Abstract
The mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of cell growth that responds to diverse environmental signals and is deregulated in many human diseases, including cancer and epilepsy1,2,3. Amino acids are a key input to this system, and act through the Rag GTPases to promote the translocation of mTORC1 to the lysosomal surface, its site of activation4. Multiple protein complexes regulate the Rag GTPases in response to amino acids, including GATOR1, a GTPase activating protein for RAGA, and GATOR2, a positive regulator of unknown molecular function. Here we identify a protein complex (KICSTOR) that is composed of four proteins, KPTN, ITFG2, C12orf66 and SZT2, and that is required for amino acid or glucose deprivation to inhibit mTORC1 in cultured human cells. In mice that lack SZT2, mTORC1 signalling is increased in several tissues, including in neurons in the brain. KICSTOR localizes to lysosomes; binds and recruits GATOR1, but not GATOR2, to the lysosomal surface; and is necessary for the interaction of GATOR1 with its substrates, the Rag GTPases, and with GATOR2. Notably, several KICSTOR components are mutated in neurological diseases associated with mutations that lead to hyperactive mTORC1 signalling5,6,7,8,9,10. Thus, KICSTOR is a lysosome-associated negative regulator of mTORC1 signalling, which, like GATOR1, is mutated in human disease11,12.
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References
Laplante, M. & Sabatini, D. M. mTOR signaling in growth control and disease. Cell 149, 274–293 (2012)
Dibble, C. C. & Manning, B. D. Signal integration by mTORC1 coordinates nutrient input with biosynthetic output. Nat. Cell Biol. 15, 555–564 (2013)
Jewell, J. L., Russell, R. C. & Guan, K.-L. Amino acid signalling upstream of mTOR. Nat. Rev. Mol. Cell Biol. 14, 133–139 (2013)
Sancak, Y. et al. The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science 320, 1496–1501 (2008)
Basel-Vanagaite, L. et al. Biallelic SZT2 mutations cause infantile encephalopathy with epilepsy and dysmorphic corpus callosum. Am. J. Hum. Genet. 93, 524–529 (2013)
Venkatesan, C., Angle, B. & Millichap, J. J. Early-life epileptic encephalopathy secondary to SZT2 pathogenic recessive variants. Epileptic Disord. 18, 195–200 (2016)
Baple, E. L. et al. Mutations in KPTN cause macrocephaly, neurodevelopmental delay, and seizures. Am. J. Hum. Genet. 94, 87–94 (2014)
Pajusalu, S., Reimand, T. & Õunap, K. Novel homozygous mutation in KPTN gene causing a familial intellectual disability-macrocephaly syndrome. Am. J. Med. Genet. A. 167A, 1913–1915 (2015)
Mc Cormack, A. et al. 12q14 microdeletions: additional case series with confirmation of a macrocephaly region. Case Rep. Genet. 2015, 192071 (2015)
Crino, P. B. mTOR: a pathogenic signaling pathway in developmental brain malformations. Trends Mol. Med. 17, 734–742 (2011)
D’Gama, A. M. et al. Mammalian target of rapamycin pathway mutations cause hemimegalencephaly and focal cortical dysplasia. Ann. Neurol. 77, 720–725 (2015)
Baldassari, S., Licchetta, L., Tinuper, P., Bisulli, F. & Pippucci, T. GATOR1 complex: the common genetic actor in focal epilepsies. J. Med. Genet. 53, 503–510 (2016)
Sancak, Y. et al. Ragulator–Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Cell 141, 290–303 (2010)
Wei, Y. & Lilly, M. A. The TORC1 inhibitors Nprl2 and Nprl3 mediate an adaptive response to amino-acid starvation in Drosophila . Cell Death Differ. 21, 1460–1468 (2014)
Wei, Y. et al. TORC1 regulators Iml1/GATOR1 and GATOR2 control meiotic entry and oocyte development in Drosophila. Proc. Natl Acad. Sci. USA 111, E5670–E5677 (2014)
Cai, W., Wei, Y., Jarnik, M., Reich, J. & Lilly, M. A. The GATOR2 component Wdr24 regulates TORC1 activity and lysosome function. PLoS Genet. 12, e1006036 (2016)
