Phosphorylation of elongation factor-2 kinase on serine 499 by cAMP-dependent protein kinase induces Ca2+/calmodulin-independent activity

Abstract

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.

P38α-MAPK Signaling Inhibition Attenuates Soleus Atrophy during Early Stages of Muscle Unloading

“…It was previously reported that eEF2 phosphorylation suppresses eEF2 binding to the ribosome and inhibits polypeptide chain growth [44]. eEF2 phosphorylation is regulated by calcium concentrations, low pH value, activities of protein kinase A, and AMP-activated protein kinase [45][46][47], which leads to the inhibition of elongation. In our experiments, eEF2 phosphorylation was increased by unloading and was not regulated by the treatment with VX-745 inhibitor, whereas unloading-induced muscle atrophy was blocked in the HSVX group.…”

Noradrenaline enhances monocarboxylate transporter 2 expression in cultured mouse cortical neurons via a translational regulation

“…Several examples whereby cAMP elevations (Lawrence et al . 1997; Morgan and Beinlich 1997), protein kinase A activation (Diggle et al . 2001) or even stimulation of β‐adrenergic receptors (McLeod et al .…”

Regulation of peptide‐chain elongation in mammalian cells

“…The first evidence for an additional mechanism for controlling eEF2 kinase activity besides its activation by Ca 2+ /calmodulin was provided by the observation that cAMP‐dependent protein kinase (PKA) can phosphorylate eEF2 kinase [36,48]. This results in eEF2 kinase becoming partially independent of Ca 2+ /calmodulin for activity [48,49], i.e. it activates eEF2 kinase at low basal Ca 2+ levels.…”

“…Diggle et al . [49] identified the site phosphorylated by PKA in rat eEF2 kinase as Ser499 (Ser500 is the human sequence), which lies outside the putative catalytic domain (Fig. 2) in a relatively poor consensus for phosphorylation by PKA.…”