Myocardial Ischemia and Increased Heart Work Modulate the Phosphorylation State of Eukaryotic Elongation Factor-2

Abstract

Protein synthesis, in particular peptide chain elongation, is an energy-consuming biosynthetic process. AMPactivated 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 timeand 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.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). Protein synthesis is regulated via the phosphorylation/dephosphorylation of translation factors and ribosomal proteins (2). The mammalian target of rapamycin (mTOR) 1 phosphorylates eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), thereby relieving its inhibitory action on eukaryotic initiation factor 4E (eIF-4E), which can then bind the mRNA cap and stimulate protein synthesis (3). The control of translation by mTOR is also exerted at elongation by regulating the phosphorylation of eukaryotic elongation factor-2 (eEF2) (4). The phosphorylation of eEF2 at Thr-56 by a specific calcium-and calmodulin-dependent eukaryotic eEF2 kinase (eEF2K) leads to its inactivation (5). The p70 ribosomal protein S6 kinase (p70S6K) lies downstream of mTOR and phosphorylates Ser-366 of eEF2K, causing inactivation (4). Therefore, mTOR activation can result in a stimulation of protein synthesis by decreasing eEF2 phosphorylation. Indeed, one of the mechanisms by which insulin stimulates protein synthesis in the heart involves mTOR activation, the effect being blocked by the mTOR inhibitor, rapamycin, and by wortmannin, which blocks phosphatidylinositol 3-kinase (PI 3-kinase) upstream of mTOR (6). The stimulation of protein synthesis by insulin also involves a mTOR-independent mechanism via the phosphorylation and inactivation of glycogen synthase kinase-3. Glycogen synthase kinase-3 phosphorylates and inactivates...

Inhibition of the mTOR/p70S6K pathway is not involved in the insulin-sensitizing effect of AMPK on cardiac glucose uptake

“…2A). In agreement with previous data obtained in perfused hearts and cardiomyocytes in suspension (2,16), no p70S6K phosphorylation could be detected in untreated cells and AMPK activation by phenformin had no effect under these basal conditions ( Fig. 2A).…”

Regulation of Transcription and Translation by Hypoxia

“…Importantly, the phosphorylation state of 4E-BP1 is not changed by anoxia. 55,56 Increased understanding of translational control under hypoxic conditions has lead to more questions concerning how cells adapt to low O 2 availability. To name a few, does O 2 availability directly regulate the activity of mTOR, a central regulator of global protein synthesis that phosphorylates both 4E-BP1 and p70S6K (Fig.…”

“…2). 55,56 The eEF2 kinase that phosphorylates and inactivates eEF2 is activated by AMPK in anoxic hepatocytes or cardiomyocytes. Importantly, the phosphorylation state of 4E-BP1 is not changed by anoxia.…”

Activation of AMP-Activated Protein Kinase Enhances Angiotensin II–Induced Proliferation in Cardiac Fibroblasts

“…4 In addition, AMPK plays a role in familial Wolff-Parkinson-White syndrome and hypertrophic cardiomyopathy via mutations in PRKAG2, a gene encoding the ␥2 subunit of AMPK. 6 -8 On the other hand, recent evidence indicates that AMPK activation inhibits proteins synthesis 9,10 and might, thus, be expected to reduce the development of hypertrophy. Although much has been learned about the role of AMPK in the development of cardiac hypertrophy, the molecular mechanism by which AMPK regulates cardiac hypertrophy is still unclear.…”