Bar-Peled, L. et al. A tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to mTORC1. Science 340, 1100–1106 (2013)
Efeyan, A. et al. Regulation of mTORC1 by the Rag GTPases is necessary for neonatal autophagy and survival. Nature 493, 679–683 (2013)
Kalender, A. et al. Metformin, independent of AMPK, inhibits mTORC1 in a Rag GTPase-dependent manner. Cell Metab. 11, 390–401 (2010)
Frankel, W. N., Yang, Y., Mahaffey, C. L., Beyer, B. J. & O’Brien, T. P. Szt2, a novel gene for seizure threshold in mice. Genes Brain Behav. 8, 568–576 (2009)
Baulac, S. mTOR signaling pathway genes in focal epilepsies. Prog. Brain Res. 226, 61–79 (2016)
Kim, D. H. et al. mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell 110, 163–175 (2002)
Boussif, O. et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl Acad. Sci. USA 92, 7297–7301 (1995)
Tsun, Z.-Y. et al. The folliculin tumor suppressor is a GAP for the RagC/D GTPases that signal amino acid levels to mTORC1. Mol. Cell 52, 495–505 (2013)
Zoncu, R. et al. mTORC1 senses lysosomal amino acids through an inside-out mechanism that requires the vacuolar H+-ATPase. Science 334, 678–683 (2011)
Petit, C. S., Roczniak-Ferguson, A. & Ferguson, S. M. Recruitment of folliculin to lysosomes supports the amino acid-dependent activation of Rag GTPases. J. Cell Biol. 202, 1107–1122 (2013)
Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012)
Yilmaz, Ö. H. et al. mTORC1 in the Paneth cell niche couples intestinal stem-cell function to calorie intake. Nature 486, 490–495 (2012)
Acknowledgements
We thank all members of the Sabatini Laboratory for helpful insights. We thank K. McKinley and I. Cheeseman for providing the retroviral GFP constructs used in this work, C. Mahaffey for technical support with the mouse experiments and J. Xie from Cell Signaling Technology Inc. for providing the DEPDC5, MIOS, NPRL2, WDR24, WDR59, C12orf66, SEH1L and SZT2 antibodies. This work was supported by grants from the NIH to D.M.S. (R01 CA103866 and R37 AI47389) and W.N.F. (R37 NS031348), Department of Defense (W81XWH-07-0448) to D.M.S. and fellowship support from the NIH to R.L.W. (T32 GM007753 and F30 CA189333), L.C. (F31 CA180271) and J.M.O. (T32 GM007753 and F30 CA210373), from NSF to K.J.C. (2016197106), from the National Defense Science & Engineering Graduate Fellowship (NDSEG) Program to G.A.W., from the Life Sciences Research Foundation to K.S. (Pfizer fellow), from the EMBO Long-Term Fellowship to M.A.-R. and from the Paul Gray UROP Fund to S.M.S. (3143900). D.M.S. is an investigator of the Howard Hughes Medical Institute.
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Contributions
R.L.W., L.C. and D.M.S. formulated the research plan and interpreted experimental results. R.L.W. and L.C. designed and performed most experiments with assistance from G.A.W., X.G., J.M.O., K.S., K.J.C., J.K., S.M.S. and M.A.-R. X.G. carried out the epistasis experiments, G.A.W. performed experiments using purified lysosomes, J.M.O. carried out experiments involving glucose signalling and K.S. performed experiments using size-exclusion chromatography. S.P. and W.N.F. generated the Szt2 gene-trap mice and collected tissues, which were analysed by R.L.W. and G.A.W. R.L.W. and D.M.S. wrote the manuscript and all authors edited it.
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D.M.S. is a founder, consultant and shareholder of Navitor Pharmaceuticals Inc., which is targeting the amino acid sensing pathway for therapeutic benefit. R.L.W., L.C. and J.M.O. are shareholders of Navitor Pharmaceuticals. R.L.W., L.C., J.M.O., D.M.S. and the Whitehead Institute have filed two provisional patents that relate to amino acid sensing by the mTOR pathway.
Extended data figures and tables
Extended Data Figure 1 GATOR1 and GATOR2 associate with endogenous KICSTOR components in an amino-acid-insensitive manner.
