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

Methods Mol Biol. 2018:1732:69-86. doi: 10.1007/978-1-4939-7598-3_5.

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.

Keywords: AMP-activated protein kinase; AMPK; Allosteric activation; Dephosphorylation; Kinase assay; Phosphorylation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • AMP-Activated Protein Kinases / chemistry
  • AMP-Activated Protein Kinases / metabolism*
  • Adenosine Diphosphate / chemistry
  • Adenosine Diphosphate / metabolism*
  • Adenosine Monophosphate / chemistry
  • Adenosine Monophosphate / metabolism*
  • Adenosine Triphosphate / chemistry
  • Adenosine Triphosphate / metabolism*
  • Allosteric Regulation
  • Binding Sites
  • Cell-Free System / metabolism
  • Enzyme Activation
  • Enzyme Assays / methods*
  • Isotope Labeling / methods
  • Ligands
  • Phosphorus Radioisotopes / chemistry
  • Phosphorylation
  • Protein Binding
  • Protein Subunits / chemistry

Substances

  • Ligands
  • Phosphorus Radioisotopes
  • Protein Subunits
  • Adenosine Monophosphate
  • Adenosine Diphosphate
  • Phosphorus-32
  • Adenosine Triphosphate
  • AMP-Activated Protein Kinases