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. 2014 Feb 13;156(4):786-99.
doi: 10.1016/j.cell.2014.01.024.

Regulation of TORC1 in response to amino acid starvation via lysosomal recruitment of TSC2

Constantinos Demetriades  1 , Nikolaos Doumpas  2 , Aurelio A Teleman  3
Affiliations

Regulation of TORC1 in response to amino acid starvation via lysosomal recruitment of TSC2

Constantinos Demetriades et al. Cell. .

Abstract

TOR complex 1 (TORC1) is a potent anabolic regulator of cellular growth and metabolism. When cells have sufficient amino acids, TORC1 is active due to its lysosomal localization mediated via the Rag GTPases. Upon amino acid removal, the Rag GTPases release TORC1, causing it to become cytoplasmic and inactive. We show here that, upon amino acid removal, the Rag GTPases also recruit TSC2 to the lysosome, where it can act on Rheb. Only when both the Rag GTPases and Rheb are inactive is TORC1 fully released from the lysosome. Upon amino acid withdrawal, cells lacking TSC2 fail to completely release TORC1 from the lysosome, fail to completely inactivate TORC1, and fail to adjust physiologically to amino acid starvation. These data suggest that regulation of TSC2 subcellular localization may be a general mechanism to control its activity and place TSC2 in the amino-acid-sensing pathway to TORC1.

