Identification of autophosphorylation sites in eukaryotic elongation factor-2 kinase
| Autor: | Gaëtan Herinckx, Sébastien Pyr dit Ruys, Ewan M. Smith, Nusrat Hussain, Mark H. Rider, Didier Vertommen, Xuemin Wang, Christopher G. Proud |
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| Přispěvatelé: | UCL - SSS/DDUV - Institut de Duve, UCL - SSS/DDUV/PHOS - Protein phosphorylation |
| Jazyk: | angličtina |
| Rok vydání: | 2012 |
| Předmět: |
Threonine
Translation calmodulin translation elongation Biochemistry 0302 clinical medicine Correction Article Catalytic Domain GST glutathione transferase Serine 2D two-dimensional Phosphorylation 0303 health sciences education.field_of_study CaM calmodulin biology ESI electrospray ionization Kinase LC liquid chromatography Autophosphorylation Transfection MHCKA myosin heavy chain kinase A 030220 oncology & carcinogenesis α-kinase eEF2K eEF2 kinase Research Article TRP transient receptor potential Elongation Factor 2 Kinase Calmodulin eukaryotic Elongation Factor 2 (eEF2) EEF2 03 medical and health sciences HEK human embryonic kidney Mass Spectrometry (MS) Humans mass spectrometry (MS) Elongation eEF2 eukaryotic elongation factor 2 education Protein kinase A Molecular Biology 030304 developmental biology Cell Biology Molecular biology Elongation factor HEK293 Cells biology.protein Elongation Factor-2 Kinase eukaryotic elongation factor 2 (eEF2) 030217 neurology & neurosurgery |
| Zdroj: | Biochemical Journal The Biochemical journal, Vol. 442, no. 3, p. 681-692 (2012) |
| ISSN: | 1470-8728 0264-6021 |
| Popis: | 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 Ca(2+) 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 Thr(348), Thr(353), Ser(366) and Ser(445), all of which are highly conserved among vertebrate eEF2Ks. We also identified a number of other sites, including Ser(78), 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 Ser(78), Thr(348) and Ser(366) to a non-phosphorylatable alanine residue decreased eEF2K activity. Phosphorylation of Thr(348) 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. Thr(348) 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]. Ser(366) 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. |
| Databáze: | OpenAIRE |
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