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

doi:10.1007/978-1-4939-7598-3_16

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)

Cell Signalling and Immunology

Research output: Chapter in Book/Report/Conference proceeding › Chapter (peer-reviewed) › peer-review

8 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 language	English
Title of host publication	AMPK
Subtitle of host publication	Methods and Protocols
Editors	Dietbert Neumann, Benoit Viollet
Place of Publication	New York
Publisher	Humana Press
Pages	239-253
Number of pages	15
ISBN (Electronic)	9781493975983
ISBN (Print)	9781493975976
DOIs	https://doi.org/10.1007/978-1-4939-7598-3_16
Publication status	Published - 2018

Publication series

Name	Methods in Molecular Biology
Publisher	Humana Press
Volume	1732
ISSN (Print)	1064-3745
ISSN (Electronic)	1940-6029

Keywords

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

ASJC Scopus subject areas

Molecular Biology
Genetics

Access to Document

10.1007/978-1-4939-7598-3_16

Role of AMPK in Nutrient Sensing and in Cancer (Investigator Award Renewal)
Hardie, G. (Investigator)
1/10/17 → 31/03/24
Project: Research
Rab Detection Initiative (Joint with Stanford School of Medicine, Max Plank Institute, Neuroscience Research Australia and Parkinsons Institute California)
Alessi, D. (Investigator) & Davies, P. (Investigator)
1/08/16 → 31/07/21
Project: Research
AMP-Activated Protein Kinase - A Tumour Suppressor that Opposes the Metabolic Changes in Proliferating Cells (Programme Grant)
Hardie, G. (Investigator)
1/01/13 → 30/09/18
Project: Research

Cite this

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

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

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.",

keywords = "Allosteric activation, AMP-activated protein kinase, AMPK, Dephosphorylation, Kinase assay, Phosphorylation",

author = "Hawley, {Simon A.} and Fyffe, {Fiona A.} and Russell, {Fiona M.} and Gowans, {Graeme J.} 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 the Cancer Research UK.",

year = "2018",

doi = "10.1007/978-1-4939-7598-3_16",

language = "English",

isbn = "9781493975976",

series = "Methods in Molecular Biology",

publisher = "Humana Press",

pages = "239--253",

editor = "Neumann, {Dietbert } and Viollet, {Benoit }",

booktitle = "AMPK",

address = "United States",

}

Intact Cell Assays to Monitor AMPK and Determine the Contribution of the AMP-Binding or ADaM Sites to Activation. / Hawley, Simon A.; Fyffe, Fiona A.; Russell, Fiona M. et al.
AMPK : Methods and Protocols. ed. / Dietbert Neumann; Benoit Viollet. New York: Humana Press, 2018. p. 239-253 (Methods in Molecular Biology; Vol. 1732).

Research output: Chapter in Book/Report/Conference proceeding › Chapter (peer-reviewed) › peer-review

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T1 - Intact Cell Assays to Monitor AMPK and Determine the Contribution of the AMP-Binding or ADaM Sites to Activation

AU - Hawley, Simon A.

AU - Fyffe, Fiona A.

AU - Russell, Fiona M.

AU - Gowans, Graeme J.

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 the Cancer Research UK.

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N2 - 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.

AB - 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.

KW - Allosteric activation

KW - AMP-activated protein kinase

KW - AMPK

KW - Dephosphorylation

KW - Kinase assay

KW - Phosphorylation

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SN - 9781493975976

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BT - AMPK

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PB - Humana Press

CY - New York

ER -

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

Abstract

Publication series

Keywords

ASJC Scopus subject areas

Access to Document

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Projects

Role of AMPK in Nutrient Sensing and in Cancer (Investigator Award Renewal)

Rab Detection Initiative (Joint with Stanford School of Medicine, Max Plank Institute, Neuroscience Research Australia and Parkinsons Institute California)

AMP-Activated Protein Kinase - A Tumour Suppressor that Opposes the Metabolic Changes in Proliferating Cells (Programme Grant)

Cite this