scholarly journals A Novel mTOR-Regulated Phosphorylation Site in Elongation Factor 2 Kinase Modulates the Activity of the Kinase and Its Binding to Calmodulin

2004 ◽  
Vol 24 (7) ◽  
pp. 2986-2997 ◽  
Author(s):  
Gareth J. Browne ◽  
Christopher G. Proud
Keyword(s):  
Amino Acids ◽  
Protein Kinase ◽  
Phosphorylation Site ◽  
Elongation Factor ◽  
Mtor Pathway ◽  
Mtor Signaling ◽  
Chain Elongation ◽  
Binding Motif ◽  
Elongation Factor 2 ◽  
Eef2 Kinase

ABSTRACT Eukaryotic elongation factor 2 (eEF2) kinase is an unusual calcium- and calmodulin-dependent protein kinase that is regulated by insulin through the rapamycin-sensitive mTOR pathway. Here we show that insulin decreases the ability of eEF2 kinase to bind calmodulin in a rapamycin-sensitive manner. We identify a novel phosphorylation site in eEF2 kinase (Ser78) that is located immediately next to its calmodulin-binding motif. Phosphorylation of this site is increased by insulin in a rapamycin-sensitive fashion. Regulation of the phosphorylation of Ser78 also requires amino acids and the protein kinase phosphoinositide-dependent kinase 1. Mutation of this site to alanine strongly attenuates the effects of insulin and rapamycin both on the binding of calmodulin to eEF2 kinase and on eEF2 kinase activity. Phosphorylation of Ser78 is thus likely to link insulin and mTOR signaling to the control of eEF2 phosphorylation and chain elongation. This site is not a target for known kinases in the mTOR pathway, e.g., the S6 kinases, implying that it is phosphorylated by a novel mTOR-linked protein kinase that serves to couple hormones and amino acids to the control of translation elongation. eEF2 kinase is thus a target for mTOR signaling independently of previously known downstream components of the pathway.

scholarly journals Stress-induced regulation of eukaryotic elongation factor 2 kinase by SB 203580-sensitive and −insensitive pathways

2002 ◽  
Vol 367 (2) ◽  
pp. 525-532 ◽  
Author(s):  
Axel KNEBEL ◽  
Claire E. HAYDON ◽  
Nick MORRICE ◽  
Philip COHEN
Keyword(s):  
Protein Kinase ◽  
Elongation Factor ◽  
Elongation Factor 2 ◽  
Eukaryotic Elongation Factor 2 ◽  
Eukaryotic Elongation Factor ◽  
Tnf Α ◽  
High Concentrations ◽  
Eef2 Kinase ◽  
Sb 203580

Eukaryotic elongation factor 2 (eEF2) kinase, the enzyme that inactivates eEF2, is controlled by phosphorylation. Previous work showed that stress-activated protein kinase 4 (SAPK4, also called p38Δ) inhibits eEF2 kinase in vitro by phosphorylating Ser-359, while ribosomal protein S6 kinases inhibit eEF2 kinase by phosphorylating Ser-366 [Knebel, Morrice and Cohen (2001) EMBO J. 20, 4360—4369; Wang, Li, Williams, Terada, Alessi and Proud (2001) EMBO J. 20, 4370—4379]. In the present study we have examined the effects of the protein synthesis inhibitor anisomycin and tumour necrosis factor-α (TNF-α) on the phosphorylation of eEF2 kinase. We demonstrate that Ser-359, Ser-366 and two novel sites (Ser-377 and Ser-396) are all phosphorylated in human epithelial KB cells, but only the phosphorylation of Ser-359 and Ser-377 increases in response to these agonists and correlates with the dephosphorylation (activation) of eEF2. Ser-377 is probably a substrate of MAPKAP-K2/K3 (mitogen-activated protein kinase-activated protein kinase 2/kinase 3) in cells, because eEF2 kinase is phosphorylated efficiently by these protein kinases in vitro and phosphorylation of this site, induced by TNF-α and low (but not high) concentrations of anisomycin, is prevented by SB 203580, which inhibits SAPK2a/p38, their ‘upstream’ activator. The phosphorylation of Ser-359 induced by high concentrations of anisomycin is probably catalysed by SAPK4/p38Δ in cells, because no other stress-activated, proline-directed protein kinase tested phosphorylates this site in vitro and phosphorylation is insensitive to SB 203580. Interestingly, the phosphorylation of Ser-359 induced by TNF-α or low concentrations of anisomycin is suppressed by SB 203580, indicating that phosphorylation is also mediated by a novel pathway. Since the phosphorylation of Ser-377 does not inhibit eEF2 kinase in vitro, our results suggest that anisomycin or TNF-α inhibit eEF2 kinase via the phosphorylation of Ser-359.

