ATP depletion increases phosphorylation of elongation factor eEF2 in adult cardiomyocytes independently of inhibition of mTOR signalling

McLeod, Laura E.; Proud, Christopher G.

doi:10.1016/s0014-5793(02)03582-2

Cited by 56 publications

(45 citation statements)

References 28 publications

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“…This suggests that AMPK can regulate eEF2 in a manner that is independent of the mTOR/eEF2K pathways, at least in the presence of EtOH. Similar observations were reported previously whereby mild ATP depletion or oxygen deprivation affected eEF2 phosphorylation independently of the mTOR/eEF2K pathway (23,35).…”

Section: Discussionsupporting

confidence: 91%

Alcohol Regulates Eukaryotic Elongation Factor 2 Phosphorylation via an AMP-activated Protein Kinase-dependent Mechanism in C2C12 Skeletal Myocytes

Hong-Brown

Brown

Huber

et al. 2007

Journal of Biological Chemistry

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Ethanol decreases protein synthesis in cells, although the underlying regulatory mechanisms of this process are not fully established. In the present study incubation of C2C12 myocytes with 100 mM EtOH decreased protein synthesis while markedly increasing the phosphorylation of eukaryotic elongation factor 2 (eEF2), a key component of the translation machinery. Both mTOR and MEK pathways were found to play a role in regulating the effect of EtOH on eEF2 phosphorylation. Rapamycin, an inhibitor of mammalian target of rapamycin, and the MEK inhibitor PD98059 blocked the EtOH-induced phosphorylation of eEF2, whereas the p38 MAPK inhibitor SB202190 had no effect. Unexpectedly, EtOH decreased the phosphorylation and activity of the eEF2 upstream regulator eEF2 kinase. Likewise, treatment of cells with the inhibitor rottlerin did not block the stimulatory effect of EtOH on eEF2, suggesting that eEF2 kinase (eEF2K) does not play a role in regulating eEF2. In contrast, increased eEF2 phosphorylation was correlated with an increase in AMP-activated protein kinase (AMPK) phosphorylation and activity. Compound C, an inhibitor of AMPK, suppressed the effects of EtOH on eEF2 phosphorylation but had no effect on eEF2K, indicating that AMPK regulates eEF2 independent of eEF2K. Finally, EtOH decreased protein phosphatase 2A activity when either eEF2 or AMPK was used as the substrate. Thus, this later action may partially account for the increased phosphorylation of eEF2 in response to EtOH and the observed sensitivity of AMPK to rapamycin and PD98059 treatments. Collectively, the induction of eEF2 phosphorylation by EtOH is controlled by an increase in AMPK and a decrease in protein phosphatase 2A activity.

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Section: Discussionsupporting

confidence: 91%

Alcohol Regulates Eukaryotic Elongation Factor 2 Phosphorylation via an AMP-activated Protein Kinase-dependent Mechanism in C2C12 Skeletal Myocytes

Hong-Brown

Brown

Huber

et al. 2007

Journal of Biological Chemistry

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“…The increase in eEF2 phosphorylation during ischemia occurred before any change in the phosphorylation state of 4E-BP1 and was not mimicked by rapamycin. This suggests that the inhibition of mTOR signaling is not involved in the AMPK-induced inhibition of basal protein synthesis, confirming previous observations in hepatocytes (17) and cardiomyocytes (26). In hepatocytes incubated with amino acids, p70S6K becomes activated, and this effect was antagonized by AMPK activation (27,28).…”

Section: Discussionsupporting

confidence: 89%

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

Horman

Beauloye²,

Vertommen

et al. 2003

Journal of Biological Chemistry

119

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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...

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“…We therefore speculated that inactivation of a translational elongation factor such as eEF2 might suppress ER stress-induced cell death pathways. In fact, it has recently become clear that cells respond to stimuli that increase energy demand or reduce its supply in adapting to the environment by translational repression at the level of translational elongation (41), suggesting that elongation factors play a protective role under cellular stresses. The finding of a direct correlation between AMPK and eEF2 kinase (4) suggests that eEF2, a downstream target of eEF2 kinase, may play a role in mediating the cardioprotective effects of AICAR.…”

Section: Discussionmentioning

confidence: 99%

AMP-Activated Protein Kinase Protects Cardiomyocytes against Hypoxic Injury through Attenuation of Endoplasmic Reticulum Stress

Terai

Hiramoto²,

Masaki³

et al. 2005

Molecular and Cellular Biology

328

253

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Oxygen deprivation leads to the accumulation of misfolded proteins in the endoplasmic reticulum (ER), causing ER stress. Under conditions of ER stress, inhibition of protein synthesis and up-regulation of ER chaperone expression reduce the misfolded proteins in the ER. AMP-activated protein kinase (AMPK) is a key regulatory enzyme involved in energy homeostasis during hypoxia. It has been shown that AMPK activation is associated with inhibition of protein synthesis via phosphorylation of elongation factor 2 (eEF2) in cardiomyocytes. We therefore examined whether AMPK attenuates hypoxia-induced ER stress in neonatal rat cardiomyocytes. We found that hypoxia induced ER stress, as assessed by the expression of CHOP and BiP and cleavage of caspase 12. Knockdown of CHOP or caspase 12 through small interfering RNA (siRNA) resulted in decreased expression of cleaved poly(ADP-ribose) polymerase following exposure to hypoxia. We also found that hypoxia-induced CHOP expression and cleavage of caspase 12 were significantly inhibited by pretreatment with 5-aminoimidazole-4-carboxyamide-1-␤-D-ribofuranoside (AICAR), a pharmacological activator of AMPK. In parallel, adenovirus expressing dominant-negative AMPK significantly attenuated the cardioprotective effects of AICAR. Knockdown of eEF2 phosphorylation using eEF2 kinase siRNA abolished these cardioprotective effects of AICAR. Taken together, these findings demonstrate that activation of AMPK contributes to protection of the heart against hypoxic injury through attenuation of ER stress and that attenuation of protein synthesis via eEF2 inactivation may be the mechanism of cardioprotection by AMPK.

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ATP depletion increases phosphorylation of elongation factor eEF2 in adult cardiomyocytes independently of inhibition of mTOR signalling

Cited by 56 publications

References 28 publications

Alcohol Regulates Eukaryotic Elongation Factor 2 Phosphorylation via an AMP-activated Protein Kinase-dependent Mechanism in C2C12 Skeletal Myocytes

Alcohol Regulates Eukaryotic Elongation Factor 2 Phosphorylation via an AMP-activated Protein Kinase-dependent Mechanism in C2C12 Skeletal Myocytes

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

AMP-Activated Protein Kinase Protects Cardiomyocytes against Hypoxic Injury through Attenuation of Endoplasmic Reticulum Stress

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