Abstract
AMP-activated protein kinase (AMPK) is extremely sensitive to cellular stress, so that nonphysiological activation of the kinase can readily occur during harvesting of cells or tissues. In this chapter we describe methods to harvest cells and tissues, and for kinase assays, that preserve the physiological activation status of AMPK as far as possible. Note that similar care with methods of cell or tissue harvesting is required when AMPK function is monitored by Western blotting, rather than by kinase assays. We also describe methods to determine whether compounds that activate AMPK in intact cells do so indirectly by interfering with cellular ATP synthesis or directly by binding to AMPK and, if the latter, whether this occurs by binding at the AMP-binding sites on the γ subunit or at the ADaM site located between the α and β subunits.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Li X, Wang L, Zhou XE, Ke J, de Waal PW, Gu X, Tan MH, Wang D, Wu D, Xu HE, Melcher K (2015) Structural basis of AMPK regulation by adenine nucleotides and glycogen. Cell Res 25(1):50–66. https://doi.org/10.1038/cr.2014.150
Xiao B, Sanders MJ, Carmena D, Bright NJ, Haire LF, Underwood E, Patel BR, Heath RB, Walker PA, Hallen S, Giordanetto F, Martin SR, Carling D, Gamblin SJ (2013) Structural basis of AMPK regulation by small molecule activators. Nat Commun 4:3017. https://doi.org/10.1038/ncomms4017
Hawley SA, Ross FA, Chevtzoff C, Green KA, Evans A, Fogarty S, Towler MC, Brown LJ, Ogunbayo OA, Evans AM, Hardie DG (2010) Use of cells expressing gamma subunit variants to identify diverse mechanisms of AMPK activation. Cell Metab 11(6):554–565. https://doi.org/10.1016/j.cmet.2010.04.001
Scott JW, Hawley SA, Green KA, Anis M, Stewart G, Scullion GA, Norman DG, Hardie DG (2004) CBS domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations. J Clin Invest 113(2):274–284
Jensen TE, Ross FA, Kleinert M, Sylow L, Knudsen JR, Gowans GJ, Hardie DG, Richter EA (2015) PT-1 selectively activates AMPK-gamma1 complexes in mouse skeletal muscle, but activates all three gamma subunit complexes in cultured human cells by inhibiting the respiratory chain. Biochem J 467(3):461–472. https://doi.org/10.1042/BJ20141142
Hawley SA, Fullerton MD, Ross FA, Schertzer JD, Chevtzoff C, Walker KJ, Peggie MW, Zibrova D, Green KA, Mustard KJ, Kemp BE, Sakamoto K, Steinberg GR, Hardie DG (2012) The ancient drug salicylate directly activates AMP-activated protein kinase. Science 336(6083):918–922. https://doi.org/10.1126/science.1215327
Ross FA, Hawley SA, Auciello FR, Gowans GJ, Atrih A, Lamont DJ, Hardie DG (2017) Mechanisms of paradoxical activation of AMP-activated protein kinase by the kinase inhibitors SU6656 and sorafenib. Cell Chem Biol 24(7):813-824. https://doi.org/10.1016/j.chembiol.2017.05.021
Gowans GJ, Hawley SA, Ross FA, Hardie DG (2013) AMP is a true physiological regulator of AMP-activated protein kinase by both allosteric activation and enhancing net phosphorylation. Cell Metab 18(4):556–566. https://doi.org/10.1016/j.cmet.2013.08.019
Easom RA, Zammit VA (1984) A cold-clamping technique for the rapid sampling of rat liver for studies on enzymes in separate cell fractions. Suitability for the study of enzymes regulated by reversible phosphorylation-dephosphorylation. Biochem J 220:733–738
Davies SP, Carling D, Munday MR, Hardie DG (1992) Diurnal rhythm of phosphorylation of rat liver acetyl-CoA carboxylase by the AMP-activated protein kinase, demonstrated using freeze-clamping. Effects of high fat diets. Eur J Biochem 203(3):615–623
Wojtaszewski JF, Nielsen P, Hansen BF, Richter EA, Kiens B (2000) Isoform-specific and exercise intensity-dependent activation of 5′-AMP-activated protein kinase in human skeletal muscle. J Physiol 528(Pt 1):221–226
Corton JM, Gillespie JG, Hardie DG (1994) Role of the AMP-activated protein kinase in the cellular stress response. Curr Biol 4(4):315–324
Ross FA, Jensen TE, Hardie DG (2016) Differential regulation by AMP and ADP of AMPK complexes containing different gamma subunit isoforms. Biochem J 473(2):189–199. https://doi.org/10.1042/BJ20150910
Acknowledgments
Studies in the Hardie Laboratory were supported by a Senior Investigator Award (097726) from the Wellcome Trust and by a Programme Grant (C37030/A15101) from the Cancer Research UK.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Hawley, S.A., Fyffe, F.A., Russell, F.M., Gowans, G.J., Grahame Hardie, D. (2018). Intact Cell Assays to Monitor AMPK and Determine the Contribution of the AMP-Binding or ADaM Sites to Activation. In: Neumann, D., Viollet, B. (eds) AMPK. Methods in Molecular Biology, vol 1732. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7598-3_16
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7598-3_16
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7597-6
Online ISBN: 978-1-4939-7598-3
eBook Packages: Springer Protocols