β‐Adrenergic agonists increase phosphorylation of elongation factor 2 in cardiomyocytes without eliciting calcium‐independent eEF2 kinase activity

McLeod, Laura E.; Wang, Lijun; Proud, Christopher G.

doi:10.1016/s0014-5793(01)02100-7

Cited by 33 publications

(39 citation statements)

References 21 publications

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“…1E) in a region that contains several known sites of phosphorylation (12,17,18). Ser-398 has not been identified previously as a phosphorylation site in eEF2 kinase.…”

Section: Ampk Phosphorylates Eef2mentioning

confidence: 96%

“…Kinase-We and others (11)(12)(13) have shown previously that conditions that activate the AMPK lead to increased phosphorylation of eEF2 and activate eEF2 kinase. We therefore studied whether eEF2 kinase was a substrate for AMPK in vitro.…”

Section: Ampk Phosphorylates Eef2mentioning

confidence: 99%

“…We have shown previously that ATP depletion or treatment of cardiomyocytes with an agent that activates AMPK (AICAR) leads to increased phosphorylation of eEF2 (12). There is some evidence that cardiac myocytes may possess a distinct form of eEF2 kinase (12), although this enzyme has neither been purified nor cloned.…”

Section: Ampk Phosphorylates Eef2mentioning

confidence: 99%

“…There is some evidence that cardiac myocytes may possess a distinct form of eEF2 kinase (12), although this enzyme has neither been purified nor cloned. To examine whether cardiac eEF2 kinase contained a phosphorylation site corresponding to Ser-398, we subjected a cardiac extract to chromatography on FPLC (Mono Q column) as described earlier (12), in order to partially purify eEF2 kinase. Fractions were monitored for eEF2 kinase activity.…”

Section: Ampk Phosphorylates Eef2mentioning

confidence: 99%

“…S6K1 is regulated through mTOR and phosphorylates eEF2 kinase at Ser-366, which decreases its activity at low Ca 2ϩ concentrations (10). We recently observed that, in cardiomyocytes and other cell types, conditions that cause depletion of ATP lead to increased phosphorylation of eEF2 (11,12). This is associated with inhibition of protein synthesis and increased activity of eEF2 kinase (11).…”

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confidence: 99%

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Stimulation of the AMP-activated Protein Kinase Leads to Activation of Eukaryotic Elongation Factor 2 Kinase and to Its Phosphorylation at a Novel Site, Serine 398

Browne

Finn

Proud

2004

Journal of Biological Chemistry

316

269

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Protein synthesis consumes a high proportion of the metabolic energy of mammalian cells, and most of this is used by peptide chain elongation. An important regulator of energy supply and demand in eukaryotic cells is the AMP-activated protein kinase (AMPK). The rate of peptide chain elongation can be modulated through the phosphorylation of eukaryotic elongation factor (eEF) 2, which inhibits its activity and is catalyzed by a specific calcium/calmodulin-dependent protein kinase termed eEF2 kinase. Here we show that AMPK directly phosphorylates eEF2 kinase, and we identify the major site of phosphorylation as Ser-398 in a regulatory domain of eEF2 kinase. AMPK also phosphorylates two other sites (Ser-78 and Ser-366) in eEF2 kinase in vitro. We develop appropriate phosphospecific antisera and show that phosphorylation of Ser-398 in eEF2 kinase is enhanced in intact cells under a range of conditions that activate AMPK and increase the phosphorylation of eEF2. Ser-78 and Ser-366 do not appear to be phosphorylated by AMPK within cells. Although cardiomyocytes appear to contain a distinct isoform of eEF2 kinase, it also contains a site corresponding to Ser-398 that is phosphorylated by AMPK in vitro. Stimuli that activate AMPK and increase eEF2 phosphorylation within cells increase the activity of eEF2 kinase. Thus, AMPK and eEF2 kinase may provide a key link between cellular energy status and the inhibition of protein synthesis, a major consumer of metabolic energy.Protein synthesis is a process of fundamental importance for all living cells. It also places substantial demands on the cell in terms of requirements for precursors, viz. amino acids and metabolic energy. Recent work (1-3) has shown that amino acids regulate the translational machinery in a number of ways, e.g. through control of the mTOR 1 signaling pathway.Protein synthesis is a major consumer of cellular energy with estimates of 30 -50% of total energy being used in this process. The vast majority of this is consumed by peptide elongation, either directly as GTP (two are hydrolyzed for each amino acid added) or indirectly during aminoacyl-tRNA charging, one ATP being hydrolyzed to AMP for each amino acid charging event.Despite this, little is known of the mechanisms by which cells couple the rate of protein synthesis to the availability of energy, i.e. ATP. In mammalian cells, peptide chain elongation requires two main elongation factors, eEF1A and eEF2. The latter mediates the translocation step of elongation in which the ribosome moves by the equivalent of one codon relative to the mRNA, and the peptidyl-tRNA shifts from the A-into the P-site on the ribosome (4). eEF2 is a phosphoprotein, and phosphorylation at a single site, Thr-56, renders it unable to bind ribosomes, thereby inactivating it (5-7). Phosphorylation is catalyzed by a specific and very unusual kinase, eEF2 kinase (8, 9). Phosphorylation of eEF2 is decreased by insulin and certain other stimuli through signaling events that require the mammalian target of rapamycin, mTOR (reviewed in ...

