title: PT-1 selectively activates AMPK-γ1 complexes in mouse skeletal muscle, but activates all three γ subunit complexes in cultured human cells by inhibiting the respiratory chain.
abstract: AMP-activated protein kinase (AMPK) occurs as heterotrimeric complexes in which a catalytic subunit (α1/α2) is bound to one of two β subunits (β1/β2) and one of three γ subunits (γ1/γ2/γ3). The ability to selectively activate specific isoforms would be a useful research tool and a promising strategy to combat diseases such as cancer and Type 2 diabetes. We report that the AMPK activator PT-1 selectively increased the activity of γ1- but not γ3-containing complexes in incubated mouse muscle. PT-1 increased the AMPK-dependent phosphorylation of the autophagy-regulating kinase ULK1 (unc-51-like autophagy-activating kinase 1) on Ser555, but not proposed AMPK-γ3 substrates such as Ser231 on TBC1 (tre-2/USP6, BUB2, cdc16) domain family, member 1 (TBC1D1) or Ser212 on acetyl-CoA carboxylase subunit 2 (ACC2), nor did it stimulate glucose transport. Surprisingly, however, in human embryonic kidney (HEK) 293 cells expressing human γ1, γ2 or γ3, PT-1 activated all three complexes equally. We were unable to reproduce previous findings suggesting that PT-1 activates AMPK by direct binding between the kinase and auto-inhibitory domains (AIDs) of the α subunit. We show instead that PT-1 activates AMPK indirectly by inhibiting the respiratory chain and increasing cellular AMP:ATP and/or ADP:ATP ratios. Consistent with this mechanism, PT-1 failed to activate AMPK in HEK293 cells expressing an AMP-insensitive R299G mutant of AMPK-γ1. We propose that the failure of PT-1 to activate γ3-containing complexes in muscle is not an intrinsic feature of such complexes, but is because PT-1 does not increase cellular AMP:ATP ratios in the specific subcellular compartment(s) in which γ3 complexes are located.
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, Dow Street, 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: Maximilian
authors.initials: M
authors.lastname: Kleinert
authors.affiliation: *Department of Nutrition, Exercise and Sports, University of Copenhagen, Universitetsparken 13, 2100 Copenhagen, Denmark.
authors.forename: Lykke
authors.initials: L
authors.lastname: Sylow
authors.affiliation: *Department of Nutrition, Exercise and Sports, University of Copenhagen, Universitetsparken 13, 2100 Copenhagen, Denmark.
authors.forename: Jonas R
authors.initials: JR
authors.lastname: Knudsen
authors.affiliation: †Division of Cell Signalling & Immunology, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
authors.forename: Graeme J
authors.initials: GJ
authors.lastname: Gowans
authors.affiliation: †Division of Cell Signalling & Immunology, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
authors.forename: D Grahame
authors.initials: DG
authors.lastname: Hardie
authors.affiliation: *Department of Nutrition, Exercise and Sports, University of Copenhagen, Universitetsparken 13, 2100 Copenhagen, Denmark.
authors.forename: Erik A
authors.initials: EA
authors.lastname: Richter
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D000103:Acetyl-CoA Carboxylase / Q000737:chemistry / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D004579:Electron Transport / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D005947:Glucose / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000378:metabolism; D018482:Muscle, Skeletal / Q000187:drug effects* / Q000378:metabolism*; D010766:Phosphorylation; D054730:Protein Interaction Domains and Motifs; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012265:Ribonucleotides / Q000494:pharmacology
chemical_list: D009097:Multienzyme Complexes; C526224:Prkag1 protein, mouse; D021122:Protein Subunits; D011994:Recombinant Proteins; D012265:Ribonucleotides; D000620:Aminoimidazole Carboxamide; D000249:Adenosine Monophosphate; C526222:PRKAG1 protein, human; C526223:PRKAG2 protein, human; C526225:PRKAG3 protein, human; D055372:AMP-Activated Protein Kinases; C498191:Acacb protein, mouse; D000103:Acetyl-CoA Carboxylase; C031143:AICA ribonucleotide; D005947:Glucose