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

Elongation factor-2 kinase (eEF-2K) negatively regulates mRNA translation v ia the phosphorylation and inactivation of elongation factor-2 (eEF-2). We have shown previously that purified eEF-2K can be phosphorylated in vitro b y cAMP-dependent protein kinase (PKA) and that this induces significant Ca2 +/calmodulin (CaM)-independent eEF-ZK activity [Redpath and Proud (1993) Bi ochem. J, 2931 31-34]. Furthermore, elevation of cAMP levels in adipocytes also increases the level of Ca2+/CaM-independent eEF-2K activity to a simil ar extent, providing a mechanistic link between elevated cAMP and the inhib ition of protein synthesis [Diggle, Redpath, Heesom and Denton (1998) Bioch em, J. 336, 575-529]. Here we describe the expression of glutathione S-tran sferase (GST)-eEF-2K fusion protein and the identification of two serine re sidues that are phosphorylated by PKA in vitro. Endoproteinase Arg-C digest ion of GST-eEF-2K produced two phosphopeptides that were separated by HPLC and sequenced. P-32 Radioactivity release from these peptides indicated tha t 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-phosphor ylatable residues indicated that both sites need to be phosphorylated to in duce Ca2+/CaM-independent eEF-2K activity in cirro. However, expression of Myc-tagged eEF-2K in HEK 293 cells, followed by treatment with chlorophenyl thio-cAMP (CPT-cAMP), showed that Ser-499 phosphorylation alone induced Ca2 +/CaM-independent eEF-2K activity in cells. Go-expression of wild-type eEF- 2K with luciferase resulted in a 2-3-fold reduction in luciferase expressio n. 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 lev els. This indicates that eEF-'K S499D is constitutively active when express ed in cells, thus leading to the suppression of its own expression. Our dat a demonstrate an important role for the phosphorylation of Ser-499 in the a ctivation of eEF-2K by PKA and the inhibition of protein synthesis.