The late endosome is essential for mTORC1 signaling

Abstract

The multisubunit mTORC1 complex integrates signals from growth factors and nutrients to regulate protein synthesis, cell growth, and autophagy. To examine how endocytic trafficking might be involved in nutrient regulation of mTORC1, we perturbed specific endocytic trafficking pathways and measured mTORC1 activity using S6K1 as a readout. When early/late endosomal conversion was blocked by either overexpression of constitutively active Rab5 (Rab5CA) or knockdown of the Rab7 GEF hVps39, insulin- and amino acid-stimulated mTORC1/S6K1 activation were inhibited, and mTOR localized to hybrid early/late endosomes. Inhibition of other stages of endocytic trafficking had no effect on mTORC1. Overexpression of Rheb, which activates mTOR independently of mTOR localization, rescued mTORC1 signaling in cells expressing Rab5CA, whereas hyperactivation of endogenous Rheb in TSC2-/- MEFs did not. These data suggest that integrity of late endosomes is essential for amino acid- and insulin-stimulated mTORC1 signaling and that blocking the early/late endosomal conversion prevents mTOR from interacting with Rheb in the late endosomal compartment.

Figure 1.

Rab5CA inhibits insulin- and amino acid—stimulated phosphorylation of S6K1. (A) HEK293E cells were transfected with HA-S6K1 and either empty vector, Rab5WT, Rab5DN, or Rab5CA constructs as indicated. Serum-starved cells were incubated without or with 1 μM human insulin for 30 min. Anti-HA immunoprecipitates and cleared lysates were blotted for pT389-S6K1, total S6K1, and Rab5. (B) The ratio of pT389 to total HA-S6K1, normalized to the ratio in vector controls. The data are the mean ± SEM from seven experiments. ***p < 0.005. (C) HEK293E cells were transfected with Myc-Akt and Rab5 constructs as indicated, and stimulated without or with 1 μM insulin for 15 min. Anti-Myc immunoprecipitates as well as cleared lysates were blotted for pT308-Akt, Myc (total Akt), and Rab5. (D) The ratio of pT308 to total Myc-Akt, normalized to the ratio in vector controls. The data are the mean ± SEM from six experiments. *p < 0.05. (E) HEK293T cells were transfected with HA-S6K1 and either empty vector or Rab5CA constructs as indicated. Amino acid–starved cells were incubated without or with 1× (final) complete amino acid solution for 10 min. Anti-HA immunoprecipitates as well as cleared lysates were blotted for pT389-S6K1, HA (total S6K1), and Rab5. (F) The ratio of pT389 to total HA-S6K1 was calculated as above. The data are the mean ± SEM from three experiments. ***p < 0.005. (G) HEK293E (left) or HEK293T (right) cells were transfected with either empty vector or Rab5CA constructs as indicated. Serum-starved HEK293E and amino acid–starved HEK293T cells were stimulated with insulin or amino acids, respectively, as described above, and whole cell lysates were blotted for endogenous pT389 S6K1, total S6K1, and overexpressed Rab5.

Figure 2.

Overexpression of Rheb, but not hyperactivation of endogenous Rheb, rescues insulin- and amino acid–stimulated S6K1 activation in Rab5CA-overexpressing cells. (A) HEK239E cells were transfected with HA-S6K1 and combinations of empty vector, Rheb, and Rab5CA, as indicated. Serum-starved cells were incubated without or with 1 μM human insulin for 30 min. Anti-HA immunoprecipitates or cleared lysates from each condition were blotted for pT389-S6K1 and HA (total S6K1). (B) The graph shows the ratio of pT389/total HA-S6K1 calculated as in Figure 1. The data are the mean ± SEM from four experiments. *p < 0.05, **p < 0.01, ***p < 0.005. (C) TSC2−/− MEFs were transfected with HA-S6K1 and combinations of empty vector, Rheb, and Rab5CA as indicated. Amino acid–starved cells were incubated without or with 1× (final) complete amino acid solution for 10 min. Anti-HA immunoprecipitates or cleared lysates from each condition were blotted for pT389-S6K1 and HA (total S6K1). (D) The graph shows the ratio of pT389/total HA-S6K1 calculated as in Figure 1. The data are the mean ± SD from two experiments. *p < 0.05, ***p < 0.005.

Figure 3.

