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Metabolism. Differential regulation of mTORC1 by leucine and glutamine.

Jewell JL, Kim YC, Russell RC, Yu FX, Park HW, Plouffe SW, Tagliabracci VS, Guan KL - Science (2015)

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Moores Cancer Center, University of California, San Diego, La Jolla, CA 92093, USA.

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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.
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Figure 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.

Mentions: In Drosophila S2 cells, TORC1 activity is inhibited in cells depleted of the Drosophila ADP ribosylation factor-Arf1 (dArf1) (21), and we observed a further decrease in amino acid–induced TORC1 activation when both dRagA and dArf1 were depleted (Fig. 4A). We used small interfering RNA (siRNA) to deplete Arf1 from HEK293A RagA/B KO cells. Gln stimulated mTORC1 activation in RagA/B KO cells treated with a control siRNA; however, it failed to induce mTORC1 activation in RagA/B KO cells depleted of Arf1 (Fig. 4B). Depletion of other Arf family members failed to inhibit Gln-induced activation of mTORC1 in RagA/B KO cells (fig. S14A). Treatment of RagA/B KO cells with brefeldin A (BFA), an Arf1 GEF inhibitor (22), at high doses blocked amino acid signaling to mTORC1, whereas BFA caused only a small decrease in mTORC1 activation in response to amino acids in control cells (Fig. 4C and fig. S14, B and C). Consistently, BFA blocked Gln-induced activation of mTORC1 in RagA/B KO cells (Fig. 4D and fig. S14D). In addition, depletion of Arf1 or BFA treatment did not inhibit Leu-induced activation of mTORC1 in control cells, nor did they affect lysosomal pH (fig. S14, E to G).


Metabolism. Differential regulation of mTORC1 by leucine and glutamine.

Jewell JL, Kim YC, Russell RC, Yu FX, Park HW, Plouffe SW, Tagliabracci VS, Guan KL - Science (2015)

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.
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Figure 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.
Mentions: In Drosophila S2 cells, TORC1 activity is inhibited in cells depleted of the Drosophila ADP ribosylation factor-Arf1 (dArf1) (21), and we observed a further decrease in amino acid–induced TORC1 activation when both dRagA and dArf1 were depleted (Fig. 4A). We used small interfering RNA (siRNA) to deplete Arf1 from HEK293A RagA/B KO cells. Gln stimulated mTORC1 activation in RagA/B KO cells treated with a control siRNA; however, it failed to induce mTORC1 activation in RagA/B KO cells depleted of Arf1 (Fig. 4B). Depletion of other Arf family members failed to inhibit Gln-induced activation of mTORC1 in RagA/B KO cells (fig. S14A). Treatment of RagA/B KO cells with brefeldin A (BFA), an Arf1 GEF inhibitor (22), at high doses blocked amino acid signaling to mTORC1, whereas BFA caused only a small decrease in mTORC1 activation in response to amino acids in control cells (Fig. 4C and fig. S14, B and C). Consistently, BFA blocked Gln-induced activation of mTORC1 in RagA/B KO cells (Fig. 4D and fig. S14D). In addition, depletion of Arf1 or BFA treatment did not inhibit Leu-induced activation of mTORC1 in control cells, nor did they affect lysosomal pH (fig. S14, E to G).

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Moores Cancer Center, University of California, San Diego, La Jolla, CA 92093, USA.

Show MeSH
Related in: MedlinePlus