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. 2014 Feb 13;156(4):771-85.
doi: 10.1016/j.cell.2013.11.049.

Spatial control of the TSC complex integrates insulin and nutrient regulation of mTORC1 at the lysosome

Suchithra Menon  1 , Christian C Dibble  2 , George Talbott  1 , Gerta Hoxhaj  1 , Alexander J Valvezan  1 , Hidenori Takahashi  3 , Lewis C Cantley  4 , Brendan D Manning  5
Affiliations

Spatial control of the TSC complex integrates insulin and nutrient regulation of mTORC1 at the lysosome

Suchithra Menon et al. Cell. .

Abstract

mTORC1 promotes cell growth in response to nutrients and growth factors. Insulin activates mTORC1 through the PI3K-Akt pathway, which inhibits the TSC1-TSC2-TBC1D7 complex (the TSC complex) to turn on Rheb, an essential activator of mTORC1. However, the mechanistic basis of how this pathway integrates with nutrient-sensing pathways is unknown. We demonstrate that insulin stimulates acute dissociation of the TSC complex from the lysosomal surface, where subpopulations of Rheb and mTORC1 reside. The TSC complex associates with the lysosome in a Rheb-dependent manner, and its dissociation in response to insulin requires Akt-mediated TSC2 phosphorylation. Loss of the PTEN tumor suppressor results in constitutive activation of mTORC1 through the Akt-dependent dissociation of the TSC complex from the lysosome. These findings provide a unifying mechanism by which independent pathways affecting the spatial recruitment of mTORC1 and the TSC complex to Rheb at the lysosomal surface serve to integrate diverse growth signals.

