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

ABSTRACT: 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 '?-kinases' which are distinct from the main eukaryotic protein kinase superfamily. In addition to the ?-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 ?-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/?-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.

SUBMITTER: Pigott CR

PROVIDER: S-EPMC3268225 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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

Pigott Craig R CR Mikolajek Halina H Moore Claire E CE Finn Stephen J SJ Phippen Curtis W CW Werner Jörn M JM Proud Christopher G CG

The Biochemical journal 20120201 1

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 S ...[more]

PMID: 22115317

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Project description: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 p38delta) 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-alpha (TNF-alpha) 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-alpha 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/p38delta 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-alpha 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-alpha inhibit eEF2 kinase via the phosphorylation of Ser-359.

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

Publications

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

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