a, An endogenously tagged GATOR1 component co-immunoprecipitates endogenous KICSTOR. Anti-Flag immunoprecipitates were prepared from HEK293T cells that express endogenously Flag-tagged DEPDC5, a GATOR1 component, that had been starved of amino acids for 50 min or starved and restimulated with amino acids for 10 min. Immunoprecipitates and cell lysates were analysed by immunoblotting for the indicated proteins. b, An endogenously tagged GATOR2 component co-immunoprecipitates endogenous GATOR1 and KICSTOR. Anti-Flag immunoprecipitates were prepared from HEK293T cells that expressed endogenously Flag-tagged WDR59, a GATOR2 component, and treated as in a. Immunoprecipitates and cell lysates were analysed by immunoblotting for the indicated proteins. c, An anti-KPTN antibody co-immunoprecipitates endogenous components of KICSTOR, GATOR1 and GATOR2. Anti-KPTN immunoprecipitates were prepared from wild-type HEK293T treated as in a. Immunoprecipitates and cell lysates were analysed by immunoblotting for the indicated proteins. Anti-GSK3β immunoprecipitates were used to monitor the non-specific binding of proteins to the beads. d, KPTN and ITFG2 form a heterodimer that requires SZT2 to associate with C12orf66. Anti-Flag immunoprecipitates and lysates prepared from HEK293T cells that expressed the indicated cDNAs were analysed by immunoblotting for the relevant epitope tags. e, Loss of KICSTOR components did not have a significant effect on the expression levels of GATOR1 or GATOR2 components. HEK293T cell clones deficient for indicated KICSTOR components or NPRL3 or WDR24 were generated via the CRISPR–Cas9 system and single-cell cloning. Cell lysates were analysed by immunoblotting for the indicated proteins. DNA sequencing of the C12orf66 gene was used to verify out-of-frame mutations in the genomic locus of the sgC12orf66 cells because an antibody that detects the C12orf66 protein in cell lysates is not available. The HEK293T cell clones analysed here were used in subsequent figures, where indicated. f, KPTN interacts with ITFG2 even in cells that lack other KICSTOR components. Immunoprecipitates and cell lysates prepared from wild-type, SZT2- or C12orf66-deficient HEK293T cell clones that expressed the indicated proteins were analysed by immunoblotting. g, Expression levels of KPTN and ITFG2 in HEK293T cells that stably express the indicated sgRNAs under amino-acid-starved or -replete conditions. Cell lysates were analysed by immunoblotting for the levels of the indicated proteins. Raptor serves as a loading control. These same cell lines were also analysed for pS6K1 and S6K1 levels in Extended Data Fig. 5a–c. h, KPTN–ITFG2 does not compete with C12orf66 for association with SZT2. HEK293T cells that expressed the indicated cDNAs were treated and analysed as in d.
Extended Data Figure 2 Size-exclusion chromatography analysis of KICSTOR components.
Lysates of wild-type or SZT2-deficient (sgSZT2) HEK293T cells were fractionated with tandem Superose 6 size-exclusion chromatography columns and the collected fractions were analysed by immunoblotting for the indicated proteins. Coloured bars indicate fractions that contain the protein indicated by that colour in the key. Fractions containing the molecular-weight standards are indicated. Note that the C12orf66 antibody exhibits significant background when used to probe total cell lysates by immunoblotting so we are only confident that the bands in the high-molecular-weight fractions, which disappear in the SZT2-deficient cells, actually represent C12orf66.
Extended Data Figure 3 SZT2 interacts with KPTN–ITFG2, C12orf66 and GATOR1.
a, SZT2 interacts with GATOR1 in the absence of other KICSTOR components. HEK293T cells that stably expressed a control sgRNA (sgAAVS1) or sgRNAs targeting the indicated KICSTOR components were transfected with the indicated cDNAs. Anti-Flag immunoprecipitates were prepared and analysed, along with cell lysates, by immunoblotting for the relevant epitope tags. The ratios of the intensities of the HA–SZT2 to Flag–DEPDC5 bands are indicated below the Flag–DEPDC5 blot. See Extended Data Fig. 1g for the expression levels of the KICSTOR components in the cell lines used here. b, SZT2 links the other KICSTOR components to the GATOR complexes. Anti-Flag immunoprecipitates prepared from wild-type or SZT2-deficient HEK293T cells that expressed the indicated cDNAs were analysed by immunoblotting for the indicated proteins. Non-specific bands are marked with an asterisk. See Extended Data Fig. 1e for the expression level of SZT2 in the SZT2-deficient HEK293T cells. c, C12orf66 interacts with SZT2 at a distinct site from KPTN–ITFG2. HEK293T cells that expressed the indicated cDNAs were analysed as in b. d, GATOR1 interacts with the first and second regions of SZT2. HEK293T cells that expressed the indicated cDNAs were analysed as in b. e, f, The association of SZT2 with KPTN–ITFG2 persists in the absence of GATOR1 (e) or GATOR2 (f). Anti-Flag immunoprecipitates were prepared from wild-type, NPRL3- or WDR24-deficient HEK293T cells that expressed the indicated cDNAs and analysed as in b.