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Figures

Figure 1
Figure 1. Insulin signaling acutely stimulates mTORC1 without effects on TSC complex stability or a block in Rheb-GAP activity
(A) Schematic of the Akt-TSC complex-Rheb circuit through which insulin activates mTORC1. (B) HeLa cells were serum starved then stimulated with a time course of insulin. (C,D) HeLa cells were serum starved then stimulated with insulin (15 min) prior to lysis and immunoprecipitation with antibodies to TSC2, TSC1, and TBC1D7 (C) or phospho-TSC2-T1462 and phospho-TSC2-S939 (D). (E) Lysates from cells treated as in (C) were fractionated using size-exclusion chromatography. Estimated molecular weights (kDa) for fractions were calculated from a standard curve (Figure S1E). (F) Endogenous TSC complexes immunoprecipitated with a TSC1 antibody, or IgG control, from lysates of cells treated as in (C) were subjected to Rheb-GAP assays using recombinant GST or GST-Rheb preloaded with GTP[α-32P]. Rheb-bound GTP and GDP were separated by thin-layer chromatography (TLC). The percentage of conversion to GDP (GDP/GTP + GDP) are graphed as the mean of three independent experiments ±SEM. *p<0.02 compared to TSC1 immunoprecipate from unstimulated cells. See supporting data in Figure S1.
Figure 2
Figure 2. Insulin acutely disrupts the lysosomal localization of the TSC complex
(A) HeLa cells were serum starved then stimulated with a time course of insulin prior to immunofluorescent labeling of endogenous TSC2 (red) and LAMP2 (green). Representative cells are shown where yellow or orange pixels indicate colocalization in the merged images. Percent colocalization and Pearson’s Correlation Coefficient (PCC) are graphed as a mean±SEM (right). *p< 1X10−6 (% colocalization) and *p< 1X10−12 (PCC), compared to unstimulated (0 min). (B) Cells were treated and labeled as in (A), but with a single 15-min insulin stimulation, and imaged by confocal microscopy. An enlarged view of the LAMP2-containing compartment from the bottom cells in the two images is shown below. Percent colocalization and PCC are presented as in (A). *p< 1X10−13 (% colocalization) and *p< 1X10−10 (PCC) compared to unstimulated (0 min). (C) HeLa cells stably expressing a control shRNA (shGFP) or one targeting TBC1D7 (shTBC1D7) were treated as in (B) and labeled for TBC1D7 (red) and LAMP1 (green). Representative cells are shown and percent colocalization is presented as in (A). *p< 1X10−10. (D) Cells treated as in (B) were labeled for TSC2 (red) and TBC1D7 (green), with percent colocalization presented as in (A). (E) Cells treated as in (B) and lysates were separated into heavy membrane and light membrane/cytosolic fractions. See supporting data in Figure S2.
Figure 3
Figure 3. Forced targeting of TSC2 to the lysosome suppresses insulin-stimulated mTORC1 signaling
(A) Schematic of FLAG-tagged WT-TSC2 and Lyso-TSC2, a fusion of TSC2 with the lysosome- targeting signal of p18/LAMTOR1. (B) Tsc2−/− MEFs were infected with lentiviruses encoding WT-TSC2 or Lyso-TSC2 and were serum starved then insulin stimulated (15 min). Representative TSC2-expressing cells are shown colabeled for TSC2 (red) and LAMP1 (green). Percent colocalization is graphed as the mean±SEM (below). *p< 1X10−12 compared to unstimulated WT-TSC2. (C) WT-TSC2 or Lyso-TSC2 were cotransfected with HA-S6K1 in Tsc2−/− MEFs, followed by serum starvation and insulin stimulation (100 nM, 15 min). Phosphorylation of the HA-S6K1 reporter was detected in anti-HA immunoprecipitates, and the relative ratios of phospho-S6K1 to total S6K1 are shown normalized to unstimulated WT-TSC2 cells. (D) 293E cells were cotransfected with empty vector (vec), WT-TSC2, or Lyso-TSC2 and HA-S6K1 and were treated and analyzed as in (C), with relative phospho-S6K1 levels normalized to the unstimulated vector cells. See supporting data in Figure S3.
Figure 4
Figure 4. Reciprocal effects of amino acids and insulin on localization of mTORC1 and the TSC complex to the lysosome
(A) HeLa cells were grown in the absence (ss) or presence of dialyzed serum (dFBS) and then starved of all amino acids (50 min) prior to stimulation with insulin (15 min), amino acids (10 min), or insulin (15 min) plus amino acids (final 10 min). Light and dark exposures are shown for phospho-S6K1. (B) HeLa cells were serum starved and stimulated with insulin (15 min) prior to immunofluorescent co-labeling of TSC2 or mTOR with LAMP2 (images shown in Figure S4A). Percent colocalization is graphed as the mean±SEM. *p< 1X10−11. (C) Cells starved of serum (16 h) and amino acids (50 min) were stimulated with insulin (15 min) prior to co-labeling of TSC2 (red) and LAMP2 (green) and quantification of colocalization as in (B) (graph at right). *p< 1X10−11. (D) Cells starved of serum (16 h) and amino acids (50 min) were stimulated with amino acids (10 min) prior to labeling and quantification of colocalization as in (B) *p< 1X10−8. (E) HeLa cells grown in 10% dFBS (16 hr) and starved of amino acids (50 min) were stimulated with amino acids (10 min) prior to labeling and quantification of colocalization as in (B). *p< 1X10−8 See supporting data in Figure S4.
