Phosphorylation of Eukaryotic Elongation Factor 2 (eEF2) by Cyclin A–Cyclin-Dependent Kinase 2 Regulates Its Inhibition by eEF2 Kinase

Abstract

Protein synthesis is highly regulated via both initiation and elongation. One mechanism that inhibits elongation is phosphorylation of eukaryotic elongation factor 2 (eEF2) on threonine 56 (T56) by eEF2 kinase (eEF2K). T56 phosphorylation inactivates eEF2 and is the only known normal eEF2 functional modification. In contrast, eEF2K undergoes extensive regulatory phosphorylations that allow diverse pathways to impact elongation. We describe a new mode of eEF2 regulation and show that its phosphorylation by cyclin A-cyclin-dependent kinase 2 (CDK2) on a novel site, serine 595 (S595), directly regulates T56 phosphorylation by eEF2K. S595 phosphorylation varies during the cell cycle and is required for efficient T56 phosphorylation in vivo. Importantly, S595 phosphorylation by cyclin A-CDK2 directly stimulates eEF2 T56 phosphorylation by eEF2K in vitro, and we suggest that S595 phosphorylation facilitates T56 phosphorylation by recruiting eEF2K to eEF2. S595 phosphorylation is thus the first known eEF2 modification that regulates its inhibition by eEF2K and provides a novel mechanism linking the cell cycle machinery to translational control. Because all known eEF2 regulation is exerted via eEF2K, S595 phosphorylation may globally couple the cell cycle machinery to regulatory pathways that impact eEF2K activity. G lobal protein synthesis is subject to complex regulation that allows cells to control this energy-intensive process in response to diverse physiologic cues. Translation is regulated at the initiation and elongation levels. For example, translation is repressed in the G 2 /M phase of the cell cycle (1, 2), in which inhibition of eukaryotic initiation factors represses mitotic translation (2-4). Translational control is also exerted at the level of elongation, and this often involves inhibition of eukaryotic elongation factor 2 (eEF2) (5-7).eEF2 is a GTP-dependent translocase that is responsible for the movement of nascent peptidyl-tRNAs from the A-site to the P-site of the ribosome. The only known normal mechanism that regulates eEF2 is an inhibitory phosphorylation at threonine 56 (T56), which falls within the eEF2 GTP-binding domain and prevents eEF2 from binding to the ribosome (8, 9). A single, atypical calmodulin-dependent kinase, termed eEF2 kinase (eEF2K), phosphorylates eEF2 on T56 (10-12). Many signals cause eEF2K to become phosphorylated on residues that inhibit or augment its activity (5,13,14). For example, the mitogen-activated protein kinase (MAPK) and mTOR pathways inhibit eEF2K in response to mitogen and nutrient signals (15-17). In contrast, AMP kinase-and protein kinase A/Ca 2ϩ -dependent signaling activates eEF2K in response to starvation, hypoxia, and oxidative stress (18-21). Thus, while many signaling pathways control eEF2 activity, this regulation is exerted exclusively via modification of eEF2K rather than eEF2. T56 phosphorylation is thus the only known eEF2 functional modification, other than its inhibition by ADP ribosylation catalyzed by bacterial toxins (6).Cyclin-dependent kinas...

Mechanism of Cytoplasmic mRNA Translation

“…This site is conserved relative to Ser595 of mammals. Interestingly, phosphorylation of Ser595 by cyclin A in mammals promotes phosphorylation of Thr56 by eEF2K (Hizli et al, 2013). The finding that the AteEF2 (LOS1) is important for protein synthesis at low temperatures (Guo et al, 2002) hints that regulation of eEF2 activity is relevant to cold acclimation and likely other stress conditions.…”

Environmental Stress Affects the Activity of Metabolic and Growth Factor Signaling Networks and Induces Autophagy Markers in MCF7 Breast Cancer Cells

“…Protein synthesis is also regulated at the level of translation elongation. eEF2 is required for the movement of peptidyltRNA along the ribosome during translation elongation, a process that is inhibited by phosphorylation of eEF2 T56 (59) and which involves the protein kinase eEF2K (60). We observed a modulation in the phosphorylation of eEF2K by environmental stress (Fig.…”

Pulsatile delivery of a leucine supplement during long-term continuous enteral feeding enhances lean growth in term neonatal pigs

“…Activation of mTORC1 results in the phosphorylation of the 70-kDa ribosomal protein S6 kinase 1 (p70S6K1) and the eukaryotic initiation factor (eIF)4E-binding protein 1 (4E-BP1), which promotes eIF4E association with eIF4G and translation initiation activation (47). Translation initiation is also controlled by phosphorylation of eIF2␣ (28), and peptide chain elongation regulation involves phosphorylation of eukaryotic elongation factor 2 (eEF2) (9,43).…”