Overexpression of Rab5CA produces hybrid early/late endosomes and alters amino acid–stimulated mTOR localization. (A) Control HeLa cells or cells transfected with Rab5CA were incubated in amino acid–free media for 50 min and then stimulated without or with 1× (final) complete amino acid solution for 10 min. Cells were fixed and immunostained for endogenous mTOR and LAMP-2 as indicated and analyzed by laser scanning confocal microscopy. Scale bars, 5 μm. (B) Control HeLa cells or cells transfected with Rab5CA were treated as in A, but were immunostained for endogenous EEA1 and mTOR. Scale bars, 5 μm. (C) HeLa cells transfected with either empty vector or Rab5CA were fixed and immunostained for Rab5, LAMP-2, and EEA1 as indicated and analyzed by laser scanning confocal microscopy. Scale bars, 5 μm. (D) Control HeLa cells or cells transfected with Rab5CA were treated as in A except that cells were immunostained for EEA1, LAMP-2, and mTOR as indicated and analyzed by laser scanning confocal microscopy. Color merges between mTOR and EEA1 and mTOR and LAMP-2 are displayed. Scale bars, 5 μm.

Figure 4.

Knockdown of hVps39 causes early/late endosomal mixing and inhibits insulin-stimulated phosphorylation of S6K1 and S6. (A) HeLa cells were transfected with either luciferase or hVps39 siRNA oligonucleotides and grown for 72 h before fixation. Cells were immunostained for endogenous LAMP-2 and EEA1 as indicated. Scale bars, 20 μm; scale bars in the high-magnification ROIs (Zoom) 5 μm. (B) HeLa cells transfected and grown as above were serum-starved and incubated without or with 1 μM human insulin for 30 min before fixation and immunostaining for phospho-S6 (pS235/236/240/244/247). Scale bars, 20 μm. (C) Total phospho-S6 fluorescence intensity was measured in 50 cells per condition in two separate experiments and is displayed as the mean ± SD. **p < 0.01. (D) Luciferase or hVps39 siRNA-treated HeLa cells treated as above were lysed in SDS sample buffer, and whole cell lysates for each condition were blotted for pT389 S6K1, total S6K1, and GAPDH as indicated. (E) The graph shows quantification of the ratio of phospho/total S6K1, normalized to the ratio in luciferase controls. The data are the mean ± SD from two experiments. ***p < 0.005. (F) Luciferase or hVps39 siRNA-treated HeLa cells were incubated in amino acid–free media for 50 min then stimulated without or with 1× (final) complete amino acid solution for 10 min. Cells were fixed and immunostained for endogenous mTOR, EEA1, and LAMP-2 as indicated and analyzed by laser scanning confocal microscopy. Color merges between mTOR and EEA1 and mTOR and LAMP-2 are shown. Scale bars, 10 μm.

Figure 4.

Knockdown of hVps39 causes early/late endosomal mixing and inhibits insulin-stimulated phosphorylation of S6K1 and S6. (A) HeLa cells were transfected with either luciferase or hVps39 siRNA oligonucleotides and grown for 72 h before fixation. Cells were immunostained for endogenous LAMP-2 and EEA1 as indicated. Scale bars, 20 μm; scale bars in the high-magnification ROIs (Zoom) 5 μm. (B) HeLa cells transfected and grown as above were serum-starved and incubated without or with 1 μM human insulin for 30 min before fixation and immunostaining for phospho-S6 (pS235/236/240/244/247). Scale bars, 20 μm. (C) Total phospho-S6 fluorescence intensity was measured in 50 cells per condition in two separate experiments and is displayed as the mean ± SD. **p < 0.01. (D) Luciferase or hVps39 siRNA-treated HeLa cells treated as above were lysed in SDS sample buffer, and whole cell lysates for each condition were blotted for pT389 S6K1, total S6K1, and GAPDH as indicated. (E) The graph shows quantification of the ratio of phospho/total S6K1, normalized to the ratio in luciferase controls. The data are the mean ± SD from two experiments. ***p < 0.005. (F) Luciferase or hVps39 siRNA-treated HeLa cells were incubated in amino acid–free media for 50 min then stimulated without or with 1× (final) complete amino acid solution for 10 min. Cells were fixed and immunostained for endogenous mTOR, EEA1, and LAMP-2 as indicated and analyzed by laser scanning confocal microscopy. Color merges between mTOR and EEA1 and mTOR and LAMP-2 are shown. Scale bars, 10 μm.

Figure 5.

Model of endosome conversion and mTORC1 signaling. The model shows a normal early-to-late endosome conversion (left) and a mixed early/late endocytic compartment (right). In order for mTOR to be properly activated in the late endocytic compartment in control cells (left), early endosomes must first undergo endosome conversion through the normal cycling of Rab5 and Rab7 and through the action of the HOPS complex, which contains hVps39. Amino acids stimulate the localization of mTOR to late endosomes, where it is proposed to interact with Rheb (Sancak et al., 2008). If early/late endosome conversion is blocked (right), either by overexpression of Rab5CA or knockdown of hVps39, then amino acids stimulate the localization of mTOR to a mixed endocytic compartment that does not contain Rheb. Under these conditions, mTORC1 is not activated.