A critical role of eEF-2K in mediating autophagy in response to multiple cellular stresses

Abstract

The phosphorylation of the subunit alpha of eukaryotic translation initiation factor 2 (eIF2alpha), a critical regulatory event in controlling protein translation, has recently been found to mediate the induction of autophagy. However, the mediators of autophagy downstream of eIF2alpha remain unknown. Here, we provide evidence that eIF2alpha phosphorylation is required for phosphorylation of eukaryotic elongation factor 2 (eEF-2) during nutrient starvation. In addition, we show that eukaryotic elongation factor 2 kinase (eEF-2K) is also required for autophagy signaling during ER stress, suggesting that phosphorylation of eEF-2 may serve as an integrator of various cell stresses for autophagy signaling. On the other hand, although the activation of eEF-2K in response to starvation requires the phosphorylation of eIF2alpha, additional pathways relying partly on Ca(2+) flux may control eEF-2K activity during ER stress, as eIF2alpha phosphorylation is dispensable for both eEF-2 phosphorylation and autophagy in this context.

Figure 1

eEF-2 phosphorylation and autophagy depend on eIF2α phosphorylation during starvation. eIF2α^A/A MEFs and their wild-type counterparts were cultured in normal medium (Earles Balanced Salt Solution (EBSS) + 10% serum and complete amino acids) or EBSS alone for the indicated time.

Figure 2

EEF-2 phosphorylation mediates autophagy during ER stress. (A) PERK^−/− MEFs, eIF2α^A/A MEFs and their respective wild-type counterparts were treated with the indicated doses of Tm for 24 h. (B) EEF-2K^−/− and wild-type MEFs were treated with the indicated doses of Tm for 24 h in the presence of the lysosomal protease inhibitor E64d (10 μg/ml). (C) EEF-2K^−/− and wild-type MEFs were treated continuously with Tm (100 ng/ml) and BAPTA-AM (20 μM) for 24 h, or transiently with thapsigargin (Tsg, 1μM) for 10 min, briefly washed and incubated in the presence of BAPTA-AM (20 μM) for 24 h. (D) Wild-type MEFs were treated with the indicated dose of cycloheximide (CHX) for 24 h. No significant cell death was noticed in treated cells.

Figure 3

A model for the role of eEF-2K in the induction of autophagy during amino acid starvation and ER stress. The phosphorylation of eIF2α activates eEF-2K, as evidenced by the phosphorylation of eEF-2 in cells treated with the PP1/GADD34 inhibitor sal. During amino acid starvation, the activation of eEF-2K is dependent on the phosphorylation of eIF2α. During ER stress, in addition to the PERK/eIF2α pathway eEF-2K is activated by at least another signaling pathway dependent on Ca²⁺.