Differential regulation by AMP and ADP of AMPK complexes containing different γ subunit isoforms

Fiona A. Ross, Thomas E. Jensen, D. Grahame Hardie (Lead / Corresponding author)

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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 γ 1, γ2 or γ 3 isoform. When assayed at a physiological ATP concentration (5 mM), γ 1- and γ 2-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, γ 3 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 Thr172 phosphorylation, when cells were incubatedwith mitochondrial inhibitors that increase cellular AMP. With γ 1 complexes, activation and Thr172 phosphorylation induced by the upstream kinase LKB1 [liver kinase B1; but not calmodulin-dependent kinase kinase (CaMKKβ)] in cellfree 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 γ 3 complexes were small or insignificant. Binding of AMP or ADP protected all three γ subunit complexes against inactivation by Thr172 dephosphorylation; with γ 2 complexes, ADP had similar potency to AMP, but with γ1 and γ 3 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.

Original languageEnglish
Pages (from-to)189-199
Number of pages11
JournalBiochemical Journal
Volume473
Issue number2
Early online date5 Jan 2016
DOIs
Publication statusPublished - 15 Jan 2016

Keywords

  • Adenine nucleotides
  • Adenosine 5′-diphosphate (ADP)
  • Adenosine 5′-phosphate (AMP)
  • Adenosine 5′-phosphate (AMP)-activated protein kinase (AMPK)
  • Adenosine 5′-triphosphate (ATP)

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