Ablation of elongation factor 2 kinase enhances heat-shock protein 90 chaperone expression and protects cells under proteotoxic stress

Abstract

Eukaryotic elongation factor 2 kinase (eEF2K) negatively regulates the elongation stage of mRNA translation and is activated under different stress conditions to slow down protein synthesis. One effect of eEF2K is to alter the repertoire of expressed proteins, perhaps to aid survival of stressed cells. Here, we applied pulsed stable isotope labeling with amino acids in cell culture (SILAC) to study changes in the synthesis of specific proteins in human lung adenocarcinoma (A549) cells in which eEF2K had been depleted by an inducible shRNA. We discovered that levels of heat-shock protein 90 (HSP90) are increased in eEF2K-depleted human cells as well as in eEF2K-knockout (eEF2K^-/-) mouse embryonic fibroblasts (MEFs). This rise in HSP90 coincided with an increase in the fraction of HSP90 mRNAs associated with translationally active polysomes, irrespective of unchanged total HSP90 levels. These results indicate that blocking eEF2K function can enhance expression of HSP90 chaperones. In eEF2K^-/- mouse embryonic fibroblasts (MEFs), inhibition of HSP90 by its specific inhibitor AUY922 promoted the accumulation of ubiquitinated proteins. Notably, HSP90 inhibition promoted apoptosis of eEF2K^-/- MEFs under proteostatic stress induced by the proteasome inhibitor MG132. Up-regulation of HSP90 likely protects cells from protein folding stress, arising, for example, from faster rates of polypeptide synthesis due to the lack of eEF2K. Our findings indicate that eEF2K and HSPs closely cooperate to maintain proper proteostasis and suggest that concomitant inhibition of HSP90 and eEF2K could be a strategy to decrease cancer cell survival.

Keywords: apoptosis; cancer; elongation; eukaryotic elongation factor 2 kinase (eEF2K); heat shock protein (HSP); mRNA translation; molecular chaperone; proteasome; protein synthesis; translation elongation factor.

Figure 1.

Knocking down or ablating eEF2K elevates HSP90 protein levels. A, lysates of A549 cells treated ±IPTG (to induce shRNA expression to knock down eEF2K) or eEF2K^+/+ and eEF2K^−/− MEFs were subjected to SDS-PAGE followed by WB analysis of the indicated proteins. B and C, quantification of HSP90 levels in A549 cells (B) or MEFs (C) from A. D, eEF2K^+/+ and eEF2K^−/− MEFs were cultured in low-glucose (5.5 mm) DMEM ± 10 mm 2-DG for 24 h before immunoblot analysis. E, quantification of HSP90 levels from D. P-eEF2, phosphorylated eEF2. *, 0.01 ≤ p < 0.05; **, 0.001 ≤ p < 0.01. Error bars represent S.D.

Figure 2.

eEF2K knockout in MEFs shifts HSP90AA1 and HSP90B1 mRNAs from polysomal to non/subpolysomal fractions. A and B, eEF2K^+/+ and eEF2K^−/− MEFs were treated as in Fig. 1E, and total cell mRNA was extracted and subjected to qPCR with primers for HSP90AA1 (A) and HSP90B1 (B). C–F, eEF2K^+/+ and eEF2K^−/− MEFs were treated as in Fig. 1E, and cell lysates were subjected to polysome analysis. A representative profile is shown in C, and positions of ribosomal/polysomal species are indicated. RT-qPCR was performed using specific primers for mouse HSP90AA1 (D), HSP90B1 (E), and B2M (F). *, 0.01 ≤ p < 0.05; **, 0.001 ≤ p < 0.01; ***, p < 0.001. Error bars represent S.D.

Figure 3.

AUY922 increases levels of ubiquitinated proteins in 1% (v/v) Triton–insoluble pellets from eEF2K^−/− MEFs. A–C, eEF2K^+/+ and eEF2K^−/− MEFs were cultured with vehicle or with 50 nm AUY922 and/or 3 μm MG132 for 24 h. Clarified cell lysates and detergent-insoluble pellets were subjected to SDS-PAGE followed by WB analysis of the indicated proteins (A). Levels of ubiquitin (Ub) in clarified lysates (B) and insoluble pellets (C) were quantified. D, MEFs were treated as in A, and cell lysates were incubated with previously heat-denatured Fluc at room temperature. Fluc refolding was assessed by measuring Fluc activity at the indicated time points. P-eEF2, phosphorylated eEF2. *, 0.01 ≤ p < 0.05; **, 0.001 ≤ p < 0.01; ***, p < 0.001. Error bars represent S.D.

Figure 4.

AUY922 induces apoptosis in eEF2K^−/− MEFs under proteolytic stress. eEF2K^+/+ and eEF2K^−/− MEFs were treated as in Fig. 3A. A, cell viability and morphology were assessed by microscopic analysis. Scale bars, 500 μm. B, cells were dispersed, stained with annexin V (FITC-A) and PI (PI-A), and then analyzed by flow cytometry. C, quantification of B. ***, p < 0.001. Error bars represent S.D.