Abstract
AMP-activated protein kinase (AMPK) inhibits several anabolic pathways such as fatty acid and protein synthesis, and identification of AMPK substrate specificity would be useful to understand its role in particular cellular processes and develop strategies to modulate AMPK activity in a substrate-specific manner. Here we show that SUMOylation of AMPKα1 attenuates AMPK activation specifically towards mTORC1 signalling. SUMOylation is also important for rapid inactivation of AMPK, to allow prompt restoration of mTORC1 signalling. PIAS4 and its SUMO E3 ligase activity are specifically required for the AMPKα1 SUMOylation and the inhibition of AMPKα1 activity towards mTORC1 signalling. The activity of a SUMOylation-deficient AMPKα1 mutant is higher than the wild type towards mTORC1 signalling when reconstituted in AMPKα-deficient cells. PIAS4 depletion reduced growth of breast cancer cells, specifically when combined with direct AMPK activator A769662, suggesting that inhibiting AMPKα1 SUMOylation can be explored to modulate AMPK activation and thereby suppress cancer cell growth.
Highlights
AMP-activated protein kinase (AMPK) inhibits several anabolic pathways such as fatty acid and protein synthesis, and identification of AMPK substrate specificity would be useful to understand its role in particular cellular processes and develop strategies to modulate AMPK activity in a substrate-specific manner
Affinity purification of the glutathione S-transferase (GST)-tagged AMPKa1 or AMPKa2 from HEK293 cells followed by western blotting (WB) indicated that both PIAS3 and PIAS4 associated with AMPKa1 and AMPKa2 (Supplementary Fig. 1a), whereas PIAS1 or PIASx were not detected
We subsequently investigated in which subcellular compartment AMPKa1 SUMOylation occurs using a proximity ligation assay (PLA)[40] in AMPKa-null mouse embryonic fibroblasts (MEFs) reconstituted with WT or SUMOylation-deficient GST–AMPKa1
Summary
AMP-activated protein kinase (AMPK) inhibits several anabolic pathways such as fatty acid and protein synthesis, and identification of AMPK substrate specificity would be useful to understand its role in particular cellular processes and develop strategies to modulate AMPK activity in a substrate-specific manner. We show that SUMOylation of AMPKa1 attenuates AMPK activation towards mTORC1 signalling. PIAS4 and its SUMO E3 ligase activity are required for the AMPKa1 SUMOylation and the inhibition of AMPKa1 activity towards mTORC1 signalling. Thereby, AMPK inhibits several cellular processes important for tumour development such as fatty acid and protein synthesis[2], and several AMPK activators including the 5-aminoimidazole-4carboxamide-1-b-D-ribofuranoside (AICAR)[3,4], metformin[5] and A769662 AMPK inhibits fatty acid synthesis by phosphorylating CoA carboxylase (ACC)[7] and protein synthesis through suppression of mammalian target of rapamycin complex 1 (mTORC1)[8]. AMPK is activated in several stages[1]: increased cellular levels of AMP and ADP bind to the AMPKg subunit, leading to stabilization of T-loop phosphorylation of AMPKa subunit provided AMPKb is myristoylated[16], and further allosteric activation following additional AMP binding. With the exception of S479 phosphorylation[19], the inhibitory mechanisms have not been characterized beyond protein–protein interactions with AMPK
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