Identification of the phosphorylation sites in elongation factor‐2 from rabbit reticulocytes

Price, Nigel T.; Redpath, Nicholas T.; Severinov, Konstantin; Campbell, David G.; Russell, Jackie; Proud, Christopher G.

doi:10.1016/0014-5793(91)80489-p

Cited by 111 publications

(68 citation statements)

References 32 publications

(22 reference statements)

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“…We found one peak consistent with a phosphorylation on the tryptic peptide FTDTR, corresponding to residues 55-59 of rat eEF-2 (Supplementary Figure 2b). This site matches exactly with a previously described phosphorylation site on eEF-2 22,36,37 and suggests that our DIGE experiment had detected a salinduced phosphorylation of eEF-2. We confirmed that sal eEF-2 phosphorylation and ER stress M Boyce et al induces eEF-2 phosphorylation, using an immunoblot for phosphorylated Thr56, the major site of eEF-2 phosphorylation in vivo.…”

Section: Resultssupporting

confidence: 91%

A pharmacoproteomic approach implicates eukaryotic elongation factor 2 kinase in ER stress-induced cell death

Boyce

Ryazanov

et al. 2008

Cell Death Differ

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Apoptosis triggered by endoplasmic reticulum (ER) stress has been implicated in many diseases but its cellular regulation remains poorly understood. Previously, we identified salubrinal (sal), a small molecule that protects cells from ER stressinduced apoptosis by selectively activating a subset of endogenous ER stress-signaling events. Here, we use sal as a probe in a proteomic approach to discover new information about the endogenous cellular response to ER stress. We show that sal induces phosphorylation of the translation elongation factor eukaryotic translation elongation factor 2 (eEF-2), an event that depends on eEF-2 kinase (eEF-2K). ER stress itself also induces eEF-2K-dependent eEF-2 phosphorylation, and this pathway promotes translational arrest and cell death in this context, identifying eEF-2K as a hitherto unknown regulator of ER stress-induced apoptosis. Finally, we use both sal and ER stress models to show that eEF-2 phosphorylation can be activated by at least two signaling mechanisms. Our work identifies eEF-2K as a new component of the ER stress response and underlines the utility of novel small molecules in discovering new cell biology. Cell Death and Differentiation (2008) 15, 589-599; doi:10.1038/sj.cdd.4402296; published online 11 January 2008 The endoplasmic reticulum (ER) serves as the primary processing site for membrane and secreted proteins. The ER recruits translating ribosomes, translocates newly synthesized polypeptides into its lumen, and promotes a variety of post-translational modifications and chaperone-facilitated protein folding. 1,2 Proper ER function is critical for numerous aspects of cell physiology, including vesicle trafficking, lipid and membrane biogenesis, and protein targeting and secretion. Accordingly, cells react rapidly to various forms of ER dysfunction -including the accumulation of unfolded, misfolded or excessive protein, ER lipid or glycolipid imbalances, or changes in the redox or ionic conditions of the ER lumenthrough a set of adaptive pathways known collectively as the ER stress response (ESR). [2][3][4][5] The ESR promotes cell survival both by increasing the capacity of the ER to fold and process client proteins and by reducing the amount of protein inside the ER. These effects are achieved through three major pathways: (1) the unfolded protein response, a transcription-dependent induction of ER lumenal chaperone proteins and many other components of the secretory apparatus, which augments the polypeptide processing capacity of the ER; 5,6 (2) the activation of proteasome-dependent ER-associated degradation to remove proteins from the ER; 7,8 and (3) the control of protein translation to modulate the polypeptide traffic into the ER. 9,10 Normally, this suite of responses succeeds in restoring ER homeostasis. However, in metazoans, persistent or intense ER stress can also trigger apoptosis. [11][12][13][14] ER stress and the apoptotic program coupled to it have been implicated in many important pathologies, including diabetes, obesity, neurodegenerativ...