scholarly journals The substrate specificity of adenosine 3′:5′-cyclic monophosphate-dependent protein kinase of rabbit skeletal muscle

1977 ◽  
Vol 162 (2) ◽  
pp. 411-421 ◽  
Author(s):  
S J Yeaman ◽  
P Cohen ◽  
D C Watson ◽  
G H Dixon
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Kinase ◽  
Phosphorylation Site ◽  
Amino Acid Sequences ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Phosphorylase Kinase ◽  
Dependent Protein Kinase ◽  
Dependent Protein

The known amino acid sequences at the two sites on phosphorylase kinase that are phosphorylated by cyclic AMP-dependent protein kinase were extended. The sequences of 42 amino acids around the phosphorylation site on the alpha-subunit and of 14 amino acids around the phosphorylation site on the beta-subunit were shown to be: alpha-subunit Phe-Arg-Arg-Leu-Ser(P)-Ile-Ser-Thr-Glu-Ser-Glx-Pro-Asx-Gly-Gly-His-Ser-Leu-Gly-Ala-Asp-Leu-Met-Ser-Pro-Ser-Phe-Leu-Ser-Pro-Gly-Thr-Ser-Val-Phe(Ser,Pro,Gly)His-Thr-Ser-Lys; beta-subunit, Ala-Arg-Thr-Lys-Arg-Ser-Gly-Ser(P)-VALIle-Tyr-Glu-Pro-Leu-Lys. The sites on histone H2B which are phosphorylated by cyclic AMP-dependent protein kinase in vitro were identified as serine-36 and serine-32. The amino acid sequence in this region is: Lys-Lys-Arg-Lys-Arg-Ser32(P)-Arg-Lys-Glu-Ser36(P)-Tyr-Ser-Val-Tyr-Val- [Iwai, K., Ishikawa, K. & Hayashi, H. (1970) Nature (London) 226, 1056-1058]. Serine-36 was phosphorylated at 50% of the rate at which the beta-subunit of phosphorylase kinase was phosphorylated, and it was phosphorylated 6-7-fold more rapidly than was serine-32. The amino acid sequences when compared with those at the phosphorylation sites of other physiological substrates suggest that the presence of two adjacent basic amino acids on the N-terminal side of the susceptible serine residue may be critical for specific substrate recognition in vivo.

scholarly journals Cyclic AMP-dependent protein kinase phosphorylates rabbit reticulocyte elongation factor-2 kinase and induces calcium-independent activity

1993 ◽  
Vol 293 (1) ◽  
pp. 31-34 ◽  
Author(s):  
N T Redpath ◽  
C G Proud
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Elongation Factor ◽  
Total Activity ◽  
Dependent Protein Kinase ◽  
Calcium Calmodulin Dependent ◽  
Elongation Factor 2 ◽  
Eukaryotic Elongation Factor ◽  
Dependent Protein ◽  
Phosphopeptide Mapping

The catalytic subunit of cyclic AMP-dependent protein kinase (PKA) phosphorylated purified calcium/calmodulin-dependent eukaryotic elongation factor-2 (eEF-2) kinase, isolated from rabbit reticulocyte lysates. It maximally incorporated about 1 mol of phosphate/mol of eEF-2 kinase. The Km of eEF-2 kinase for PKA was calculated to be 7 microM. Phosphorylation of eEF-2 kinase by PKA induced calcium-independent activity which amounted to 40-50% of the total activity measured in the presence of calcium. Furthermore, the level of calcium-independent activity induced by phosphorylation by PKA was similar to that induced by the calcium-stimulated autophosphorylation of eEF-2 kinase. Phosphopeptide mapping of eEF-2 kinase labelled by autophosphorylation and by PKA revealed a number of common phosphopeptides. This suggests that PKA may phosphorylate the same site(s) which are phosphorylated autocatalytically and which are responsible for the induction of calcium-independent activity. The possible implications these findings have for the control of translation are discussed.

scholarly journals Insights into the regulation of eukaryotic elongation factor 2 kinase and the interplay between its domains

