Dynamin-dependent amino acid endocytosis activates mechanistic target of rapamycin complex 1 (mTORC1)

Abstract

The mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of protein synthesis and potential target for modifying cellular metabolism in various conditions, including cancer and aging. mTORC1 activity is tightly regulated by the availability of extracellular amino acids, and previous studies have revealed that amino acids in the extracellular fluid are transported to the lysosomal lumen. There, amino acids induce recruitment of cytoplasmic mTORC1 to the lysosome by the Rag GTPases, followed by mTORC1 activation by the small GTPase Ras homolog enriched in brain (Rheb). However, how the extracellular amino acids reach the lysosomal lumen and activate mTORC1 remains unclear. Here, we show that amino acid uptake by dynamin-dependent endocytosis plays a critical role in mTORC1 activation. We found that mTORC1 is inactivated when endocytosis is inhibited by overexpression of a dominant-negative form of dynamin 2 or by pharmacological inhibition of dynamin or clathrin. Consistently, the recruitment of mTORC1 to the lysosome was suppressed by the dynamin inhibition. The activity and lysosomal recruitment of mTORC1 were rescued by increasing intracellular amino acids via cycloheximide exposure or by Rag overexpression, indicating that amino acid deprivation is the main cause of mTORC1 inactivation via the dynamin inhibition. We further show that endocytosis inhibition does not induce autophagy even though mTORC1 inactivation is known to strongly induce autophagy. These findings open new perspectives for the use of endocytosis inhibitors as potential agents that can effectively inhibit nutrient utilization and shut down the upstream signals that activate mTORC1.

Keywords: amino acid; autophagy; dynamin; endocytosis; mTORC1; mammalian target of rapamycin (mTOR); mechanistic target of rapamycin (mTOR).

Figure 1.

Endocytosis inhibition results in mTORC1 inactivation. A, 293T cells were transfected with pcDNA3.1 vector (mock) or pcDNA3.1/dynamin 2 K44A. Cells were harvested at 24 h after transfection. B, quantification of the immunoblot data in A. Each bar indicates mean ± S.D. of three independent experiments. *, p < 0.05. C, 293T cells were treated with 0.1% DMSO (vehicle), 80 μm Dynasore, or 30 μm PitStop 2 for the indicated time.

Figure 2.

Endocytosis inhibitors and amino acid deprivation lead to inactivation of mTORC1 but not mTORC2. A, 293T cells were treated for 1 or 4 h with 0.1% DMSO, 80 μm Dynasore, 30 μm PitStop 2, or 250 nm Torin 1 in RPMI1640 medium containing 10% Nu-Serum, or with amino acid- and serum-free RPMI1640 medium. B, quantification of immunoblot data in A. Each bar indicates mean ± S.D. of three independent experiments. *, p < 0.05; ***, p < 0.001; ****, p < 0.0001 compared with the vehicle treatment group.

Figure 3.

Amino acid-induced activation and lysosomal localization of mTORC1 is prevented by endocytosis inhibition. A, 293T cells were starved for amino acids in amino acid-free RPMI1640 medium containing 10% dialyzed Nu-Serum in combination with 0.1% DMSO or 80 μm Dynasore. After 50 min starvation, cells were stimulated for 40 min with amino acids by adding 1/50 volume of ×50 amino acid solution containing l-glutamine. B, quantification of immunoblot data in A. Mean values of two independent experiments are shown. C, 293T cells were treated as in A and subjected to immunofluorescence with anti-mTOR and anti-Lamp1 antibodies. Scale bar = 10 μm. D, quantification of cells that exhibit lysosomal localization of mTOR by analysis of image data in C. Each bar indicates mean ± S.D. of three independent experiments. ***, p < 0.001. E, quantification of fluorescence intensity of mTOR on lysosomes by analysis of image data in C. Each bar indicates mean ± S.D. of three independent experiments. ****, p < 0.0001.

Figure 4.

Lysosomal localization and activation of mTORC1 are prevented by endocytosis inhibition and rescued by CHX. A, 293T cells were treated for 1 h with 0.2% DMSO, 80 μm Dynasore, or 80 μm Dynasore and 10 μg/ml of CHX, fixed, and stained with anti-mTOR and anti-Lamp1 antibodies. The results shown are representative of three independent experiments. Scale bar = 10 μm. B, quantification of cells that exhibit lysosomal localization of mTOR by analysis of image data in A. Each bar indicates mean ± S.D. of three independent experiments. **, p < 0.01. C, quantification of fluorescence intensity of mTOR on lysosomes by analysis of image data in A. Each bar indicates mean ± S.D. of three independent experiments. *, p < 0.05; **, p < 0.01. D, 293T cells were treated for 1 or 4 h with 0.2% DMSO, 10 μg/ml of CHX, 80 μm Dynasore, or 80 μm Dynasore and 10 μg/ml of CHX. E, quantification of immunoblot data in D. Each bar indicates mean ± S.D. of four independent experiments. *, p < 0.05; **, p < 0.01 compared with the vehicle treatment group.

Figure 5.

Decreased mTORC1 activity in endocytosis-inhibited cells is rescued by overexpression of Rheb and constitutively active Rag. A, 293T cells were transfected with pRK5/HA-GST (mock); pRK5/HA-GST-RagB(99L) and pRK5/HA-GST(75L) (RagB^GTP/RagC^GDP); or pRK5/HA-GST-Rheb1 (Rheb). At 24 h post-transfection, cells were treated for 4 h with 0.1% DMSO (vehicle) or 80 μm Dynasore. B, quantification of immunoblot data in A. Each bar indicates mean ± S.D. of three independent experiments. **, p < 0.01. C, 293T cells were transfected as in A and subjected to immunofluorescence with anti-mTOR and anti-HA antibodies. Arrows indicate colocalization of mTOR and RagB/C. Scale bar = 10 μm. D, quantification of cells that exhibit punctate mTOR localization by analysis of image data in C. Only transfected (HA-positive) cells were examined. Each bar indicates mean ± S.D. of three independent experiments. ****, p < 0.0001.

Figure 6.

Endocytosis inhibition leads to decreased autophagic activity even with mTORC1 inactivation. A, left: 293T cells were treated for 4 h with DMSO, 80 μm Dynasore, 30 μm PitStop 2, 250 nm Torin 1, and 100 nm bafilomycin A1 singly or in combination. All combinations contained 0.175% DMSO as solvent. Right: DMSO, 80 μm Dynasore, 30 μm PitStop 2, and 100 nm bafilomycin A1 were added singly or in combination to RPMI1640 medium containing 10% Nu-Serum or to amino acid-free RPMI1640 medium containing 10% dialyzed Nu-Serum, and 293T cells were treated with the indicated medium for 4 h. All combinations contained 0.15% DMSO as solvent. B, 293T cells were treated as in A and subjected to immunofluorescence with anti-LC3 and anti-WIPI2 antibodies. Arrows indicate WIPI2 dots. Scale bar = 10 μm. C, quantification of immunofluorescence data in B. Each bar indicates mean ± S.D. of three independent experiments. *, p < 0.05; **, p < 0.01; ****, p < 0.0001.