The eEF2 kinase coordinates the DNA damage response to cisplatin by supporting p53 activation

Abstract

Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K) is a stress-responsive hub that inhibits the translation elongation factor eEF2, and consequently mRNA translation elongation, in response to hypoxia and nutrient deprivation. EEF2K is also involved in the response to DNA damage but its role in response to DNA crosslinks, as induced by cisplatin, is not known. Here we found that eEF2K is critical to mediate the cellular response to cisplatin. We uncovered that eEF2K deficient cells are more resistant to cisplatin treatment. Mechanistically, eEF2K deficiency blunts the activation of the DNA damage response associated ATM and ATR pathways, in turn preventing p53 activation and therefore compromising induction of cisplatin-induced apoptosis. We also report that loss of eEF2K delays the resolution of DNA damage triggered by cisplatin, suggesting that eEF2K contributes to DNA damage repair in response to cisplatin. In support of this, our data shows that eEF2K promotes the expression of the DNA repair protein ERCC1, critical for the repair of cisplatin-caused DNA damage. Finally, using Caenorhabditis elegans as an in vivo model, we find that deletion of efk-1, the worm eEF2K ortholog, mitigates the induction of germ cell death in response to cisplatin. Together, our data highlight that eEF2K represents an evolutionary conserved mediator of the DNA damage response to cisplatin which promotes p53 activation to induce cell death, or alternatively facilitates DNA repair, depending on the extent of DNA damage.

Fig. 1. eEF2K mediates cellular susceptibility towards cisplatin.

A Viability of Eef2k^+/+ and Eef2k^−/− MEFs treated with the indicated concentrations of cisplatin (CisPt) or vehicle control for 48 h as measured by an MTT assay (n = 3). Untreated condition was set to 100% for each cell line. B Viability of HEK293 cells stably expressing individual shRNAs targeting eEF2K (sh-eEF2K1 and sh-eEF2K2) or scrambled control (sh-scr) treated with the indicated concentrations of CisPt or vehicle control for 72 h as measured by an MTT assay (n = 3). Untreated condition was set to 100% for each cell line. C Cell death rates of Eef2k^+/+ and Eef2k^−/− MEFs treated with the indicated concentrations of CisPt or vehicle control for 48 h as indicated, as measured using trypan blue staining (n = 3). D Level of apoptosis markers in Eef2k^+/+ and Eef2k^−/− MEFs treated with CisPt (5 μM) for the indicated times, as measured with immunoblot analysis of cleaved-PARP and cleaved-Caspase-3. The immunoblots are representative of three independent experiments. E Level of apoptosis markers in HEK293 cells stably expressing sh-eEF2K1, sh-eEF2K2, or sh-scr treated with CisPt (5 μM) for the indicated times, as measured with immunoblot analysis of cleaved-PARP and cleaved-Caspase-3. The immunoblots are representative of three independent experiments. Data are expressed as mean ± SD; *P < 0.05, ***P < 0.005, ns = non-significant.

Fig. 2. eEF2K supports the induction of the DDR pathways in response to cisplatin.

A Scheme illustrating ATM/ATR signalling in response to DNA damage. B Level of activity of the ATM/ATR-dependent DDR pathways in Eef2k^+/+ and Eef2k^−/− MEFs treated with CisPt (5 μM) for the indicated times, as measured with immunoblot analysis using the indicated antibodies. The immunoblots are representative of three independent experiments. C Level of activity of the ATM/ATR-dependent DDR pathways in MEFs transfected with siRNA targeting eEF2K (si-Eef2k) or scrambled control (scr) treated with CisPt (5 μM) for the indicated times, as measured with immunoblot analysis using the indicated antibodies. The immunoblots are representative of three independent experiments. * indicates unspecific bands (as previously reported [32]). D Level of γH2AX foci formation in Eef2k^+/+ and Eef2k^−/− MEFs treated with CisPt (50 μM) or vehicle control for 2 h, as determined by immunofluorescence using a γH2AX antibody (n = 3). E Level of 53BP1 foci formation in Eef2k^+/+ and Eef2k^−/− MEFs treated with CisPt (50 μM) or vehicle control for 2 h, as determined by immunofluorescence using a 53BP1 antibody (n = 3). Data are expressed as mean ± SD; ***P < 0.005.

Fig. 3. eEF2K promotes enhanced DNA damage repair in response to cisplatin.

A Level of DNA damage in Eef2k^+/+ and Eef2k^−/− MEFs treated with vehicle (-) or with CisPt (5 μM) for 2 h followed by 2 or 24 h period of recovery, as measured by modified alkali comet assay (n = 3). B Level of DNA damage in HEK293 cells stably expressing sh-eEF2K1 or sh-scr treated with vehicle (-) or with CisPt (5 μM) for 2 h followed by 2 or 24 h period of recovery, as measured by modified alkali comet assay (n = 3). C Level of γH2AX foci formation and resolution in Eef2k^+/+ and Eef2k^−/− MEFs treated with vehicle (-) or with CisPt (5 μM) for 2 h followed by 0 to 48 h period of recovery, as determined by immunofluorescence using a γH2AX antibody (n = 3). D Micronuclei formation in Eef2k^+/+ and Eef2k^−/− MEFs treated with vehicle (-) or with CisPt (5 μM) for 2 h followed by 16 or 24 h period of recovery, as measured using DAPI staining. Red arrows indicating micronuclei positive cells and grey arrows indicating micronuclei negative cells (n = 3). E Level of ERCC1 and XPF expression in Eef2k^+/+ and Eef2k^−/− MEFs treated with CisPt (5 μM) for the indicated times, as measured with immunoblot analysis using the indicated antibodies. Data are expressed as mean ± SD; *P < 0.05, ***P < 0.005.

Fig. 4. eEF2K-mediated cellular susceptibility towards cisplatin is dependent on p53 activation and expression.

A Level of p53 activation in Eef2k^+/+ and Eef2k^−/− MEFs treated with CisPt (50 μM) for the indicated times, as measured with immunoblot analysis using the indicated antibodies. B Level of p53 activation in HEK293 cells stably expressing sh-eEF2K1, sh-eEF2K2, or sh-scr treated with CisPt (50 μM) for the indicated times, as measured with immunoblot using the indicated antibodies. C, D Viability of Eef2k^+/+ MEFs (C) or Eef2k^−/− MEFs (D) transfected with individual siRNA targeting p53 (si-p53-1 and si-p53-2) or scrambled control (si-scr) treated with the indicated concentrations of cisplatin (CisPt) or vehicle control for 48 h, using an MTT assay (n = 3). Data are expressed as mean ± SD; *P < 0.05, ***P < 0.005.

Fig. 5. The eEF2K ortholog, efk-1, mediates germ cell death in Caenorhabditis elegans in response to cisplatin.

A Level of germ cell death in wild type (wt) and efk-1(ok3609) (efk-1) adult C. elegans treated with the indicated concentrations of CisPt for 48 h, as measured by number of germ cell corpses per gonad arm. B Egg production in wild type (wt) and efk-1 knockout (efk-1) adult C. elegans treated with the indicated concentrations of CisPt for 48 h, as measured by number of eggs produced per worm per hour. C Percentage of viable eggs produced by wt and efk-1 knockout (efk-1) adult C. elegans treated as in (B). D Proposed model for the role of eEF2K in the DDR triggered by cisplatin. Data are expressed as mean ± SD; ***P < 0.001.