Figure 5
Figure 5. A subpopulation of Rheb localizes to the lysosome where its colocalization with the TSC complex and mTORC1 is respectively regulated by insulin and amino acids
(A) HeLa cells with siRNA-mediated knockdown of Rheb1 and 2, or control siRNAs, were serum starved (16 hr) with or without FTI-277 (10 μM) prior to co-labeling of Rheb1 (red) and LAMP1 (green). Representative cells are shown, along with an immunoblot of parallel lysates (right) showing effects on Rheb levels and modification (farnesylated (F) and un-farnesylated (UF)) and the percent colocalization graphed as a mean±SEM. *p< 1X10−7. (B) HeLa cells starved of serum (16 hr) and amino acids (50 min) were stimulated with amino acids (10 min) prior to co-labeling of Rheb1 and LAMP1 (images in Figure S5A). The percent colocalization is graphed as in (A). (C) Cells were serum starved then stimulated with insulin (15 min) prior to co-labeling as in (B) (images in Figure S5B). The percent colocalization is graphed as in (A). (D) Cells were treated as in (B) prior to co-labeling of mTOR or TSC2 (red) and Rheb1 (green). Representative cells are shown, and the percent colocalization is graphed as in (A). *p< 1X10−9. (E) Cells were treated as in (C) prior to the labeling and analysis described in (D). *p< 1X10−9. See supporting data in Figure S5.
Figure 6
Figure 6. Localization of the TSC complex, but not mTORC1, to the lysosome is dependent on farnesylated Rheb
(A) HeLa cells with siRNA-mediated knockdown of Rheb1 and 2, or control siRNAs (siC), were serum starved then stimulated with insulin (100 nM, 15 min). Note: the siC and siRheb1/2 samples are from the same blot and exposure. (B) Cells treated as in (A) were co-labeled for TSC2 (red) and LAMP2 (green). Representative cells are shown and percent colocalization is graphed as a mean±SEM (right). *p< 1X10−12 for comparison with unstimulated siC cells. (C) Cells treated as in (A) were co-labeled for mTOR and LAMP2 (images in Figure S6A) and quantified as in (B). (D) HeLa cells serum starved (16 hr) with or without FTI-277 (FTI; 10 μM) were left unstimulated or were stimulated with insulin (15 min). (E) Cells were treated as in (D) prior to labeling and quantification of colocalization as in (B) (graph to right). *p< 1X10−10 for comparison with untreated, serum-starved cells. (F) Cells treated as in (D) were co-labeled for TSC2 and Rheb1 (images in Figure S6B). Colocalization was quantified as in (B). *p< 1X10−9 for comparison with untreated cells. (G) Cells treated as in (D) were co-labeled for mTOR and LAMP2 (images in Figure S6C). Colocalization was quantified as in (B). (H) Cells treated as in (D) were co-labeled for mTOR and Rheb1 (images in Figure S6D). Colocalization was quantified as in (B). *p< 1X10−8 for comparison with untreated cells. (I) Endogenous TSC complex components were pulled down from lysates of serum starved HeLa cells with recombinant purified GST-Rheb preloaded with no nucleotide, GTPγS, or GDPβS compared to control pull downs with GST or GST-Rab5A preloaded with GDPβS. GST-fused bait proteins were detected with Ponceau S stain. (J) GST-Rheb pulldowns were performed as in (I), but cells were stimulated with insulin (15 min). See supporting data in Figure S6.
Figure 7
Figure 7. Akt-mediated phosphorylation of TSC2 results in dissociation of the TSC complex from the lysosome in response to insulin or PTEN loss
(A) Serum starved HeLa cells were pretreated (30 min) with vehicle (DMSO), wortmannin (100 nM), or MK2206 (2 μM) and then stimulated with insulin (15 min). (B) Cells treated as in (A) were co-labeled for TSC2 (red) and LAMP2 (green). Representative cells are shown and the percent colocalization is graphed as a mean±SEM. *p< 1X10−8 for comparison with insulin stimulation in vehicle-treated cells. (C) Cells were treated as in (A) and lysates were separated into heavy membrane and light membrane/cytosolic fractions. (D) Tsc2−/− MEFs expressing empty vector (V), wild-type TSC2 (WT), or the Akt-phosphorylation-site mutant of TSC2 (5A) were serum starved and stimulated with insulin (15 min). (E) Tsc2−/− MEFs were treated as in (D) prior to co-labeling for reconstituted TSC2 (red) and endogenous LAMP1 (green). Colocalization was quantified as in (B). *p< 1X10−8 for comparison with insulin-stimulated, TSC2-WT-expressing cells. (F) Tsc2−/− MEFs reconstituted with wild-type TSC2 (WT) or mutant TSC2 (5A) were treated as in (D) prior to lysis in hypotonic buffer with chemical crosslinking followed by immunoprecipitation with protein A/G agarose alone (C) or with TSC2 antibody. (G) Serum starved (16 hr) PC3 cells were treated (30 min) with vehicle (DMSO) or MK2206 (2 μM). (H) PC3 cells were treated as in (G) prior to co-labeling for TSC2 (red) and LAMP2 (green). Colocalization was quantified as in (B). *p< 1X10−10. (I) Model of the spatial regulation of mTORC1 and the TSC complex at the lysosome. See text for details. See supporting data in Figure S7.
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