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. 2013 Aug:61:61-71.
doi: 10.1016/j.freeradbiomed.2013.03.016. Epub 2013 Mar 29.

Molecular control of the amount, subcellular location, and activity state of translation elongation factor 2 in neurons experiencing stress

Sandro Argüelles  1 , Simonetta Camandola  2 , Emmette R Hutchison  3 , Roy G Cutler  2 , Antonio Ayala  4 , Mark P Mattson  5
Affiliations

Molecular control of the amount, subcellular location, and activity state of translation elongation factor 2 in neurons experiencing stress

Sandro Argüelles et al. Free Radic Biol Med. 2013 Aug.

Abstract

Eukaryotic elongation factor 2 (eEF-2) is an important regulator of the protein translation machinery whereby it controls the movement of the ribosome along the mRNA. The activity of eEF-2 is regulated by changes in cellular energy status and nutrient availability and by posttranslational modifications such as phosphorylation and mono-ADP-ribosylation. However, the mechanisms regulating protein translation under conditions of cellular stress in neurons are unknown. Here we show that when rat hippocampal neurons experience oxidative stress (lipid peroxidation induced by exposure to cumene hydroperoxide; CH), eEF-2 is hyperphosphorylated and ribosylated, resulting in reduced translational activity. The degradation of eEF-2 requires calpain proteolytic activity and is accompanied by accumulation of eEF-2 in the nuclear compartment. The subcellular localization of both native and phosphorylated forms of eEF-2 is influenced by CRM1 and 14.3.3, respectively. In hippocampal neurons p53 interacts with nonphosphorylated (active) eEF-2, but not with its phosphorylated form. The p53-eEF-2 complexes are present in cytoplasm and nucleus, and their abundance increases when neurons experience oxidative stress. The nuclear localization of active eEF-2 depends upon its interaction with p53, as cells lacking p53 contain less active eEF-2 in the nuclear compartment. Overexpression of eEF-2 in hippocampal neurons results in increased nuclear levels of eEF-2 and decreased cell death after exposure to CH. Our results reveal novel molecular mechanisms controlling the differential subcellular localization and activity state of eEF-2 that may influence the survival status of neurons during periods of elevated oxidative stress.

Keywords: 14.3.3; CRM1; Eukaryotic elongation factor 2; Free radicals; Hippocampal neurons; Lipid peroxidation; eEF-2; p53.

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Conflict of interest statement

