Abstract
Protein synthesis consumes a high proportion of the metabolic energy of mammalian cells, and most of this is used by peptide chain elongation. An important regulator of energy supply and demand in eukaryotic cells is the AMP-activated protein kinase (AMPK). The rate of peptide chain elongation can be modulated through the phosphorylation of eukaryotic elongation factor (eEF) 2, which inhibits its activity and is catalyzed by a specific calcium/calmodulin-dependent protein kinase termed eEF2 kinase. Here we show that AMPK directly phosphorylates eEF2 kinase, and we identify the major site of phosphorylation as Ser-398 in a regulatory domain of eEF2 kinase. AMPK also phosphorylates two other sites (Ser-78 and Ser-366) in eEF2 kinase in vitro. We develop appropriate phosphospecific antisera and show that phosphorylation of Ser-398 in eEF2 kinase is enhanced in intact cells under a range of conditions that activate AMPK and increase the phosphorylation of eEF2. Ser-78 and Ser-366 do not appear to be phosphorylated by AMPK within cells. Although cardiomyocytes appear to contain a distinct isoform of eEF2 kinase, it also contains a site corresponding to Ser-398 that is phosphorylated by AMPK in vitro. Stimuli that activate AMPK and increase eEF2 phosphorylation within cells increase the activity of eEF2 kinase. Thus, AMPK and eEF2 kinase may provide a key link between cellular energy status and the inhibition of protein synthesis, a major consumer of metabolic energy.
Highlights
Protein synthesis is a major consumer of cellular energy with estimates of 30 –50% of total energy being used in this process
Given that protein synthesis uses a high proportion of cellular energy, it is perhaps surprising that so little work has previously been devoted to understanding how cells might coordinate the rate of protein synthesis with the availability of metabolic energy
This fits well with our findings that suggest that AMPK is a key link between cellular status and the regulation of protein synthesis; AMPK would be activated under the latter condition but not under the former
Summary
Protein synthesis is a major consumer of cellular energy with estimates of 30 –50% of total energy being used in this process. When GST-eEF2 kinase was incubated with AMPK and cold ATP and subsequently analyzed by SDS-PAGE and Western blotting, clear signals were seen using the anti-Ser(P)-78 and anti-Ser(P)398 antisera, as well as for a phosphospecific antiserum for Ser-366 that had been developed by us previously [10] (Fig. 2, A–C).
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