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Section: Resultssupporting

confidence: 91%

A pharmacoproteomic approach implicates eukaryotic elongation factor 2 kinase in ER stress-induced cell death

Boyce

Ryazanov

et al. 2008

Cell Death Differ

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“…eEF2 is phosphorylated at Thr56 by eEF2 kinase (eEF2K), impairing its interaction with the ribosome and thus inactivating elongation. 64 The activity of eEF2K can be regulated by S6 kinases. 35 Phosphorylation of eEF2K at Ser366 by S6Ks impairs its activity and hence favors activation of translation.…”

Section: Discussionmentioning

confidence: 99%

Evidence of Placental Translation Inhibition and Endoplasmic Reticulum Stress in the Etiology of Human Intrauterine Growth Restriction

Yung

Calabrese

Hynx

et al. 2008

The American Journal of Pathology

335

304

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Unexplained intrauterine growth restriction of the fetus (IUGR) results from impaired placental development, frequently associated with maternal malperfusion. Some cases are complicated further by preeclampsia (PE؉IUGR). Here, we provide the first evidence that placental protein synthesis inhibition and endoplasmic reticulum (ER) stress play key roles in IUGR pathophysiology. Increased phosphorylation of eukaryotic initiation factor 2␣ suggests suppression of translation initiation in IUGR placentas, with a further increase in PE؉IUGR cases. Consequently, AKT levels were reduced at the protein, but not mRNA, level. Additionally, levels of other proteins in the AKT-mammalian target of rapamycin pathway were decreased, and there was associated dephosphorylation of 4E-binding protein 1 and activation of glycogen synthase kinase 3␤. Cyclin D1 and the eukaryotic initiation factor 2B epsilon subunit were also down-regulated, providing additional evidence for this placental phenotype. The central role of AKT signaling in placental growth regulation was confirmed in Akt1 null mice, which display IUGR. In addition, we demonstrated ultrastructural and molecular evidence of ER stress in human IUGR and PE؉IUGR placentas, providing a potential mechanism for eukaryotic initiation factor 2␣ phosphorylation. In confirmation, induction of low-grade ER stress in trophoblast-like cell lines reduced cellular proliferation. PE؉IUGR placentas showed elevated ER stress with the additional expression of the pro-apoptotic protein C/EBP-homologous protein/growth arrest and DNA damage 153. These findings may account for the increased microparticulate placental debris in the maternal circulation of these cases, leading to endothelial cell activation and impairing placental development.

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“…eEF-2 is the sole substrate of this kinase and does not appear to be phosphorylated by any other kinase (Mitsui et al, 1993;. Three threonyl residues located at the NH 2 terminus of eEF-2 can be phosphorylated by eEF-2 kinase in vitro (Ovchinnikov et al, 1990;Price et al, 1991;. Phosphorylation of Thr 56 alone seems to be responsible for the inhibition of mRNA translation in various cell-free systems Ryazanov et al, 1988;.…”

Section: Abstract: Elongation Factor-2; Phosphorylation; Calcium; Prmentioning

confidence: 99%

Glutamate-Dependent Phosphorylation of Elongation Factor-2 and Inhibition of Protein Synthesis in Neurons

et al. 1997

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Postischemic delayed neuronal death is attributed to excitotoxic activation of glutamate receptors. It is preceded by a persistent inhibition of protein synthesis, the molecular basis of which is not known. Here we have examined in cortical neurons in culture the regulation by glutamate of phosphorylation of eukaryotic elongation factor-2 (eEF-2) by eEF-2 kinase, a Ca 2ϩ / calmodulin-dependent enzyme. Using a phosphorylation statespecific antibody, we show that glutamate, which triggers a large influx of Ca 2ϩ , enhances dramatically the phosphorylation of eEF-2. On the basis of kinetic and pharmacological analysis, we demonstrate a close correlation among the increase in cytosolic Ca 2ϩ concentration, the degree of eEF-2 phosphorylation, and the inhibition of protein synthesis. A 30 min treatment with NMDA induced a transient phosphorylation of eEF-2 and delayed neuronal death. However, pharmacological inhibition of protein translation was not neurotoxic by itself and protected neurons against the toxicity evoked by low concentrations of NMDA. Thus, phosphorylation of eEF-2 and the resulting depression of protein translation may have protective effects against excitotoxicity and open new perspectives for understanding long-term effects of glutamate.

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Identification of the phosphorylation sites in elongation factor‐2 from rabbit reticulocytes

Cited by 111 publications

References 32 publications

A pharmacoproteomic approach implicates eukaryotic elongation factor 2 kinase in ER stress-induced cell death

A pharmacoproteomic approach implicates eukaryotic elongation factor 2 kinase in ER stress-induced cell death

Evidence of Placental Translation Inhibition and Endoplasmic Reticulum Stress in the Etiology of Human Intrauterine Growth Restriction

Glutamate-Dependent Phosphorylation of Elongation Factor-2 and Inhibition of Protein Synthesis in Neurons

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