GCN2 contributes to mTORC1 inhibition by leucine deprivation through an ATF4 independent mechanism

Abstract

It is well known that the GCN2 and mTORC1 signaling pathways are regulated by amino acids and share common functions, in particular the control of translation. The regulation of GCN2 activity by amino acid availability relies on the capacity of GCN2 to sense the increased levels of uncharged tRNAs upon amino acid scarcity. In contrast, despite recent progress in the understanding of the regulation of mTORC1 by amino acids, key aspects of this process remain unsolved. In particular, while leucine is well known to be a potent regulator of mTORC1, the mechanisms by which this amino acid is sensed and control mTORC1 activity are not well defined. Our data establish that GCN2 is involved in the inhibition of mTORC1 upon leucine or arginine deprivation. However, the activation of GCN2 alone is not sufficient to inhibit mTORC1 activity, indicating that leucine and arginine exert regulation via additional mechanisms. While the mechanism by which GCN2 contributes to the initial step of mTORC1 inhibition involves the phosphorylation of eIF2α, we show that it is independent of the downstream transcription factor ATF4. These data point to a novel role for GCN2 and phosphorylation of eIF2α in the control of mTORC1 by certain amino acids.

Figure 1. GCN2 is necessary for mTORC1 regulation by leucine.

(A) Wild-Type (WT) MEFs were transferred to fresh medium containing dialysed serum and all amino acids except leucine, and then maintained in the presence or in absence of leucine (Leu) for the times indicated (minutes). Immunoblot analyses were performed on the resulting cell lysates using the indicated antibodies. The ratio of phosphorylated S6K1 (Thr389) to total S6K1 was determined by densitometry analysis, differences between control cells (dark bars)and leucine starved cells (light bars) at each time point were assessed by 1-way ANOVA. Bars with (*) are significantly different from each other (P < 0.05). (B) WT or GCN2-KO (−/−) MEFs were maintained in all amino acids including or excluding leucine (Leu) for the times indicated. The ratio of phosphorylated S6K1 (Thr389) to total S6K1 was determined by densitometry analysis, differences between control cells and leucine starved cells at each time point were assessed by 1-way ANOVA. Bars with (*) are significantly different from each other (P < 0.05). (C) WT or GCN2^−/− MEFs were cultured in presence or in absence of all amino acids (AA) for the times shown. (D) WT and GCN2^−/− MEFs were kept in the presence (+AA) or absence (−AA) of AA for 75 min; or in the absence of AA for 60 min prior to addition for 15 min of a medium containing all AA (−AA → + AA) or a medium containing all AA except leucine (−AA → -Leu). In all panels, immunoblot analyses were performed on the resulting cell lysates using the indicated antibodies.

Figure 2. ATF4 is not involved at early times of mTORC1 regulation by leucine.

(A) WT or GCN2^−/− MEFs were cultured in presence or in absence of leucine (Leu) with all the other AAs for 30 min, 60 min or 120 min. The ratio of phosphorylated S6K1 (Thr389) to total S6K1 was determined by densitometry analysis, differences between control cells (dark bars) and leucine starved cells (light bars) at each time point were assessed by 1-way ANOVA. Bars with (*) are significantly different from each other (P < 0.05). (B) WT MEFs and ATF4^−/− MEFs were cultured in the presence or absence of AA for 75 min; or in absence of AA for 60 min prior to addition for 15 min of a medium containing all AA (−AA → + AA) or a medium containing all AA except leucine (−AA → -Leu). In all panels, immunoblot analyses were performed on the resulting cell lysates using the indicated antibodies.

Figure 3. The phosphorylation of eIF2α is necessary for the regulation of mTORC1 by leucine.

(A) WT or eIF2α[Ser51Ala] MEFs were maintained in presence or in absence of leucine (Leu) with all the other AAs for the times shown. A positive control has been included in the immunoblot analysis for the phosphorylated eIF2α in the eIF2α[Ser51Ala] MEFs. The ratio of phosphorylated S6K1 (Thr389) to total S6K1 was determined by densitometry analysis, differences between control cells (dark bars) and leucine starved cells (light bars) at each time point were assessed by 1-way ANOVA. Bars with (*) are significantly different from each other (P < 0.05). (B) WT or eIF2α[Ser51Ala] MEFs were kept in the presence or absence of AA for 75 min + AA); or in the absence of AA (−AA) for 60 min prior to addition for 15 min of a medium containing all AA (−AA → + AA) or a medium containing all AA except leucine (−AA → -Leu). A positive control has been included in the immunoblot analysis for the phosphorylated eIF2α in the eIF2α[Ser51Ala] MEFs. Immunoblot analyses were performed to measure the amounts of the indicated proteins and their levels of phosphorylation.

Figure 4. The phosphorylation of eIF2α is not sufficient to inhibit mTORC1.

(A) WT MEFs were treated without (Ctl) or with tunicamycin (Tun) for the times shown. (B) WT MEFs were treated without (Ctl) or with histidinol (Hol) for the times shown. (C) WT MEFs were cultured in the presence or in absence of lysine (Lys) with all the other AAs for the indicated times. (D) WT MEFs were cultured in the presence or absence of AA for 75 min (+AA); or in absence of AA for 60 min prior to addition for 15 min of a medium containing all AA (−AA → + AA) or a medium containing all AA except leucine (−AA → -Leu) or lysine (−AA → -Lys). Immunoblot analyses were performed to measure the amounts of the indicated proteins and their levels of phosphorylation.

Figure 5. GCN2 is necessary for the regulation of mTORC1 by arginine.

(A) WT or GCN2^−/− MEFs were cultured in presence or in absence of arginine (Arg) with all the other AAs for 30, 60 or 120 min. The ratio of phosphorylated S6K1 (Thr389) to total S6K1 was determined by densitometry analysis, differences between control cells (dark bars) and arginine starved cells (light bars) at each time point were assessed by 1-way ANOVA. Bars with (*) are significantly different from each other (P < 0.05). (B) WT MEFs and GCN2^−/− MEFs were cultured in the presence or absence of AA for 75 min; or in absence of AA for 60 min prior to addition for 15 min of a medium containing all AA (−AA → + AA) or a medium containing all AA except arginine (−AA → -Arg). Immunoblot analyses were performed to assess the amounts of the indicated proteins and their levels of phosphorylation.