Metabolism. Differential regulation of mTORC1 by leucine and glutamine

Abstract

The mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) integrates environmental and intracellular signals to regulate cell growth. Amino acids stimulate mTORC1 activation at the lysosome in a manner thought to be dependent on the Rag small guanosine triphosphatases (GTPases), the Ragulator complex, and the vacuolar H(+)-adenosine triphosphatase (v-ATPase). We report that leucine and glutamine stimulate mTORC1 by Rag GTPase-dependent and -independent mechanisms, respectively. Glutamine promoted mTORC1 translocation to the lysosome in RagA and RagB knockout cells and required the v-ATPase but not the Ragulator. Furthermore, we identified the adenosine diphosphate ribosylation factor-1 GTPase to be required for mTORC1 activation and lysosomal localization by glutamine. Our results uncover a signaling cascade to mTORC1 activation independent of the Rag GTPases and suggest that mTORC1 is differentially regulated by specific amino acids.

Fig. 1. Gln, but not Leu, activates mTORC1 independently of RagA and RagB

mTORC1 activity was analyzed by the phosphorylation of S6K1 (pS6K1), S6 (pS6), and 4EBP1 (p4EBP1) and the mobility shift of 4EBP1. AA, amino acids. (A) mTORC1 activity was analyzed in control (CON) and RagA/B KO MEFs under normal conditions (NC). mTOR, Raptor, RagA, RagB, and RagC protein were also analyzed. (B) mTORC1 activity was analyzed in CON and RagA/B KO MEFs under NC, in the absence of amino acids (–AA) and at the indicated times after the addition of amino acids (+AA) (left). Relative abundance of pS6K1 is plotted (right). (C) mTORC1 activity after stimulation with Leu (top) or Gln (bottom) in CON and RagA/B KO MEFs. (D) mTORC1 activity was analyzed after stimulation with Leu or Gln in RagA/B KO MEFs stably expressing Flag-tagged RagA at the indicated times. (E) mTORC1 activity was analyzed in CON, RagA KO (A1) and RagA/B KO (AB1) HEK293A cells that were starved of amino acids or stimulated with Leu or Gln for 150 min.

Fig. 2. Gln-induced mTORC1 lysosomal localization in the absence of RagA and RagB

(A) Immunofluorescence (IF) analysis depicting mTORC1 activation by phosphorylation of S6 (pS6; orange) in RagA/B KO MEFs. mTOR (green) and LAMP2 (red) are also shown. Quantification of the percentage of pS6 cells with mTOR and LAMP2 colocalization (top right) and the percentage of cells with mTOR not at lysosome that also contain S6 phosphorylation (bottom right). (B and C) IF analysis depicting mTOR and LAMP2 in CON (B) or RagA/B KO MEFs (C), without amino acids or stimulated with Leu or Gln for 150 min (top). Quantification of the percent of cells with mTOR at the lysosome without amino acids or stimulated with Leu or Gln (bottom). Higher-magnification images (A) to (C) of the area depicted by the inset and their overlays are shown on the right.

Fig. 3. Gln-induced mTORC1 activation requires the v-ATPase but not the Ragulator

mTORC1 activity was analyzed by the phosphorylation of S6K1 (pS6K) and the mobility shift of 4EBP1. (A) mTORC1 activity was analyzed in CON and p14 KO MEFs that were starved of amino acids, then stimulated with Leu (top) or Gln (bottom) at the indicated times. (B and C) Analysis of mTORC1 activity in CON and RagA/B KO cells that were starved of amino acids; pretreated with or without 1 μM Baf A; followed by amino acid, Leu, or Gln stimulation for 150 min. (D) CON and RagA/B KO MEFs were treated with shRNA CON (shCON) or shRNA targeting the v-ATPase V0c subunit (shV0c). CON and RagA/B KO MEFs were starved of amino acids, followed by amino acid stimulation, and mTORC1 activity was assessed.

Fig. 4. Arf1 is required for Gln signaling to mTORC1 in the absence of RagA and RagB

(A) TORC1 activity was analyzed for the phosphorylation of Drosophila S6K1 (pdS6K1) in Drosophila S2 cells treated with double-stranded RNA (dsRNA) targeting GFP, RagA, or Arf1. Cells were starved of amino acids for 1 hour, then stimulated with or without amino acids for 30 min. (B) mTORC1 activity was analyzed by the phosphorylation of S6K1 (pS6K1) or mobility shift of 4EBP1 in RagA/B KO HEK293A cells treated with control (siCON) or Arf1 siRNA (siArf1). Cells were starved of amino acids then stimulated with Gln for 150 min. (C) mTORC1 activity was analyzed as in (B) in CON and RagA/B KO MEFs starved of amino acids, then pretreated with the indicated concentrations of BFA, and stimulated with amino acids for 150 min. Labels s.e. and l.e. denote shorter exposure and longer exposure, respectively. (D) mTORC1 activity was analyzed as in (B) in RagA/B KO HEK293A cells starved of amino acids, then pretreated with or without 1 μM BFA, and stimulated with Leu or Gln for 150 min. (E) IFanalysis depicting mTORC1 activation (pS6; orange) and lysosomal localization (LAMP2; red, mTOR; green) in RagA/B KO MEFs. Cells were starved of amino acids, pretreated with or without 1 μM BFA, followed by stimulation with Gln for 150 min. Higher magnification images of the area depicted by the inset and their overlays are shown on the right of the images. Quantification of the percentage of cells with mTOR at the lysosome under different conditions and corresponding Western blot (right). (F) mTORC1 activity was analyzed as in (B) in RagA/B KO HEK293A cells transfected with HA-Raptor or HA-Raptor containing the C-terminal CAAX motif of Rheb (HA-Raptor-C-Rheb). Cells were starved of amino acids, pretreated with or without 1 μM BFA, and stimulated with Gln for 150 min.