Phosphorylation of elongation factor-2 kinase differentially regulates the enzyme's stability under stress conditions

Abstract

Eukaryotic elongation factor-2 kinase (eEF-2K) is a Ca(2+)/calmodulin-dependent enzyme that negatively regulates protein synthesis. eEF-2K has been shown to be up-regulated in cancer, and to play an important role in cell survival through inhibition of protein synthesis. Post-translational modification of protein synthesis machinery is important for its regulation and could be critical for survival of cancer cells encountering stress. The purpose of our study was to examine the regulation of eEF-2K during stress with a focus on the roles of phosphorylation in determining the stability of eEF-2K. We found that stress conditions (nutrient deprivation and hypoxia) increase eEF-2K protein. mRNA levels are only transiently increased and shortly return to normal, while eEF-2K protein levels continue to increase after further exposure to stress. A seemingly paradoxical decrease in eEF-2K stability was found when glioma cells were subjected to stress despite increased protein expression. We further demonstrated that phosphorylation of eEF-2K differentially affects the enzyme's turnover under both normal and stress conditions, as evidenced by the different half-lives of phosphorylation-defective mutants of eEF-2K. We further found that the eEF-2K site (Ser398) phosphorylated by AMPK is pivotal to the protein's stability, as the half-life of S398A mutant increases to greater than 24h under both normal and stress conditions. These data indicate that eEF-2K is regulated at multiple levels with phosphorylation playing a critical role in the enzyme's turnover under stressful conditions. The complexity of eEF-2K phosphorylation highlights the intricacies of protein synthesis control during cellular stress.

Fig. 1

Cellular stresses increase eEF-2K expression while decreasing eEF-2K protein stability. (A) T98G and LN229 cells were cultured in media deficient in serum, glutamine, or oxygen for 24 h. (B) T98G and LN229 cells were cultured in serum-containing or serum-free media for 24, 48, or 72 h. (C) T98G cells were subjected to various stresses for 24 or 48 h. At the end of treatment, eEF-2K protein and mRNA levels were measured. LN229 results (not shown) were similar. The bars represent the mean value ± SD from three independent experiments.

Fig. 2

Phosphorylation sites on eEF-2K differentially affect its turnover. (A) T98G^shVector and LN229^shVector cells were subjected to various stresses for 24 h, followed by treatment with 20 μM of cycloheximide (CHX). Cell samples were collected at time points from 0 to 24 h. LN299 results (not shown) are similar. (B) T98G ^shVector and LN229^shVector cells were treated with various stresses for 24 h. The levels of the respective proteins were examined by Western Blot. (C) LN229^shEF2K and T98G^shEF2K cells were transfected with the S78A/S366A eEF-2K phosphorylation-defective mutants for 24 h, followed by 24 h of various stress treatments. Cells were then treated with 20 μM of cycloheximide (CHX) and collected at time points from 0 to 24 h. (D) LN229^shEF2K and T98G^shEF2K cells were transfected with the S398A eEF-2K phosphorylation mutants for 24 h, followed by 24 h of various stress treatments. Cells were then treated with 20 μM of CHX and collected at time points from 0 to 24 h. *p < 0.05.

Fig. 3

Inhibition of the mTOR pathway with rapamycin increases eEF-2K stability regardless of stress. (A) T98G ^shVector and LN229^shVector cells were treated with 100 ng/ml rapamycin and exposed to various stresses for 48 h. eEF-2K protein expression was then examined. (B) T98G ^shVector and LN229^shVector cells were treated with 100 ng/ml rapamycin and exposed to various stresses for 24 h. Cells were then treated with 20 μM of CHX and collected at time points from 0 to 24 h. Results shown are the representative of three similar experiments.

Fig. 4

Inhibition of AMPK pathway with compound C decreases eEF-2K stability under both stressful and non-stressful conditions. (A) T98G ^shVector and LN229^shVector cells were treated with 10 μM of compound C and exposed to various stresses for 48 h. eEF-2K protein expression was then examined. (B) T98G ^shVector and LN229^shVector cells were treated with 20 μM compound C and exposed to various stresses for 24 h. Cells were then treated with 20 μM of CHX and collected at time points from 0 to 24 h. Results shown are the representative of three similar experiments.