Functional properties of phosphorylated elongation factor 2

Carlberg, U.; Nilsson, Anders; Nygård, Odd

doi:10.1111/j.1432-1033.1990.tb19169.x

Cited by 252 publications

(209 citation statements)

References 39 publications

(13 reference statements)

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“…If the interaction of the phosphorylated factor were tighter than that of non-phosphorylated eEF-2 it could then exert a dominant inhibitory effect by blocking the binding, and thus the action, of nonphosphorylated eEF-2. However, as mentioned above, Nyg h d and co-workers have reported that phosphorylated eEF-2 (apparently mono-phosphorylated) failed to bind efficiently to ribosomes [19]. This was due to a 10-100-fold decrease in affinity for ribosomes in complexes resembling the pretranslocational complexes which arise in elongation.…”

Section: Discussionmentioning

confidence: 93%

Regulation of elongation factor‐2 by multisite phosphorylation

Redpath

Price

Severinov

et al. 1993

European Journal of Biochemistry

172

149

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We have studied the phosphorylation of protein synthesis elongation factor eEF-2, the effects of phosphorylation on its activity and the dephosphorylation of phosphorylated eEF-2 by protein phosphatases-2A and -2C. Extensive analysis of phosphopeptides generated from eEF-2 phosphorylated in v i m by subsequent digestion with CNBr and trypsin indicated that Thr56 and Thr58 are the only residues significantly phosphorylated, consistent with our earlier report. They are also the only two residues to be significantly phosphorylated in reticulocyte lysates : in this system monophosphorylated eEF-2 corresponded only to phosphorylation of Thr56, no factor phosphorylated at only Thr58 being detected. Phosphorylation of Thr56 and Thr58 was found to be an ordered process, modification of Thr.56 preceding, and apparently being required for, phosphorylation of Thr58. This presumably explains why the only species of mono-phosphorylated eEF-2 detected are phosphorylated at Thr56.The eEF-2 kinase could phosphorylate a synthetic peptide based on residues 49-60 of eEF-2 (RAGETRFTDTRK), albeit only at a very low rate, and with a very high K,, compared to eEF-2 itself. The kinase phosphorylated the residues corresponding to Thr56 and Thr58, apparently in a random manner, but not Thr53.In the light of the existence of two phosphorylation sites in eEF-2, the relationship between phosphorylation and activity was investigated. Activity was measured in the poly(U)-directed synthesis of polyphenylalanine, where both the bis-and mono-phosphorylated (mono at Thr56) forms of the factor were found to be completely inactive. Indeed, the phosphorylated species appeared to be able to impair the activity of non-phosphorylated eEF-2 in this system. Experiments using reticulocyte lysates also indicated that both phosphorylated forms of eEF-2 were inactive in the translation of physiological templates, but no evidence for dominant inhibition by these species was obtained.Protein phosphatases-2A and -2C (PP-2A and PP-2C) can each efficiently dephosphorylate phosphorylated eEF-2. While bis-phosphorylated eEF-2 was a better substrate for PP-2A than monophosphorylated factor (phosphorylated at Thr56), the converse was true for PP-2C. This seemed to be due, at least in part, to the inhibition of dephosphorylation of Thr56 by PP-2C by the presence of phosphate on Thr58. Nevertheless, PP-2C exhibited a preference for dephosphorylation of Thr56 in bis-phosphorylated eEF-2, while PP-2A showed no such preference. These findings are discussed in terms of current knowledge of the specificity of these two protein phosphatases.Elongation factor-2 (eEF-2) mediates the translocation step of peptide-chain elongation in eukaryotic cells. It is a monomeric protein of apparent M, 100000 which binds guanine nucleotides and also possesses ribosome-stimulated GTPase activity [l].eEF-2 is phosphorylated by a specific eEF-2 kinase on threonine residues [2, 31. The eEF-2 kinase is absolutely dependent on Ca2+/calmodulin for activity and is widely distributed in mammalian t...

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Section: Discussionmentioning

confidence: 93%

Regulation of elongation factor‐2 by multisite phosphorylation

Redpath

Price

Severinov

et al. 1993

European Journal of Biochemistry

172

149

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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%

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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“…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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Functional properties of phosphorylated elongation factor 2

Cited by 252 publications

References 39 publications

Regulation of elongation factor‐2 by multisite phosphorylation

Regulation of elongation factor‐2 by multisite phosphorylation

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

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

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