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.
Highlights
Eukaryotic elongation factor 2 kinase negatively regulates the elongation stage of mRNA translation and is activated under different stress conditions to slow down protein synthesis
We discovered that levels of heat-shock protein 90 (HSP90) are increased in Eukaryotic elongation factor 2 kinase (eEF2K)-depleted human cells as well as in eEF2K-knockout mouse embryonic fibroblasts (MEFs)
By reducing the availability of active Eukaryotic elongation factor 2 (eEF2), eEF2K slows down the elongation stage of protein synthesis [1], which consumes the vast majority of the energy used (Ͼ99%) by protein synthesis. eEF2K is activated under diverse cell stresses such as hypoxia [2], low pH [3], nutrient deprivation [4], and glycolytic stress [5], which are hallmarks of tumor microenvironments, and where saving energy or nutrients such as amino acids would benefit tumor cell survival. eEF2K-knockout mice are healthy and viable [2], showing that eEF2K is not required for normal cell function under standard animal husbandry conditions
Summary
Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K) is an atypical protein kinase that phosphorylates eEF2 on Thr-56 and. 2-DG treatment led to a reduction in HSP90 protein levels in MEFs (Fig. 1, D and E) [6] It was shown previously [9] that TOP mRNAs (mRNAs that contain a 5Ј-terminal oligopyrimidine tract, many of which encode ribosomal proteins and translation factors) are recruited onto polysomes upon activation of eEF2K, thereby causing an increase in the relative synthesis of TOP mRNA– encoded proteins compared with others. Inhibiting HSP90 activity by AUY922 increased levels of ubiquitinated proteins in the pellet fraction in eEF2Kϩ/ϩ MEFs, but the effect was considerably enlarged in eEF2KϪ/Ϫ cells (Fig. 3, A–C), indicating a greater buildup of incorrectly folded proteins. This was accompanied with poly(ADP-ribose) polymerase cleavage (Fig. 3A), indicating apoptosis [12]. These data indicate that when elongation rates are enhanced, subsequent up-regulation of HSP90 chaperone expression plays an important role in maintaining proteostasis
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