Conflict of Interest; The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
Subtoxic levels of lipid peroxidation increase phosphorylation and ADP-ribosylation of eEF-2 levels in primary hippocampal neurons. Cultures of hippocampal neurons were incubated with increasing concentrations of CH for 3 h. A. Lipid peroxidation assay was determined using the FOX method. B. Cell viability was measured by MTS assay. C. Necrosis was measured by lactate dehydrogenase (LDH) release assay. The results for values in A, B and C are the mean and SEM from five experiments. **p<0.01 and ***p<0.001 vs. control. D. The levels of total eEF-2, phosphorylated eEF-2, ADP ribosylated eEF-2 and β-actin were detected as described in the Materials and Methods. E and F. Optical densities of the phosphorylated/total eEF-2 bands (E) and ADP ribosylated/total eEF-2 (F). Values are the mean and SEM of four experiments. *p< 0.05 and ***p<0.001 vs. control.
Figure 2
Figure 2
Evidence that calpains mediate degradation of eEF-2 in neurons subjected to lipidperoxidation. A and B. Hippocampal primary cultures were pre-treated with 50 μMMDL28170 (MDL) (A) or 50 μM E64d (B) and were then exposed to 10 or 15 μM CH for 3 h. Graphs show quantitation of optical density of the phosphorylated/total eEF-2 bands. Values are the mean and SEM of four experiments. *p< 0.05, **p<0.01 and ***p<0.001 vs. control.
Figure 3
Figure 3
Evidence of subcellular localization of eEF-2 in primary hippocampal neurons. A. Subcellular fractionation and immunoblot analysis of total and phosphorylated eEF-2, β-actin (cytoplasmic marker) and hnRNP (nuclear marker) were performed as described in the Materials and Methods on hippocampal primary cultures untreated, or treated with 5 or 10 μM CH for 3 h before separation into a cytoplasmic and a nuclear fraction. B. Confocal images of eEF-2 immunoreactivity in cultured hippocampal neurons (green). Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue) to identify nuclei. Data are representative of three independent neuronal cultures. C. Optical densities of the phosphorylated/total eEF-2 bands in cytoplasm and nucleus. Values are the mean and SEM of four experiments. **p<0.01 vs. control in cytoplasm and †p<0.05 vs. control in nucleus.
Figure 4
Figure 4
p53 interacts with eEF-2 in cytoplasm and nucleus of primary hippocampal neurons. A – C. Whole-cell (A), cytoplasmic (B) and nuclear (C) extracts from hippocampal primary cultures treated with CH for 3 h were subjected to immunoprecipitation (IP) with p53 antibody or with control IgG and analyzed by immunoblotting for eEF-2 or phosphorylated eEF-2. D. The levels of β-actin (cytosolic marker) and hnRNP (nuclear marker) were detected by immunoblot analysis to ensure optimal fractionation. Data are representative of observations from four independent experiments.
Figure 5
Figure 5
Evidence that p53-deficient cells are more sensitive to lipid peroxidation. Wild-type (WT) or p53-deficient cells (p53-) were incubated for 3 h in the presence of CH at indicated concentrations. A. Cell viability was measured by the MTS assay and cell death was measured based on lactate dehydrogenase (LDH) release assay as described in the Materials and Methods. Values are the mean and SEM of 4 experiments. ***p<0.001 (cell viability),†p<0.05, ††p<0.01and †††p<0.001 (cell death); wild-type vs. p53-deficient cells. B. Lipidperoxidation levels were determined using the FOX method. Values are the mean and SEM of 5 experiments. *p< 0.05 and **p<0.01 vs. wild-type control. ††p<0.01 vs. p53-deficient cell control. C – E. To directly determine the effects of CH on nascent proteins, wild-type (WT) and p53-deficient cells (p53-) were pretreated with CH, supplemented with methionine-free D-MEM and L-azidohomoalanine (AHA) to label the newly synthesized proteins. C. After electrophoresis the gels were analyzed using a Typhoon 9400 scanner (GE Healthcare), fluorescence excitation (545 nm) and emission (580 nm). D. Nascent protein synthesis in WT and p53-deficient cells (p53-) treated with CH. Values are the mean and SEM of 4 experiments and the bars represent the band density integrated over all bands. E. Nitrocellulose membrane was stained with Ponceau Red.**p< 0.01 and ***p<0.001 vs. Wild-type control, ††p<0.01and †††p<0.001 vs. p53-deficient cells control, ‡‡ p<0.01 vs. Wild-type 5 μM CH and \\ p< 0.01vs. Wild-type 10 μM CH.
Figure 6
Figure 6
