Identification of autophosphorylation sites in eukaryotic elongation factor-2 kinase


Pyr Dit Ruys, Sebastien, Wang, Xuemin, Smith, Ewan M., Herinckx, Gaetan, Hussain, Nusrat, Rider, Mark H., Vertommen, Didier and Proud, Christopher G. (2012) Identification of autophosphorylation sites in eukaryotic elongation factor-2 kinase. The Biochemical Journal, 442, (3), 681-692. (doi:10.1042/BJ20111530).

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Description/Abstract

eEF2K [eEF2 (eukaryotic elongation factor 2) kinase] phosphorylates and inactivates the translation elongation factor eEF2. eEF2K is not a member of the main eukaryotic protein kinase superfamily, but instead belongs to a small group of so-called α-kinases. The activity of eEF2K is normally dependent upon Ca2+ and calmodulin. eEF2K has previously been shown to undergo autophosphorylation, the stoichiometry of which suggested the existence of multiple sites. In the present study we have identified several autophosphorylation sites, including Thr348, Thr353, Ser366 and Ser445, all of which are highly conserved among vertebrate eEF2Ks. We also identified a number of other sites, including Ser78, a known site of phosphorylation, and others, some of which are less well conserved. None of the sites lies in the catalytic domain, but three affect eEF2K activity. Mutation of Ser78, Thr348 and Ser366 to a non-phosphorylatable alanine residue decreased eEF2K activity. Phosphorylation of Thr348 was detected by immunoblotting after transfecting wild-type eEF2K into HEK (human embryonic kidney)-293 cells, but not after transfection with a kinase-inactive construct, confirming that this is indeed a site of autophosphorylation. Thr348 appears to be constitutively autophosphorylated in vitro. Interestingly, other recent data suggest that the corresponding residue in other α-kinases is also autophosphorylated and contributes to the activation of these enzymes [Crawley, Gharaei, Ye, Yang, Raveh, London, Schueler-Furman, Jia and Cote (2011) J. Biol. Chem. 286, 2607–2616]. Ser366 phosphorylation was also detected in intact cells, but was still observed in the kinase-inactive construct, demonstrating that this site is phosphorylated not only autocatalytically but also in trans by other kinases.

Item Type: Article
ISSNs: 0264-6021 (print)
1470-8728 (electronic)
Subjects: Q Science > QH Natural history > QH301 Biology
Divisions: Faculty of Natural and Environmental Sciences > Biological Sciences > Molecular & Cellular
ePrint ID: 337172
Date Deposited: 20 Apr 2012 08:52
Last Modified: 27 Mar 2014 20:20
URI: http://eprints.soton.ac.uk/id/eprint/337172

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