2012 ◽  
Vol 442 (1) ◽  
pp. 105-118 ◽  
Author(s):  
Craig R. Pigott ◽  
Halina Mikolajek ◽  
Claire E. Moore ◽  
Stephen J. Finn ◽  
Curtis W. Phippen ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Catalytic Domain ◽  
Functional Organization ◽  
Elongation Factor ◽  
Kinase Domain ◽  
Conserved Residues ◽  
Elongation Factor 2 ◽  
Eukaryotic Elongation Factor 2 ◽  
Kinase Catalytic Domain ◽  
Eukaryotic Elongation Factor

eEF2K (eukaryotic elongation factor 2 kinase) is a Ca2+/CaM (calmodulin)-dependent protein kinase which regulates the translation elongation machinery. eEF2K belongs to the small group of so-called ‘α-kinases’ which are distinct from the main eukaryotic protein kinase superfamily. In addition to the α-kinase catalytic domain, other domains have been identified in eEF2K: a CaM-binding region, N-terminal to the kinase domain; a C-terminal region containing several predicted α-helices (resembling SEL1 domains); and a probably rather unstructured ‘linker’ region connecting them. In the present paper, we demonstrate: (i) that several highly conserved residues, implicated in binding ATP or metal ions, are critical for eEF2K activity; (ii) that Ca2+/CaM enhance the ability of eEF2K to bind to ATP, providing the first insight into the allosteric control of eEF2K; (iii) that the CaM-binding/α-kinase domain of eEF2K itself possesses autokinase activity, but is unable to phosphorylate substrates in trans; (iv) that phosphorylation of these substrates requires the SEL1-like domains of eEF2K; and (v) that highly conserved residues in the C-terminal tip of eEF2K are essential for the phosphorylation of eEF2, but not a peptide substrate. On the basis of these findings, we propose a model for the functional organization and control of eEF2K.

scholarly journals Insights Into the Pathologic Roles and Regulation of Eukaryotic Elongation Factor-2 Kinase

2021 ◽  
Vol 8 ◽  
Author(s):  
Darby J. Ballard ◽  
Hao-Yun Peng ◽  
Jugal Kishore Das ◽  
Anil Kumar ◽  
Liqing Wang ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase ◽  
Elongation Factor ◽  
Protein Translation ◽  
Negative Regulator ◽  
Translation Elongation ◽  
Elongation Factor 2 ◽  
Global Rate ◽  
Eukaryotic Elongation Factor 2 ◽  
Eukaryotic Elongation Factor

Eukaryotic Elongation Factor-2 Kinase (eEF2K) acts as a negative regulator of protein synthesis, translation, and cell growth. As a structurally unique member of the alpha-kinase family, eEF2K is essential to cell survival under stressful conditions, as it contributes to both cell viability and proliferation. Known as the modulator of the global rate of protein translation, eEF2K inhibits eEF2 (eukaryotic Elongation Factor 2) and decreases translation elongation when active. eEF2K is regulated by various mechanisms, including phosphorylation through residues and autophosphorylation. Specifically, this protein kinase is downregulated through the phosphorylation of multiple sites via mTOR signaling and upregulated via the AMPK pathway. eEF2K plays important roles in numerous biological systems, including neurology, cardiology, myology, and immunology. This review provides further insights into the current roles of eEF2K and its potential to be explored as a therapeutic target for drug development.

scholarly journals Inhibition by ricin of protein synthesis in vitro. Inhibition of the binding of elongation factor 2 and of adenosine diphosphate-ribosylated elongation factor 2 to ribosomes

1975 ◽  
Vol 146 (1) ◽  
pp. 127-131 ◽  
Author(s):  
L Montanaro ◽  
S Sperti ◽  
A Mattioli ◽  
G Testoni ◽  
F Stirpe
Keyword(s):  
Protein Synthesis ◽  
Binding Sites ◽  
Polypeptide Chain ◽  
Elongation Factor ◽  
Adenosine Diphosphate ◽  
Chain Elongation ◽  
Elongation Factor 2 ◽  
In Vitro Inhibition ◽  
Liver Ribosomes

The binding of EF2 (elongation factor 2) and of ADP-ribosyl-EF 2 to rat liver ribosomes is inhibited by ricin. This result suggests that the native enzyme and its ADP-ribose derivative have the same or closely related binding sites on the ribosome. The inhibition by ricin of the binding of EF 2 to ribosomes is consistent with the previous observation that ricin affects EF 2-catalysed translocation during polypeptide chain elongation.

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