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

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

1 Citation (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

Fingerprint

AMP-Activated Protein Kinases
Adenosine Triphosphate
Adenosine Monophosphate
Ligands
Adenosine Diphosphate
Phosphoprotein Phosphatases
Phosphotransferases
Phosphorylation

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). New York: Humana Press. https://doi.org/10.1007/978-1-4939-7598-3_5
Fyffe, Fiona A. ; Hawley, Simon A. ; Gray, Alexander ; Hardie, D. Grahame. / Cell-Free Assays to Measure Effects of Regulatory Ligands on AMPK. AMPK : Methods and Protocols. editor / Dietbert Neumann ; Benoit Viollet. New York : Humana Press, 2018. pp. 69-86 (Methods in Molecular Biology).
@inbook{0ed7945695524232b21ca60df4967a4a,
title = "Cell-Free Assays to Measure Effects of Regulatory Ligands on AMPK",
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 = "Allosteric activation, AMP-activated protein kinase, AMPK, Dephosphorylation, Kinase assay, Phosphorylation",
author = "Fyffe, {Fiona A.} and Hawley, {Simon A.} and Alexander Gray and Hardie, {D. Grahame}",
note = "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 Cancer Research UK.",
year = "2018",
doi = "10.1007/978-1-4939-7598-3_5",
language = "English",
isbn = "9781493975976",
series = "Methods in Molecular Biology",
publisher = "Humana Press",
pages = "69--86",
editor = "Neumann, {Dietbert } and Viollet, {Benoit }",
booktitle = "AMPK",

}

Fyffe, FA, Hawley, SA, Gray, A & Hardie, DG 2018, Cell-Free Assays to Measure Effects of Regulatory Ligands on AMPK. in D Neumann & B Viollet (eds), AMPK : Methods and Protocols. Methods in Molecular Biology, vol. 1732, Humana Press, New York, pp. 69-86. https://doi.org/10.1007/978-1-4939-7598-3_5

Cell-Free Assays to Measure Effects of Regulatory Ligands on AMPK. / Fyffe, Fiona A.; Hawley, Simon A.; Gray, Alexander; Hardie, D. Grahame (Lead / Corresponding author).

AMPK : Methods and Protocols. ed. / Dietbert Neumann; Benoit Viollet. New York : Humana Press, 2018. p. 69-86 (Methods in Molecular Biology; Vol. 1732).

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

TY - CHAP

T1 - Cell-Free Assays to Measure Effects of Regulatory Ligands on AMPK

AU - Fyffe, Fiona A.

AU - Hawley, Simon A.

AU - Gray, Alexander

AU - Hardie, D. Grahame

N1 - 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 Cancer Research UK.

PY - 2018

Y1 - 2018

N2 - 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.

AB - 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.

KW - Allosteric activation

KW - AMP-activated protein kinase

KW - AMPK

KW - Dephosphorylation

KW - Kinase assay

KW - Phosphorylation

UR - http://www.scopus.com/inward/record.url?scp=85042707613&partnerID=8YFLogxK

U2 - 10.1007/978-1-4939-7598-3_5

DO - 10.1007/978-1-4939-7598-3_5

M3 - Chapter (peer-reviewed)

SN - 9781493975976

T3 - Methods in Molecular Biology

SP - 69

EP - 86

BT - AMPK

A2 - Neumann, Dietbert

A2 - Viollet, Benoit

PB - Humana Press

CY - New York

ER -

Fyffe FA, Hawley SA, Gray A, Hardie DG. Cell-Free Assays to Measure Effects of Regulatory Ligands on AMPK. In Neumann D, Viollet B, editors, AMPK : Methods and Protocols. New York: Humana Press. 2018. p. 69-86. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-4939-7598-3_5