Abstract
AMP-activated protein kinase (AMPK) is a metabolic stress-sensing enzyme responsible for maintaining cellular energy homeostasis. Activation of AMPK by salicylate and the thienopyridone A-769662 is critically dependent on phosphorylation of Ser108 in the β1 regulatory subunit. Here, we show a possible role for Ser108 phosphorylation in cell cycle regulation and promotion of pro-survival pathways in response to energy stress. We identify the autophagy initiator Unc-51-like kinase 1 (ULK1) as a β1-Ser108 kinase in cells. Cellular β1-Ser108 phosphorylation by ULK1 was dependent on AMPK β-subunit myristoylation, metabolic stress associated with elevated AMP/ATP ratio, and the intrinsic energy sensing capacity of AMPK; features consistent with an AMP-induced myristoyl switch mechanism. We further demonstrate cellular AMPK signaling independent of activation loop Thr172 phosphorylation, providing potential insight into physiological roles for Ser108 phosphorylation. These findings uncover new mechanisms by which AMPK could potentially maintain cellular energy homeostasis independently of Thr172 phosphorylation.
Highlights
AMP-activated protein kinase (AMPK) is a metabolic stress-sensing enzyme responsible for maintaining cellular energy homeostasis
Ligand-induced allosteric regulation of AMPK is governed by distinct phosphorylation events that either sensitize AMPK to nucleotides/drugs binding at γ-subunit sites (phosphorylation of Thr[172] in the α-subunit activation loop8–10), or small compounds binding at the ADaM site (phosphorylation of β-Ser[108] in the β-CBM11–13)
We found that H2O2 and phenformin treatments of HEK293T cells that induced Ser[108] phosphorylation (Fig. 3b) produced significant falls in adenylate energy charge (AEC), with H2O2 incubation having the greater effect (AMP/ATP ratios: basal 0.0093 ± 0.0015; phenformin 0.0177 ± 0.0041; H2O2 0.0418 ± 0.0033) (Fig. 7a)
Summary
An AMP myristoyl switch triggers β1-Ser[108] transphosphorylation. H2O2 and phenformin indirectly activate AMPK through perturbation of adenine nucleotide ratios (increased AMP/ATP ratio, reduced adenylate charge). Phenformin treatment of HEK293T cells failed to induce Ser[108] phosphorylation in KI-α1β1γ1 carrying a mutation in the γ1 nucleotide site 3 (γ1-D245A) that renders AMPK insensitive to stimulation by AMP3 (Fig. 7b). Incubation of HEK293T cells with 1 μM AZD8055 or INK128 induced almost complete loss of ULK-pSer[757], without significantly affecting adenylate charge or stimulating phosphorylation of Ser[108] in KI-α1β1γ1 expressed in these cells (Fig. 7a, c). Phenformin/A-769662 co-incubation resulted in a further 5.8-fold increase in pACC compared to phenformin alone, producing >30% the pACC signal in WT α2-expressing cells treated with phenformin (Fig. 8a) Neither of these effects on pACC were detected in α1/2-dKO iMEFs transduced with empty lentivirus (Supplementary Fig. 6), confirming that AMPK was the only kinase phosphorylating ACC-Ser[79] under these conditions. Phenformin/A-769662 co-incubation was the only condition that induced a detectable increase in ULK1-Ser[555] phosphorylation, an AMPK substrate that is important for ULK1 activation[32] (Fig. 8b)
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