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

Thomas E. Jensen (Lead / Corresponding author), Fiona A. Ross, Maximilian Kleinert, Lykke Sylow, Jonas R. Knudsen, Graeme J. Gowans, D. Grahame Hardie, Erik A. Richter

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Abstract

AMP-activated protein kinase (AMPK) occurs as heterotrimeric complexes in which a catalytic subunit (α 1/α 2) is bound to one of two β subunits (β 1/β 2) and one of three γ subunits (γ 1/γ 2/γ 3). 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 γ 1- but not γ 3-containing complexes in incubated mouse muscle. PT-1 increased the AMPK-dependent phosphorylation of the autophagy-regulating kinase ULK1 (unc-51-like autophagyactivating kinase 1) on Ser<sup>555</sup> , but not proposed AMPK-γ 3 substrates such as Ser<sup>231</sup> on TBC1 (tre-2/USP6, BUB2, cdc16) domain family, member 1 (TBC1D1) or Ser<sup>212</sup> on acetyl-CoA carboxylase subunit 2 (ACC2), nor did it stimulate glucose transport. Surprisingly, however, in human embryonic kidney (HEK) 293 cells expressing human γ 1, γ 2 or γ 3, PT-1 activated all three complexes equally.Wewere 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-γ 1. We propose that the failure of PT-1 to activate γ 3-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 γ 3 complexes are located.

Original languageEnglish
Pages (from-to)461-472
Number of pages12
JournalBiochemical Journal
Volume467
Issue number3
DOIs
Publication statusPublished - May 2015

Keywords

  • Acetyl-CoA carboxylase subunit 2 (ACC2)
  • AMP-activated protein kinase (AMPK)
  • Glucose
  • Skeletal muscle
  • TBC1D1
  • Unc-51-like autophagy-activating kinase 1 (ULK1)

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