wellcome Trust and Royal Society Sir Henry Dale fellowship to MGG (104194/Z/14/Z), a Wellcome Trust studentship to RSD, and a BBSRC studentship to AJC.

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Authors and Affiliations

  1. Department of Neuroscience, Physiology & Pharmacology, University College London, London, UK

    Ashton J. Curtis, Ryan S. Dowsell & Matthew G. Gold

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  1. Ashton J. Curtis
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  2. Ryan S. Dowsell
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  3. Matthew G. Gold
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Correspondence to Matthew G. Gold .

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Editors and Affiliations

  1. Department of Physiology, Anatomy and Genetics, Oxford University, Oxford, UK

    Manuela Zaccolo

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Curtis, A.J., Dowsell, R.S., Gold, M.G. (2022). Assaying Protein Kinase A Activity Using a FRET-Based Sensor Purified from Mammalian Cells. In: Zaccolo, M. (eds) cAMP Signaling. Methods in Molecular Biology, vol 2483. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2245-2_2

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  • DOI: https://doi.org/10.1007/978-1-0716-2245-2_2

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2244-5

  • Online ISBN: 978-1-0716-2245-2

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