Elongation factor-2 kinase (eEF-2K) negatively regulates mRNA translation via the phosphorylation and inactivation of elongation factor-2 (eEF-2). We have shown previously that purified eEF-2K can be phosphorylated in vitro by cAMP-dependent protein kinase (PKA) and that this induces significant Ca2+/calmodulin (CaM)-independent eEF-2K activity [Redpath and Proud (1993) Biochem. J. 293, 31–34]. Furthermore, elevation of cAMP levels in adipocytes also increases the level of Ca2+/CaM-independent eEF-2K activity to a similar extent, providing a mechanistic link between elevated cAMP and the inhibition of protein synthesis [Diggle, Redpath, Heesom and Denton (1998) Biochem. J. 336, 525–529]. Here we describe the expression of glutathione S-transferase (GST)-eEF-2K fusion protein and the identification of two serine residues that are phosphorylated by PKA in vitro. Endoproteinase Arg-C digestion of GST-eEF-2K produced two phosphopeptides that were separated by HPLC and sequenced. 32P Radioactivity release from these peptides indicated that the sites of phosphorylation were Ser-365 and Ser-499, both of which lie C-terminal to the catalytic domain. Mutation of these sites to non-phosphorylatable residues indicated that both sites need to be phosphorylated to induce Ca2+/CaM-independent eEF-2K activity in vitro. However, expression of Myc-tagged eEF-2K in HEK 293 cells, followed by treatment with chlorophenylthio-cAMP (CPT-cAMP), showed that Ser-499 phosphorylation alone induced Ca2+/CaM-independent eEF-2K activity in cells. Co-expression of wild-type eEF-2K with luciferase resulted in a 2–3-fold reduction in luciferase expression. Expression of eEF-2K S499D resulted in a 10-fold reduction in luciferase expression despite the fact that this mutant was expressed at very low levels. This indicates that eEF-2K S499D is constitutively active when expressed in cells, thus leading to the suppression of its own expression. Our data demonstrate an important role for the phosphorylation of Ser-499 in the activation of eEF-2K by PKA and the inhibition of protein synthesis.
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February 2001
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Research Article|
January 25 2001
Phosphorylation of elongation factor-2 kinase on serine 499 by cAMP-dependent protein kinase induces Ca2+/calmodulin-independent activity
Tricia A. DIGGLE;
Tricia A. DIGGLE
1
*Department of Biochemistry, University of Leicester, University Road, Leicester LE1 7RH, U.K.
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Tatiana SUBKHANKULOVA;
Tatiana SUBKHANKULOVA
*Department of Biochemistry, University of Leicester, University Road, Leicester LE1 7RH, U.K.
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Kathryn S. LILLEY;
Kathryn S. LILLEY
†Protein and Nucleic Acid Laboratory, University of Leicester, University Road, Leicester LE1 7RH, U.K.
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Nita SHIKOTRA;
Nita SHIKOTRA
*Department of Biochemistry, University of Leicester, University Road, Leicester LE1 7RH, U.K.
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Anne E. WILLIS;
Anne E. WILLIS
*Department of Biochemistry, University of Leicester, University Road, Leicester LE1 7RH, U.K.
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Nicholas T. REDPATH
Nicholas T. REDPATH
2
‡MRC Laboratory for Molecular Cell Biology, University College London, Gower Street, London WC1E 6BT, U.K.
2To whom correspondence should be addressed (e-mail n.redpath@ucl.ac.uk).
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Publisher: Portland Press Ltd
Received:
August 11 2000
Revision Received:
October 16 2000
Accepted:
November 07 2000
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 2001
2001
Biochem J (2001) 353 (3): 621–626.
Article history
Received:
August 11 2000
Revision Received:
October 16 2000
Accepted:
November 07 2000
Citation
Tricia A. DIGGLE, Tatiana SUBKHANKULOVA, Kathryn S. LILLEY, Nita SHIKOTRA, Anne E. WILLIS, Nicholas T. REDPATH; Phosphorylation of elongation factor-2 kinase on serine 499 by cAMP-dependent protein kinase induces Ca2+/calmodulin-independent activity. Biochem J 1 February 2001; 353 (3): 621–626. doi: https://doi.org/10.1042/bj3530621
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