title: Differential regulation by AMP and ADP of AMPK complexes containing different γ subunit isoforms.
abstract: The γ subunits of heterotrimeric AMPK complexes contain the binding sites for the regulatory adenine nucleotides AMP, ADP and ATP. We addressed whether complexes containing different γ isoforms display different responses to adenine nucleotides by generating cells stably expressing FLAG-tagged versions of the γ1, γ2 or γ3 isoform. When assayed at a physiological ATP concentration (5 mM), γ1- and γ2-containing complexes were allosterically activated almost 10-fold by AMP, with EC50 values one to two orders of magnitude lower than the ATP concentration. By contrast, γ3 complexes were barely activated by AMP under these conditions, although we did observe some activation at lower ATP concentrations. Despite this, all three complexes were activated, due to increased Thr(172) phosphorylation, when cells were incubated with mitochondrial inhibitors that increase cellular AMP. With γ1 complexes, activation and Thr(172) phosphorylation induced by the upstream kinase LKB1 [liver kinase B1; but not calmodulin-dependent kinase kinase (CaMKKβ)] in cell-free assays was markedly promoted by AMP and, to a smaller extent and less potently, by ADP. However, effects of AMP or ADP on activation and phosphorylation of the γ2 and γ3 complexes were small or insignificant. Binding of AMP or ADP protected all three γ subunit complexes against inactivation by Thr(172) dephosphorylation; with γ2 complexes, ADP had similar potency to AMP, but with γ1 and γ3 complexes, ADP was less potent than AMP. Thus, AMPK complexes containing different γ subunit isoforms respond differently to changes in AMP, ADP or ATP. These differences may tune the responses of the isoforms to fit their differing physiological roles.
authors.affiliation: Division of Cell Signalling & Immunology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, U.K.
authors.forename: Fiona A
authors.initials: FA
authors.lastname: Ross
authors.affiliation: Department of Nutrition, Exercise and Sports, University of Copenhagen, Universitetsparken 13, 2100 Copenhagen, Denmark.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Jensen
authors.affiliation: Division of Cell Signalling & Immunology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, U.K. d.g.hardie@dundee.ac.uk.
authors.forename: D Grahame
authors.initials: DG
authors.lastname: Hardie
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism* / Q000502:physiology; D000244:Adenosine Diphosphate / Q000494:pharmacology / Q000502:physiology*; D000249:Adenosine Monophosphate / Q000494:pharmacology / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D057809:HEK293 Cells; D006801:Humans; D020033:Protein Isoforms / Q000502:physiology; D021122:Protein Subunits / Q000502:physiology
chemical_list: D020033:Protein Isoforms; D021122:Protein Subunits; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; C526222:PRKAG1 protein, human; C526223:PRKAG2 protein, human; C526225:PRKAG3 protein, human; D055372:AMP-Activated Protein Kinases
keywords: adenine nucleotides; adenosine 5′-diphosphate (ADP); adenosine 5′-phosphate (AMP); adenosine 5′-phosphate (AMP)-activated protein kinase (AMPK); adenosine 5′-triphosphate (ATP)