Cell-Free Assays to Measure Effects of Regulatory Ligands on AMPK

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

3 Citations (Scopus)

Abstract

AMP-activated protein kinase (AMPK) is an energy sensor that is activated by increases in the cellular AMP/ATP and ADP/ATP ratios by three mechanisms: (1) allosteric activation, (2) promotion of phosphorylation at Thr172 on the α subunit by upstream kinases, and (3) inhibition of dephosphorylation of Thr172 by protein phosphatases. All of these effects are triggered by the binding of AMP or ADP at one or more of three sites on the γ subunit, where they displace ATP. AMPK is also activated by ligands that bind in the ADaM site, which is located between the α and β subunits. In this chapter we describe cell-free assays that can be used to study these varied activation mechanisms.

Original languageEnglish
Title of host publicationAMPK
Subtitle of host publicationMethods and Protocols
EditorsDietbert Neumann, Benoit Viollet
Place of PublicationNew York
PublisherHumana Press
Pages69-86
Number of pages18
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

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Keywords

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

Cite this

Fyffe, F. A., Hawley, S. A., Gray, A., & Hardie, D. G. (2018). Cell-Free Assays to Measure Effects of Regulatory Ligands on AMPK. In D. Neumann, & B. Viollet (Eds.), AMPK : Methods and Protocols (pp. 69-86). (Methods in Molecular Biology; Vol. 1732). Humana Press. https://doi.org/10.1007/978-1-4939-7598-3_5