AMPK phosphorylates and inactivates liver glycogen synthase but does not phosphorylate myosin regulatory light chains

The AMP-activated protein kinase (AMPK) is an important protein kinase that controls cellular and whole body energy homeostasis. During energy stress, AMPK is activated by an increase in AMP/ATP and/or ADP/ATP ratio. In order to restore energy balance, AMPK activation stimulates catabolic pathways to produce ATP and while at the same time inhibiting ATP-consuming anabolic pathways. The search for new AMPK targets is thus fundamental to understanding the mechanisms by which organisms can adapt their metabolism to deal with physiological and pathophysiological stress conditions. This thesis was devoted to the study of two potential AMPK targets, myosin regulatory light chains (MLCs) and the liver isoenzyme of glycogen synthase (GYS2). Firstly, MLCs have been proposed to be directly phosphorylated by AMPK (Lee et al., 2007). This observation contradicted results previously obtained in our laboratory. The first publication compared the kinetics of MLC phosphorylation by recombinant AMPK with that catalyzed by a commercial purified preparation of AMPK from rat liver (used by Lee et al., 2007) and by MLC kinase (MLCK), the dedicated kinase well-known to phosphorylate MLCs. Our results show that i) the commercial AMPK preparation was contaminated by kinase(s) able to phosphorylate MLC, explaining the result obtained by Lee et al. (2007) ii) MLCs are not a physiological substrate for AMPK. In this publication, it was also demonstrated that AMPK activation in epithelial cells by A769662, a pharmacological activator of AMPK, led to MLC phosphorylation. Moreover, this phosphorylation was reduced by H1152, a Rho kinase inhibitor, suggesting that Rho kinase could be involved in the control of MLC phosphorylation by AMPK in intact cells. In the second publication, we found that both recombinant GYS1, the muscle isoenzyme already known to be an AMPK substrate, and recombinant GYS2 were phosphorylated to a similar extent by recombinant AMPK. We also identified Ser7 as the AMPK phosphorylation site on GYS2. GYS1 and GYS2 phosphorylation by AMPK led to enzyme inactivation by virtue of a decrease in affinity for UDP-glucose (assayed in the absence of glucose 6-phosphate (G6P)) and a decrease in affinity for G6P (assayed at low UDP-glucose concentrations). In freshly isolated hepatocytes incubated with two different AMPK activators, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) and A769662, GYS2 Ser7 phosphorylation increased and GYS was inactivated. Pharmacological GYS2 inactivation was significantly blunted in hepatocytes from mice in which the two AMPK catalytic α1/α2 subunits had been genetically deleted in liver, suggesting that GYS2 inactivation by treatment with AICAR and A769662 is at least partly due to AMPK activation.(BIOL 3) -- UCL, 201

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The Salt-Inducible Kinases : Emerging Metabolic Regulators

The discovery of liver kinase B1 (LKB1) as an upstream kinase for AMP-activated protein kinase (AMPK) led to the identification of several related kinases that also rely on LKB1 for their catalytic activity. Among these, the salt-inducible kinases (SIKs) have emerged as key regulators of metabolism. Unlike AMPK, SIKs do not respond to nucleotides, but their function is regulated by extracellular signals, such as hormones, through complex LKB1-independent mechanisms. While AMPK acts on multiple targets, including metabolic enzymes, to maintain cellular ATP levels, SIKs primarily regulate gene expression, by acting on transcriptional regulators, such as the cAMP response element-binding protein-regulated transcription coactivators and class IIa histone deacetylases. This review describes the development of research on SIKs, from their discovery to the most recent findings on metabolic regulation