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“…1E) in a region that contains several known sites of phosphorylation (12,17,18). Ser-398 has not been identified previously as a phosphorylation site in eEF2 kinase.…”

Section: Ampk Phosphorylates Eef2mentioning

confidence: 96%

Section: Ampk Phosphorylates Eef2mentioning

confidence: 99%

Section: Ampk Phosphorylates Eef2mentioning

confidence: 99%

Section: Ampk Phosphorylates Eef2mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Stimulation of the AMP-activated Protein Kinase Leads to Activation of Eukaryotic Elongation Factor 2 Kinase and to Its Phosphorylation at a Novel Site, Serine 398

Browne

Finn

Proud

2004

Journal of Biological Chemistry

316

269

View full text Add to dashboard Cite

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Eukaryotic elongation factor‐2 (eEF2): its regulation and peptide chain elongation

Kaul

Pattan

Rafeequi

2011

Cell Biochemistry & Function

177

119

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Regulation at the level of translation in eukaryotes is feasible because of the longer lifetime of eukaryotic mRNAs in the cell. The elongation stage of mRNA translation requires a substantial amount of energy and also eukaryotic elongation factors (eEFs). The important component of eEFs, i.e. eEF2 promotes the GTP-dependent translocation of the nascent protein chain from the A-site to the P-site of the ribosome. Mostly the eEF2 is regulated by phosphorylation and dephosphorylation by a specific kinase known as eEF2 kinase, which itself is up-regulated by various mechanisms in the eukaryotic cell. The activity of this kinase is dependent on calcium ions and calmodulin. Recently it has been shown that the activity of eEF2 kinase is regulated by MAP kinase signalling and mTOR signalling pathway. There are also various stimuli that control the peptide chain elongation in eukaryotic cell; some stimuli inhibit and some activate eEF2. These reports provide the mechanisms by which cells likely serve to slow down protein synthesis and conserve energy under nutrient deprived conditions via regulation of eEF2. The regulation via eEF2 has also been seen in mammary tissue of lactating cows, suggesting that eEF2 may be a limiting factor in milk protein synthesis. Regulation at this level provides the molecular understanding about the control of protein translocation reactions in eukaryotes, which is critical for numerous biological phenomenons. Further the elongation factors could be potential targets for regulation of protein synthesis like milk protein synthesis and hence probably its foreseeable application to synthetic biology.

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High hydrostatic pressure inhibits the biosynthesis of eukaryotic elongation factor‐2

Elo

Karjalainen

Sironen

et al. 2004

J of Cellular Biochemistry

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High continuous hydrostatic pressure is known to inhibit the total cellular protein synthesis. In this study, our goal was to identify pressure-regulated proteins by using two dimensional gel electrophoresis and mass spectrometry. This analysis showed that under 30 MPa continuous hydrostatic pressure the biosynthesis of eukaryotic elongation factor-2 (eEF-2) was inhibited both in HeLa carcinoma and T/C28a4 chondrocytic cell lines. Western blot analysis of HeLa cells revealed that the cellular protein level of eEF-2 decreased by 40%-50% within 12 h of the pressure treatment. However, the steady-state mRNA level of eEF-2 was not affected by the pressure. Cycloheximide addition after 4 h-pressure treatment suggested that the half-life of eEF-2 protein was shorter in pressurized cells. eEF-2 is responsible for the translocation of ribosome along the specific mRNA during translation, and its phosphorylation prevents the ribosomal translocation. Therefore, increased phosphorylation of eEF-2 was considered as one mechanism that could explain the reduced level of protein synthesis in pressurized HeLa cell cultures. However, Western blot analysis with an antibody recognizing the Thr56-phosphorylated form of eEF-2 showed that phosphorylation of eEF-2 was not elevated in pressurized samples. In conclusion, the inhibition of protein synthesis under high pressure occurs independent of the phosphorylation of eEF-2. However, this inhibition may result from the decrease of cellular eEF-2 protein.

show abstract

β‐Adrenergic agonists increase phosphorylation of elongation factor 2 in cardiomyocytes without eliciting calcium‐independent eEF2 kinase activity

Cited by 33 publications

References 21 publications

Stimulation of the AMP-activated Protein Kinase Leads to Activation of Eukaryotic Elongation Factor 2 Kinase and to Its Phosphorylation at a Novel Site, Serine 398

Stimulation of the AMP-activated Protein Kinase Leads to Activation of Eukaryotic Elongation Factor 2 Kinase and to Its Phosphorylation at a Novel Site, Serine 398

Eukaryotic elongation factor‐2 (eEF2): its regulation and peptide chain elongation

High hydrostatic pressure inhibits the biosynthesis of eukaryotic elongation factor‐2

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