Elongation Factor 2 Kinase Is Regulated by Proline Hydroxylation and Protects Cells during Hypoxia

Abstract

Protein synthesis, especially translation elongation, requires large amounts of energy, which is often generated by oxidative metabolism. Elongation is controlled by phosphorylation of eukaryotic elongation factor 2 (eEF2), which inhibits its activity and is catalyzed by eEF2 kinase (eEF2K), a calcium/calmodulin-dependent ␣-kinase. Hypoxia causes the activation of eEF2K and induces eEF2 phosphorylation independently of previously known inputs into eEF2K. Here, we show that eEF2K is subject to hydroxylation on proline-98. Proline hydroxylation is catalyzed by proline hydroxylases, oxygen-dependent enzymes which are inactivated during hypoxia. Pharmacological inhibition of proline hydroxylases also stimulates eEF2 phosphorylation. Pro98 lies in a universally conserved linker between the calmodulin-binding and catalytic domains of eEF2K. Its hydroxylation partially impairs the binding of calmodulin to eEF2K and markedly limits the calmodulin-stimulated activity of eEF2K. Neuronal cells depend on oxygen, and eEF2K helps to protect them from hypoxia. eEF2K is the first example of a protein directly involved in a major energy-consuming process to be regulated by proline hydroxylation. Since eEF2K is cytoprotective during hypoxia and other conditions of nutrient insufficiency, it may be a valuable target for therapy of poorly vascularized solid tumors. Many cells require aerobic metabolism to generate energy, necessitating an adequate supply of oxygen. Protein synthesis, especially translation elongation, is a major energy-consuming process, and translation elongation uses both ATP (for aminoacyl-tRNA charging) and GTP (at least two GTP equivalents are used during each round of the elongation process). Overall, at least four ATP equivalents are used for each amino acid added to the growing chain during elongation. Elongation rates can be regulated through the phosphorylation of eukaryotic elongation factor 2 (eEF2) (1). Phosphorylation of eEF2 on Thr56 by eEF2 kinase (eEF2K) inhibits its ability to interact with ribosomes (2), thereby impairing translation elongation. Indeed, a range of studies has shown that increased phosphorylation of eEF2 is associated with slower ribosomal movement along the mRNA (e.g., see references 3 to 5).eEF2K interacts with calmodulin (CaM) through a binding site which lies almost immediately N terminal to its catalytic domain (6, 7). The catalytic domain belongs to the small group of (six) mammalian ␣-kinases, rather than the main protein kinase superfamily; ␣-kinases show no sequence homology and only limited three-dimensional structural homology to other protein kinases (8, 9). eEF2K activity is regulated through several signaling pathways linked, e.g., to nutrient availability; these include signaling through the mammalian target of rapamycin complex 1 (mTORC1), which represses eEF2K activity, and the AMPactivated protein kinase (AMPK), a key cellular energy sensor (10) which causes activation of eEF2K (11, 12), probably in part by inhibiting mTORC1 signaling. Both inputs operate such th...

Structural Basis for the Recognition of Eukaryotic Elongation Factor 2 Kinase by Calmodulin

“…Given that N-lobe based interactions are weaker with respect to those involving the C-lobe, and themselves do not appear to be critical, hydroxylation of P98 should have a relatively small effect on CaM binding, in agreement with the data of Moore et. al (Moore et al, 2015). In the same study it was also shown that mutation of W99 ( W99A , W99L ) does not lead to a significant reduction in CaM binding, as expected from our structure.…”

“…Additionally, the W85S mutant is severely compromised in its ability to phosphorylate eEF-2 in cells. Recent studies have shown that hydroxylation on P98 (also fully conserved, Figure S8) by hydroxylases, that are inactivated during hypoxic conditions, lead to activation of eEF-2K and impaired protein translation (Moore et al, 2015). We have shown that P98 contributes to the engagement of the N-lobe and based on our structure it is conceivable that the introduction of a polar group on P98 would reduce its hydrophobic interactions with the N-lobe of CaM.…”

Eukaryotic elongation factor 2 kinase as a drug target in cancer, and in cardiovascular and neurodegenerative diseases

“…During hypoxia, eEF2K is activated by a mechanism that involves proline hydroxylases, which require oxygen as a co-substrate and are involved in other effects of hypoxia, such as the regulation of the transcription factor hypoxia-inducible factor 1α (HIF1α). However, the regulation of eEF2K by oxygen appears not to involve HIF1α, but rather, the direct hydroxylation of eEF2K at a highly conserved proline (Pro98; Figure 1, 2) adjacent to the CaM-binding site [22] ; this residue is in a strongly conserved motif in the 'linker' between the CaMbinding motif and the catalytic domain of eEF2K. This modification limits the ability of CaM to activate eEF2K, keeping its activity low in oxygenated conditions.…”

New Insights into Protein Hydroxylation and Its Important Role in Human Diseases

“…eEF2K is a calcium/calmodulin-dependent (CaM) α kinase. Hypoxia or prolyl hydroxylase inhibition was reported to activate eEF2K kinase activity, thereby contributing to increased eEF2 phosphorylation [106]. As a result, cells demonstrate decreased translation elongation and protein synthesis under hypoxia, which protects them from excessive usage of ATP.…”