p53 is involved in subcellular trafficking of non-phosphorylated eEF-2 into the nucleus. A. Subcellular fractionation and analysis of total and phosphorylated eEF-2 levels in nuclear and cytoplasmic fractions of wild type and p53-deficient cells treated with CH. α-tubulin and hnRNP were used as cytoplasmic and nuclear markers. B. Optical density of the eEF-2 bands. Values are the mean and SEM of 4 experiments. **p< 0.01 vs. cytoplasmic extracts from wild-type control, ††p<0.01 vs. nuclear extracts from wild-type control, ‡‡‡ p<0.001 vs. cytoplasmic extracts from p53-deficient cells control, § p<0.05 vs. nuclear extracts from p53-deficient cells control, ¶¶ p< 0.01 vs. nuclear extracts from wild-type control and \\ p< 0.01 vs. nuclear extracts from wild-type 5 μM. C. Optical density of the phosphorylated/total eEF-2 bands. Values for band intensity ratios are the mean and SEM of 4 experiments. *p< 0.05 vs. cytoplasmic extracts from wild-type control, ††p<0.01 vs. nuclear extracts from wild-type control, ‡ p<0.05 vs. cytoplasmic extracts from p53-deficient cells control, §§§ p<0.001 vs. nuclear extracts from p53-deficient cells control, ¶¶¶ p< 0.001 vs. nuclear extracts from wild-type control and \\\ p< 0.001 vs. nuclear extracts from wild-type 5 μM.HCT116 cells were transfected with a fixed amount of plasmid expressing Flag-p53. D. Immunoblots for p53 and α-tubulin are shown. E. Analysis of the total and phosphorylated eEF-2 levels in nuclear and cytoplasmic fractions of wild-type and p53-deficient cells after transfection (α-tubulin and hnRNP were used as cytoplasmic and nuclear markers, respectively. F and G. Optical densities of the eEF-2 total bands (F) and phosphorylated/total eEF-2 ratio (G). Values are the mean and SEM from three independent transfections. ***p< 0.001 vs. cytoplasmic extracts from p53-deficient cells. †††p<0.001 vs. nuclear extracts from p53-deficient cells.
Figure 7
Figure 7
Evidence for the differential involvement of 14-3-3 protein and CRM1 in the subcellular localization of total and phosphorylated eEF-2. A– C. Hippocampal primary cultures (A), and HCT116 cells expressing (B) or lacking (C) p53, were fractionated (cytoplasm and nucleus), pre-treated for 25 min at 30°C in phosphatase buffer supplemented with protein phosphatase 2A (PP2A) or with protein phosphatase 2A and okadaic acid (OA), subjected to immunoprecipitation (IP) with eEF-2antibody or with control IgG, and analyzed by immunoblotting with a 14-3-3 antibody. β-actin and hnRNP were used as cytoplasmic and nuclear markers (bottom). Data are representative of four independent experiments. D. Hippocampal neurons were treated with 50 nM Leptomycin B. After 3 or 6 h, neurons were collected and separated into cytoplasmic and nuclear fractions, and analyzed by SDS-PAGE followed by immunoblot. β-actin and hnRNP were used as cytoplasmic and nuclear markers.E. Optical density of the ratio nucleus/cytoplasm total eEF-2bands. Values are the mean and SEM of four experiments. *p<0.05, ***p<0.001 vs. control. F. Optical density of the ratio nucleus/cytoplasm phosphorylated eEF-2 bands.
Figure 8
Figure 8
The nuclear increase of functional (non-phosphorylated) eEF-2 may play a role in mediating neuronal survival following mild oxidative stress. A and B. Hippocampal neurons 72 h postinfection with control or eEF-2-expressing lentiviruses were treated for 3 h (A) or 24 h (B) with increasing concentrations of CH(10, 15 and 20 μM). Cell viability was measured by MTS assay and necrotic cell death was measured based on lactate dehydrogenase (LDH) release assay. Values are the mean and SEM of four independent infections. ***p<0.001, *p<0.05 (cell viability), ††p<0.01and †††p<0.001(cell death) Control (C) vs. eEF-2 overexpression (O.E). C – E. Hippocampal neurons were infected with control or eEF-2-expressing lentiviruses and treated with 15 μM CH or vehicle for 3h. Immunoblots for total and phosphorylated eEF-2 (C), and quantification of total eEF-2 (D) and phosphorylated/total eEF-2 (E). Values are the mean and SEM of four independent infections. ***p<0.001 Control vs. eEF-2 overexpression (O.E.), †††p<0.001 and ††p<0.01 vs. Control non-treated with CH, ‡‡‡p<0.001 and ‡‡ p<0.01 vs. eEF-2 overexpression (O.E.) non-treated with CH.
Figure 9
Figure 9
Evidence of efficiency of the over-expression and its effect on eEF-2 subcellular localization in hippocampal neurons subjected to oxidative stress. A. Immunoblots for total and phosphorylated eEF-2 in nuclear and cytoplasmic fractions are shown. β-actin and hnRNP were used as cytoplasmic and nuclear markers. B and C. Optical densities of the total eEF-2 band (B) and the phosphorylated/total eEF-2 bands (C) in nuclear and cytoplasmic fractions. Values are the mean and SEM of four independent infections. ***p<0.001, ** p<0.01, *p<0.05 Control (C) vs. eEF-2 overexpression (O.E.), ††p<0.01 vs. the value for cytoplasm of control cells not treated with CH. ‡‡‡ p<0.001 vs. the value for nucleus of control cells not treated with CH.
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