Intact Cell Assays to Monitor AMPK and Determine the Contribution of the AMP-Binding or ADaM Sites to Activation

Simon A. Hawley, Fiona A. Fyffe, Fiona M. Russell, Graeme J. Gowans, D. Grahame Hardie (Lead / Corresponding author)

Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)

2 Citations (Scopus)

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.

Original languageEnglish
Title of host publicationAMPK
Subtitle of host publicationMethods and Protocols
EditorsDietbert Neumann, Benoit Viollet
Place of PublicationNew York
PublisherHumana Press
Pages239-253
Number of pages15
ISBN (Electronic)9781493975983
ISBN (Print)9781493975976
DOIs
Publication statusPublished - 2018

Publication series

NameMethods in Molecular Biology
PublisherHumana Press
Volume1732
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Allosteric activation
  • AMP-activated protein kinase
  • AMPK
  • Dephosphorylation
  • Kinase assay
  • Phosphorylation

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    Hawley, S. A., Fyffe, F. A., Russell, F. M., Gowans, G. J., & Hardie, D. G. (2018). Intact Cell Assays to Monitor AMPK and Determine the Contribution of the AMP-Binding or ADaM Sites to Activation. In D. Neumann, & B. Viollet (Eds.), AMPK : Methods and Protocols (pp. 239-253). (Methods in Molecular Biology; Vol. 1732). Humana Press. https://doi.org/10.1007/978-1-4939-7598-3_16