Mechanism of elongation factor 2 (EF‐2) inactivation upon phosphorylation Phosphorylated EF‐2 is unable to catalyze translocation

Ryazanov, Alexey G.; Davydova, Elena K.

doi:10.1016/0014-5793(89)81452-8

Cited by 151 publications

(121 citation statements)

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“…[22][23][24]49 We therefore asked whether eEF-2K is necessary for the full downregulation of translation observed during ER stress. We treated wild-type and eEF-2K À/À MEFs with Tg to induce rapid, synchronous ER stress and then metabolically labeled newly synthesized proteins with radioactive amino acids.…”

Section: Resultsmentioning

confidence: 99%

“…21 The activity of eEF-2 can be regulated post-translationally: phosphorylation of eEF-2 on Thr56 inhibits its translational function by blocking its ability to promote ribosome translocation. [22][23][24] The phosphorylation of eEF-2 is catalyzed by eEF-2 kinase (eEF-2K), an unusual calcium/calmodulin-dependent enzyme belonging to the alpha kinase family of atypical protein kinases. 25 EEF-2 phosphorylation has been shown to play an important role in coupling protein synthesis to energy metabolism in response to calcium flux, 26 cyclic adenosine monophosphate (AMP)/protein kinase A and AMP-activated protein kinase signaling, [27][28][29][30] amino acid or glucose limitation, 31,32 and cytoplasmic pH changes, 33 but no role for eEF-2 phosphorylation in ER stress has been reported.…”

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confidence: 99%

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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: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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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“…30 -34 The dephosphorylated form of eEF2 is the active protein that gets inactivated on threonine phosphorylation. 33 HMVECs were pretreated with 1 mmol/L Cys, an inhibitor of TGM2, or 5 mmol/L PAA, a PCB inhibitor before resveratrol treatment and tested in the scratch and 3 H thymidine proliferation assays. PAA and Cys failed to neutralize the effect of resveratrol on migration or proliferation of HMVECs (see supplemental Figure S3, D-F at http://ajp.amjpathol.org).…”

Section: Pcb and Tgm2 Do Not Mediate Resveratrol-induced Effects On Ementioning

confidence: 99%

Resveratrol Regulates Pathologic Angiogenesis by a Eukaryotic Elongation Factor-2 Kinase-Regulated Pathway

Khan

Dace

Ryazanov

et al. 2010

The American Journal of Pathology

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Abnormal angiogenesis is central to the pathophysiology of diverse disease processes including cancers, ischemic and atherosclerotic heart disease, and visually debilitating eye disease. Resveratrol is a naturally occurring phytoalexin that has been demonstrated to ameliorate and decelerate the aging process as well as blunt end organ damage from obesity. These effects of resveratrol are largely mediated by members of the sirtuin family of proteins. We demonstrate that resveratrol can inhibit pathological angiogenesis in vivo and in vitro by a sirtuin-independent pathway. Resveratrol inhibits the proliferation and migration of vascular endothelial cells by activating eukaryotic elongation factor-2 kinase. The active kinase in turn phosphorylates and inactivates elongation factor-2, a key mediator of ribosomal transfer and protein translation. Functional inhibition of the kinase by gene deletion in vivo or RNA as well as pharmacological inhibition in vitro is able to completely reverse the effects of resveratrol on blood vessel growth. These studies have identified a novel and critical pathway that promotes aberrant vascular proliferation and one that is amenable to modulation by pharmacological means. In addition, these results have uncovered a sirtuin-independent pathway by which resveratrol regulates angiogenesis.

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“…It is phosphorylated on threonine residues by a specific eEF-2 kinase, (previously termed Caa+/calmodulin.dependent protein kinase III [3,4]) and its phosphorylation is increased in intact ceils in response to stimuli which increase intracellular Ca z÷'ion concentrations [6][7][8][9][10][11][12][13]. Phosphorylation of the endogenous eEF-2 impairs the translation of mRNA in the reticulocyte lysate celt-free system [5] and several other lines of evidence show that phosphorylated eEF-2 is inactive, or has only low activity [3,4,14,15].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 1991

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The sites in ~ukaryolic elongation factor eEF-2 phosphorylated by the Ca~*tcalmodulin.depcnd¢nt dZF.2 ktnas© in vitro have been identified, The kinase eatalysed the rapid mcorperation of one real ol'phosphate par real cEF.2 .nd the slower incorporation of a second mol. All the phosphorylation sit,:s in eE F-2 are contained in the CN Br fragment ¢orrespondinil to residues 22~ 155. TP/ptie discstion cf phosphorylnted QEF.2 yielded 3 phospkopeptides, one being tlnique to monophosphoryl~ted eF.F.2, The phosphorylation sites w©re identified as threonine residues 56 and 58. tha fona~r bein8 more rapidly phosphorylated, Ala.Gly.Glu-Thr.Phc-Th#*-Asp.Thr'~.Arl~. The same sites are labelled in eEF-2 isolated from reticulocyte lysates,

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Mechanism of elongation factor 2 (EF‐2) inactivation upon phosphorylation Phosphorylated EF‐2 is unable to catalyze translocation

Cited by 151 publications

References 12 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

Resveratrol Regulates Pathologic Angiogenesis by a Eukaryotic Elongation Factor-2 Kinase-Regulated Pathway

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

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