Abstract
Protein synthesis, in particular peptide chain elongation, is an energy-consuming biosynthetic process. AMP-activated protein kinase (AMPK) is a key regulatory enzyme involved in cellular energy homeostasis. Therefore, we tested the hypothesis that, as in liver, it could mediate the inhibition of protein synthesis by oxygen deprivation in heart by modulating the phosphorylation of eukaryotic elongation factor-2 (eEF2), which becomes inactive in its phosphorylated form. In anoxic cardiomyocytes, AMPK activation was associated with an inhibition of protein synthesis and an increase in phosphorylation of eEF2. Rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), did not mimic the effect of oxygen deprivation to inhibit protein synthesis in cardiomyocytes or lead to eEF2 phosphorylation in perfused hearts, suggesting that AMPK activation did not inhibit mTOR/p70 ribosomal protein S6 kinase (p70S6K) signaling. Human recombinant eEF2 kinase (eEF2K) was phosphorylated by AMPK in a time- and AMP-dependent fashion, and phosphorylation led to eEF2K activation, similar to that observed in extracts from ischemic hearts. In contrast, increasing the workload resulted in a dephosphorylation of eEF2, which was rapamycin-insensitive, thus excluding a role for mTOR in this effect. eEF2K activity was unchanged by increasing the workload, suggesting that the decrease in eEF2 phosphorylation could result from the activation of an eEF2 phosphatase.
Highlights
Protein synthesis, in particular peptide chain elongation, is an energy-consuming biosynthetic process, accounting for a large proportion of the oxygen requirements of cells [1]
Oxygen Deprivation Inhibits Protein Synthesis, Activates AMPactivated protein kinase (AMPK), and Increases eukaryotic elongation factor-2 (eEF2) Phosphorylation in Rat Cardiomyocytes—Isolated cardiomyocytes were incubated for 10 min with 1 M oligomycin, an inhibitor of oxidative phosphorylation, or under anoxic conditions (N2 replacing O2 in the gas phase)
We show that AMPK activation correlates with an increase in eEF2 phosphorylation in anoxic cardiomyocytes and ischemic heart
Summary
In particular peptide chain elongation, is an energy-consuming biosynthetic process, accounting for a large proportion of the oxygen requirements of cells [1]. Activation of PI 3-kinase and mTOR could be implicated in the stimulation of protein synthesis by increasing the workload, which was one of the aspects studied in this report. An inhibition of mTOR/p70S6K signaling following AMP-activated protein kinase (AMPK) activation has been proposed to explain the inhibition of protein synthesis by oxygen deprivation [13]. AMPK stimulates ATP-producing pathways and inhibits energy-consuming processes (14 –16). In this factor-2; eEF2K, eEF2 kinase; 4E-BP1, 4E-binding protein-1; eIF2B, eukaryotic initiation factor 2B; p70S6K, p70 ribosomal protein S6 kinase; Mes, 4-morpholineethanesulfonic acid; nano-ESI-MS/MS, nanoelectrospray ionization tandem mass spectrometry; Cr, creatine; PCr, phosphocreatine; PI, phosphatidylinositol; ERK, extracellular signalregulated kinase; MEK, mitogen-activated protein kinase/ERK kinase. We show that myocardial ischemia and increased workload modulate eEF2 phosphorylation by mechanisms independent of mTOR/p70S6K signaling
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