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Figures

Figure 1
Figure 1. Conserved binding of TSC2 to the Rag GTPase complex
(A) Drosophila Rag proteins are required for complete TORC1 inactivation upon amino acid withdrawal. Immunoblots of S2 cells treated with dsRNAs against LacZ (control), RagA or RagC, incubated with media lacking only amino acids in the presence of dialyzed serum for indicated times. (B) Shot-gun mass spectroscopy analysis identifies Tsc2 as a Rag binding protein. Two statistics of peptide abundance are shown for Tsc2 and control proteins from two independent FLAG-RagA + FLAG-RagC immunoprecipitates (IP) from S2 cells. (C) Confirmation by coIP that Rag proteins bind Tsc2 but not an unrelated protein, Medea. Immunoblots of anti-FLAG IPs from Drosophila S2 cells expressing FLAG-tagged RagA + RagC and V5-tagged Tsc2 or Medea, as indicated. (D) Human TSC2 binds human Rag dimeric complexes. Immunoblots of anti-FLAG IPs from HEK293FT cells expressing FLAG-tagged TSC2 and HA-GST-tagged Rag proteins or an HA-GST control, as indicated.
Figure 2
Figure 2. TSC2 binding to the Rag proteins depends on cellular amino acid signaling
(A) Schematic diagram of RagA, RagB, and various mutants used in panels B-D to delineate the binding interfaces between TSC2 and RagA. (B) Both N-terminal and C-terminal regions of RagA are involved in TSC2 binding. CoIP of FLAG-TSC2 with RagA, RagB, and hybrid versions of the two proteins as shown in panel A, showing that both the N-terminal 33 amino acids as well as the 5 C-terminal amino acids specific for RagA impact TSC2 binding. (C) All five C-terminal amino acids specific for RagA, as shown in panel A, contribute towards TSC2 binding, as each one improves the binding of RagB to TSC2 when introduced singly. CoIP experiments were performed as in (B). (D) The N-terminal 424 amino acids of TSC2 bind strongly to Rag proteins, although the remaining amino acids 425-1784 also contribute towards Rag binding. CoIP in HEK293FT cells of truncated versions of FLAG-TSC2 with HA-RagA+C. ‘Inactive’-locked RagAlow-nucleotide and RagCGTP were used as this complex binds TSC2 most strongly, as in panel F. (E) Amino acid starvation increases binding of endogenous TSC2 to the Rag GTPases. Immunoblots of anti-FLAG IPs from HEK293FT cells expressing FLAG-tagged RagA + RagC, treated +/− amino acids in the presence of dialyzed FBS for 1h. (F) Endogenous TSC2 and TSC1 bind most strongly to Rag dimers in the ‘inactive’ state, which antiparallels Raptor binding. Immunoblots of anti-FLAG IPs from HEK293FT cells expressing FLAG-tagged WT RagA + RagC and mutants locked into the active RagAGTP/RagClow-nucleotide or inactive RagAlow-nucleotide/RagCGTP states.
Figure 3
Figure 3. Amino acid starvation causes TSC2 lysosomal localization in a Rag-dependent manner
(A,C) TSC2 accumulates on lysosomes upon amino acid deprivation. Confocal micrograph of HEK293FT cells (A) or MEFs (C) treated +/− all amino acids in the presence of dialyzed FBS for the indicated times, stained for TSC2 (green) and the lysosomal marker LAMP2 (red). LAMP2 aggregates do not show TSC2 accumulation in the +aa condition. (B) TSC2 quickly relocalizes away from lysosomes upon amino acid readdition. HEK293FT cells, starved for amino acids in the presence of dialyzed serum for 4h, then re-supplied with amino acid-containing media for 5 minutes. Cells stained and imaged as in (A).
Figure 4
Figure 4. Lysosomal recruitment of TSC2 depends on the Rag proteins
(A-A’) Lysosomal localization of TSC2 upon amino acid removal requires the LAMTOR complex. Confocal micrograph of p14/LAMTOR2 knock-out MEFs (A), or control p14 knock-out MEFs reconstituted to express an EGFP-p14 fusion (A’), treated +/− amino acids for 1h in the presence of dialyzed FBS. Lysosomes marked either with anti-LAMP2 antibody or with the lysosomaly localized EGFP-p14. (B) Lysosomal localization of TSC2 upon amino acid removal requires the Rag proteins. MEFs transfected with control siRNA against Renilla luciferase or siRNAs targeting the various Rag proteins, treated +/− amino acids for 1h in the presence of dialyzed FBS. Cells stained and imaged as in (A). (C) Lysosomal localization of TSC2 upon amino acid removal requires that the Rag proteins change to the inactive conformation. MEFs transfected with control siRNA or siRNAs targeting components of the GATOR1 complex DEPDC5, NPRL2, or NPRL3 required for the Rag proteins to become inactive upon amino acid removal. Cells stained and imaged as in (A).
Figure 5
Figure 5. TSC2 loss causes insensitivity to amino acid removal in Drosophila and mammalian cells
(A-B) Tsc2 but not PTEN knock-down makes Drosophila cells largely insensitive to amino acid removal. (A) Immunoblots of Drosophila S2 cells treated with no dsRNA, dsRNA against GFP (control), or dsRNA against Tsc2, treated with media containing (+) or lacking amino acids for the indicated times. (B) PTEN knock-down S2 cells efficiently inactivate TORC1 upon amino acid removal, despite starting with elevated TORC1 activity levels. (C-D) TSC2 knock-out MEFs do not completely shut off TORC1 upon amino acid withdrawal, whereas PTEN knock-out MEFs do. Immunoblots from (C) TSC2−/− and the respective control TSC2+/+ MEFs, or (D) PTEN knock-out MEFs, treated with medium +/− amino acids for the indicated times. Lanes 6 and 12 (C), cells treated +20nM Rapamycin for 1h in the presence of amino acids (‘Rapa’). Quantified with a LI-COR imaging system.
Figure 6
Figure 6. TSC2 is required for mTOR to localize away from lysosomes upon amino acid removal in a Rheb dependent manner
(A-B) mTOR is released from lysosomes upon amino acid removal in control (A) but not TSC2-null MEFs (A’). This is rescued by re-expressing TSC2 + EGFP (to mark transfected cells) but not EGFP alone in the TSC-null MEFs (B). Note that the cell expressing TSC2 and GFP no longer has mTOR accumulated on lysosomes, whereas the surrounding, non-transfected cells retain lysosomally localized mTOR. (C) Defective release of mTOR from lysosomes upon amino acid withdrawal in TSC2-null MEFs is rescued by knocking down Rheb. TSC2-null MEFs transfected with either Rheb siRNAs (upper panels) or control siRNAs (lower panels) treated 1h with +/− amino acid containing medium in the presence of dialyzed FBS.
Figure 7
Figure 7. TSC2 null cells are impaired in their response to amino acid starvation, leading to their death
(A-B) Amino acid removal causes cell death in TSC2 null cells. TSC2−/− and the respective TSC2+/+ control MEFs were treated on day 1 with medium +/− amino acids in the presence of dialyzed FBS. On day 4, all samples were given fresh medium containing amino acids. (A) DIC images captured on days 1, 4, and 7. (B) Cell viability determined using the Cell-Titer Glo kit (Promega). (C) TSC2-null MEFs show defective autophagy induction upon amino acid withdrawal. TSC2−/− and the respective TSC2+/+ control MEFs were treated with medium containing (+) or lacking amino acids in the presence of dialyzed FBS for the indicated times. Activation of autophagy visualized on an immunoblot via increased conversion of LC3A to the faster migrating lipidated form II and degradation of p62 protein. (D-E) Pharmacological inhibition of TORC1 rescues survival of TSC2-null MEFs upon amino acid withdrawal. TSC2−/− mouse embryonic fibroblasts were treated with medium containing (+aa) or lacking (−aa) amino acids with or without 20nM Rapamycin (−aa/+Rapa) for 3 days in the presence of dialyzed FBS. DIC images (D) and respective cell viability titers determined using the Cell-Titer Glo kit (Promega) (E). Error bars: std. dev.
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