β-Subunit myristoylation is the gatekeeper for initiating metabolic stress sensing by AMP-activated protein kinase (AMPK)

Abstract

The AMP-activated protein kinase (AMPK) is an αβγ heterotrimer that acts as a master metabolic regulator to maintain cellular energy balance following increased energy demand and increases in the AMP∕ATP ratio. This regulation provides dynamic control of energy metabolism, matching energy supply with demand that is essential for the function and survival of organisms. AMPK is inactive unless phosphorylated on Thr172 in the α-catalytic subunit activation loop by upstream kinases (LKB1 or calcium-calmodulindependent protein kinase kinase β). How a rise in AMP levels triggers AMPK α-Thr172 phosphorylation and activation is incompletely understood. Here we demonstrate unequivocally that AMP directly stimulates α-Thr172 phosphorylation provided the AMPK β-subunit is myristoylated. Loss of the myristoyl group abolishes AMP activation and reduces the extent of α-Thr172 phosphorylation. Once AMPK is phosphorylated, AMP further activates allosterically but this activation does not require β-subunit myristoylation. AMP and glucose deprivation also promote membrane association of myristoylated AMPK, indicative of a myristoyl-switch mechanism. Our results show that AMP regulates AMPK activation at the initial phosphorylation step, and that β-subunit myristoylation is important for transducing the metabolic stress signal.myristome | signal transduction | adenylate charge | γ-subunit T he AMP-activated protein kinase (AMPK) is a key regulator of cellular and whole-body energy homeostasis that coordinates metabolic pathways in order to balance nutrient supply with energy demand. AMPK protects cells from physiological and pathological stresses (e.g., nutrient starvation, hypoxia/ischemia, and exercise) that lower cellular energy charge (increase AMP∕ ATP ratio) by directing metabolism toward ATP production and inhibiting anabolic pathways that utilize ATP∕NADPH (1, 2). This regulation is achieved by acute phosphorylation of key enzymes in major branches of metabolism including fat synthesis, protein synthesis, and carbohydrate metabolism, as well as phosphorylation of transcription factors to have longer-term regulatory effects. AMPK also functions in regulating whole-body energy homeostasis, food intake, and body weight in response to a variety of hormones including leptin, adiponectin, and ghrelin. These properties have made AMPK a promising drug target to treat the growing incidence of metabolic diseases including obesity, type 2 diabetes, cancer growth and metastasis, and cardiovascular disease (1, 2).AMPK is an αβγ heterotrimer consisting of an α catalytic subunit and β and γ regulatory subunits, with corresponding homologues in all eukaryotes. Multiple isoforms exist for each subunit in mammals (α1, α2, β1, β2, γ1, γ2, and γ3). The α-subunits consist of an N-terminal kinase catalytic domain followed by an autoinhibitory sequence and a C-terminal β-subunit binding domain (3). The β-subunits contain an internal carbohydrate-binding module and a conserved C-terminal sequence that functions to tether α-and γ-subunits (4, 5). ...

Myristoylation confers noncanonical AMPK functions in autophagy selectivity and mitochondrial surveillance

“…3A,B), morphologically resembling both mitochondrial aggregates and autophagosomerelated p62 bodies 19,21 . Similar subcellular distribution has been reported for AMPKb subunits 48,49 . Indeed, transfection of AMPKb1-GFP exhibited co-localization with MitoTracker Red (MTR) in Saos2 cells and the co-localization was increased by a short exposure to CCCP (Supplementary Fig.…”

Heterotrimer-independent regulation of activation-loop phosphorylation of Snf1 protein kinase involves two protein phosphatases

“…The AMPK γ-subunit contributes to the regulation of activation-loop phosphorylation through nucleotide binding (16)(17)(18)(19)(20)(21)(22)(23), and recent evidence supports a similar role for the Snf4 subunit of SNF1 (26,27). In addition, mutations in all three SNF1 subunits cause Thr-210 phosphorylation during growth on high levels of glucose, suggesting that they perturb the heterotrimer and that its native conformation prevents Thr-210 phosphorylation or promotes its dephosphorylation (37,38).…”

“…The Snf4/γ-subunit regulates activation-loop phosphorylation through adenine nucleotide binding. The AMPK γ-subunit binds AMP (16,17), which causes allosteric activation (18), promotes phosphorylation of the activation loop of the α-subunit (19), and inhibits its dephosphorylation in vitro (20,21); binding of ADP is also involved in regulation of AMPK phosphorylation (22,23). In the case of SNF1, AMP does not cause allosteric activation (24,25) or protect the activation loop from dephosphorylation (20), but ADP protects against dephosphorylation of SNF1 in vitro (26,27).…”

PI3K/AKT, MAPK and AMPK signalling: protein kinases in glucose homeostasis

“…It has been recently proposed that binding of AMP triggers exposure of a myristoyl group at the AMPK β-subunit, which promotes membrane association and primes AMPK for activation by upstream kinases (Ref. 117). In addition, it was shown that binding of ADP to AMPK protects against dephosphorylation of Thr172, but does not induce allosteric activation of AMPK (Ref.…”