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- Title
PT-1 selectively activates AMPK-γ 1 complexes in mouse skeletal muscle, but activates all three γ subunit complexes in cultured human cells by inhibiting the respiratory chain.
- Authors
Jensen, Thomas E.; Ross, Fiona A.; Kleinert, Maximilian; Sylow, Lykke; Knudsen, Jonas R.; Gowans, Graeme J.; Hardie, D. Grahame; Richter, Erik A.
- 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 (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 Ser555, but not proposed AMPK-γ 3 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 γ 1, γ2 or γ 3, 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-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.
- Subjects
PROTEIN kinases; SKELETAL muscle; CELL culture; LABORATORY mice; RESPIRATION
- Publication
Biochemical Journal, 2015, Vol 467, Issue 3, p461
- ISSN
0264-6021
- Publication type
Article
- DOI
10.1042/BJ20141142