1. Control by AMPK.
AMPK is a serine/threonine protein kinase involved in the control of cellular energy homeostasis. It stimulates ATP-producing pathways and inhibits energy consuming processes. We contributed to this field by finding new AMPK targets. We demonstrated that the activation of 6-phosphofructo-2-kinase (PFK-2), the enzyme responsible for the synthesis of fructose 2,6-bisphosphate, by AMPK participates in the stimulation of heart glycolysis by ischaemia (Pasteur Effect).
We also showed that AMPK activation inhibits protein synthesis both in anoxic rat hepatocytes and in ischaemic hearts. Protein synthesis inhibition results from the phosphorylation-induced activation of eEF2 kinase by AMPK; eEF2 kinase in turn phosphorylates and inactivates eEF2 (eukaryotic elongation factor-2), which controls the predominant energy-consuming step in protein synthesis. In electrically stimulated rat skeletal muscle, we showed that although protein synthesis inhibition was associated with an increase in eEF2 phosphorylation, this was independent of AMPK activation. We also studied the role of AMPK in shutting down protein synthesis in animals that adapt to extreme energy stress. Although AMPK does not appear to play a major role in decreasing energy consumption in tissues during mammalian hibernation, it could play an important role in adaptation to freeze-tolerance in frogs and adaptation to anoxia tolerance in turtles.
Control of glycolysis and protein synthesis by ischaemia in heart
Metabolic targets of AMPK
We identified smooth muscle myosin light chain kinase (smMLCK) as a new AMPK target. Phosphorylation desensitizes smMLCK to stimulation by Ca/calmodulin (CaM). AMPK is activated in aortic smooth muscle cells in response to vasoconstrictors via the CaM-dependent protein kinase kinase-β pathway. Moreover, aortic rings from AMPK α1 (the predominant AMPK isoform in vascular smooth muscle) knockout mice display increased contractility in response to vasoconstrictors and KCl-induced depolarization. Our hypothesis is that AMPK activation by vasoconstrictors leads to phosphorylation and inactivation of smMLCK, thereby contributing to reduced ATP turnover in the tonic phase of contraction.
We were unable to confirm published data claiming that myosin regulatory light chains (LC20) can be directly phosphorylated by AMPK at the smMLCK Ser19 site. Our results indicate that LC20 is not a physiological AMPK substrate to explain energy-dependent changes in cell structure and that this published observation was rather due to commercial AMPK contamination by kinases capable of LC20 phosphorylation.
Control of vascular tone by AMPK
We also found that AMPK activation leads to a dramatic reorganization of the actin cytoskeleton in epithelial cells, and increases cell migration. AMPK activation is associated with RhoA activation and increased phosphorylation of ezrin/moesin/radixin. We are focusing on the mechanisms of RhoA activation by AMPK that might mediate these effets.
Another new AMPK target we are studying is the liver isoform of glycogen synthase (GS). In isolated rat hepatocytes, AMPK activation leads to GS inactivation. Purified liver GS is an AMPK substrate and its phosphorylation inactivates the enzyme. Phosphorylation occurs at a single site (Ser7) and the involvement of AMPK in GS inactivation/phosphorylation in hepatocytes from liver-specific double AMPK α1/α2 knockout mice is being studied.
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