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Cited by 764PI3K/AKT/mTOR signaling in cancerAutophagy in Disease and TherapyMetabolism, Diabetes, and Cancer

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Differential regulation of mTORC1 by leucine and glutamine

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Jewell, Jenna L., Kim, Young Chul, Russell, Ryan C., Yu, Fa-Xing, Park, Hyun Woo, Plouffe, Steven W., Tagliabracci, Vincent S., Guan, Kun-Liang·2015·Science
DOI 10.1126/science.1259472PMC PMC4384888
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Abstract

Getting specific about amino acid sensing: The protein kinase complex mTORC1 regulates growth and metabolism, and its activity is controlled in response to the abundance of cellular amino acids. Jewell et al. report that control of mTORC1 in response to glutamine does not require the Rag guanosine triphosphatase (GTPase) implicated in the sensing of other amino acids such as leucine (see the Perspective by Abraham). For sensing of glutamine, another GTPase, Arf1, was required. Distinct mechanisms thus appear to couple various amino acids to signaling by the mTORC1 complex. Science , this issue p. 194 ; see also p. 128

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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. 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.CC BY-NC · PubMed Central
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. 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.CC BY-NC · PubMed Central
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. 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.CC BY-NC · PubMed Central
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.
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.CC BY-NC · PubMed Central

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Metadata

Authors
Jewell, J. L., Kim, Y. C., Russell, R. C., Yu, F.-X., Park, H. W., Plouffe, S. W., Tagliabracci, V. S., & Guan, K.-L.
Affiliations
  • University of California San Diego (US)
  • Fudan University (CN)
Corresponding author
  • Kim, Young Chul
  • Russell, Ryan C.
  • Guan, Kun-Liang
ORCID
Venue
Science
Publisher
American Association for the Advancement of Science (AAAS)
Published
2015-01-09
Volume
347
Issue
6218
Pages
194-198
ISSN
  • 0036-8075 (ISSN-L)
  • 1095-9203
DOI
10.1126/science.1259472
PMC
PMC4384888
OpenAlex
W2157760261
Cited by
757
References
35
Citations by year
2026: 37 · 2025: 45 · 2024: 58 · 2023: 62 · 2022: 75 · 2021: 102 · 2020: 81 · 2019: 54 · 2018: 60 · 2017: 57 · 2016: 65 · 2015: 60
FWCI
35.04
Type
journal article
Language
en
Open access
Licence
Locations
  • Science (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion, cc-by-nc)
  • PubMed Central (acceptedVersion, cc-by-nc)
  • eScholarship (California Digital Library) (submittedVersion, cc-by-nc)
  • eScholarship (California Digital Library) (submittedVersion)
Funders
  • NIH (R01GM051586, R01CA108941, T32CA121938, T32GM007752, K99DK099254)
  • Department of Defense (W81XWH-13-1-055)
  • The Hartwell Foundation
  • Canadian Institutes of Health Research
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › PI3K/AKT/mTOR signaling in cancer
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Autophagy in Disease and Therapy
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Metabolism, Diabetes, and Cancer
Keywords
mTORC1, leucine, glutamine, Rag GTPases, Ragulator complex, V-ATPase, lysosomal translocation, Arf1, amino acid signaling, cell growth regulation, raga
MeSH terms
Mechanistic Target of Rapamycin Complex 1, Regulatory-Associated Protein of mTOR, Animals, Enzyme Activation, Glutamine, Humans, Leucine, Lysosomes, Mice, Knockout, Monomeric GTP-Binding Proteins, ADP-Ribosylation Factor 1, Protein Transport, Multiprotein Complexes, Adaptor Proteins, Signal Transducing, Mice, Gene Knockdown Techniques, HEK293 Cells, TOR Serine-Threonine Kinases

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