Extended Data Figure 4 Subcellular localization of the GATOR and KICSTOR complexes.
a, Expression levels of GFP-tagged GATOR1, GATOR2 or KICSTOR components used in the localization experiments. NPRL2-deficient or wild-type HeLa cells that stably expressed the indicating GFP-tagged proteins were single-cell sorted for the low-GFP population and single-cell clones were analysed by immunoblotting for levels of the indicated proteins. b, Quantification of the imaging data in Fig. 1g–i. Data are mean ± s.e.m. c, Amino acids do not control the localization of GATOR2 at the lysosomal surface. Wild-type HeLa cells that stably expressed GFP-tagged WDR24, a component of GATOR2, were starved, or starved and restimulated with amino acids for the indicated times before processing for immunofluorescence detection of GFP and LAMP2. Scale bar, 10 μm. Quantification is shown in the bar graph. d, Amino acids do not regulate the amounts of GATOR1, GATOR2 or KICSTOR components on purified lysosomes. Lysosomes immunopurified with anti-HA beads from wild-type HEK293T cells that expressed HA-tagged LAMP1 and treated as in c were analysed by immunoblotting for the levels of the indicated proteins.
Extended Data Figure 5 Loss of KICSTOR affects the sensitivity of the mTORC1 pathway to nutrients but not growth factors.
a–c, CRISPR–Cas9-mediated depletion of KPTN (a), ITFG2 (b) or C12orf66 (c) renders mTORC1 signalling insensitive to amino acid deprivation. HEK293T cells that stably expressed the indicated sgRNAs were starved of amino acids for 50 min, or starved and restimulated with amino acids for 10 min. Cell lysates were analysed by immunoblotting for the levels and phosphorylation states of the indicated proteins. See Extended Data Fig. 1g for the expression levels of the KICSTOR components in the cell lines used here. d, Analysis of indicated SZT2 sgRNA-treated HeLa cell clones for levels of SZT2 and RAPTOR by immunoblotting. Note that not all clones have complete loss of the SZT2 protein. e, mTORC1 signalling in SZT2-deficient cells is insensitive to amino acid deprivation. Indicated HeLa cell SZT2-deficient clones from d were treated and analysed as in a. f, CRISPR–Cas9-mediated depletion of ITFG2 renders mTORC1 signalling partially insensitive to amino acid deprivation. HeLa cells that stably expressed the indicated ITFG2 sgRNAs were treated and analysed as in a. g, Glucose levels do not affect GATOR1 localization, as indicated by GFP–NPRL2. HeLa cells that expressed GFP–NPRL2 from Fig. 1h were starved of glucose for 50 min, or starved and restimulated with glucose for 10 min before processing for immunofluorescence for GFP and LAMP2. Scale bars represent 10 μm. Quantification is shown in the bar graph. h, In SZT2-deficient cells mTORC1 signalling is still sensitive to serum starvation and insulin stimulation. Indicated HeLa cell SZT2-deficient clones from d were serum starved for 50 min, or serum starved and restimulated with insulin for 10 min. Cell lysates were analysed by immunoblotting for the levels and phosphorylation states of the indicated proteins. i, In cells with CRISPR–Cas9-mediated depletion of ITFG2, mTORC1 signalling is still sensitive to serum starvation and insulin stimulation. HeLa cells that stably expressed the indicated ITFG2 sgRNAs were treated and analysed as in h. j, Quantification of pS6K1 blots in h and i. Data are mean ± s.d.
Extended Data Figure 6 Activation of mTORC1 signalling in tissues from Szt2GT/GT mice.
a, SZT2 inhibits mTORC1 signalling in mouse liver and muscle. Mice with the indicated genotypes were treated and analysed by immunoblotting for the levels and phosphorylation state of S6 as in Fig. 2d. The animals examined here represent an additional two animals of each genotype beyond those analysed in Fig. 2d, e. b, Quantification of the ratio of pS6 to S6 bands in a and in Fig. 2d, e. Data are mean ± s.d. of n = 4. c, SZT2 inhibits mTORC1 signalling in hepatocytes and cardiomyocytes in vivo. Liver and heart sections prepared from mice treated as in Fig. 2d were analysed by immunohistochemistry for S6 pS235/S236 levels and serial sections were stained with haematoxylin and eosin (H&E). Liver image is centred over a central vein. Scale bar, 40 μm.
Extended Data Figure 7 GATOR1, like KICSTOR, functions downstream of or parallel to GATOR2 in the mTORC1 pathway.
a, GATOR1, like KICSTOR, is epistatic to GATOR2. Wild-type, WDR24- or NPRL3- and WDR24-deficient HEK293T cells were starved of amino acids for 50 min, or starved and restimulated with amino acids for 10 min. Cell lysates were analysed by immunoblotting for the indicated proteins and phosphorylation states. b, Quantification of the imaging data in Fig. 3d. Data are mean ± s.e.m.
Extended Data Figure 8 KICSTOR regulates the lysosomal localization of GATOR1 but not of GATOR2 or the Rag GTPases.
a, Quantification of the imaging data in Fig. 4a, b. Data are mean ± s.e.m. b, Loss of KICSTOR components does not affect the lysosomal localization of GATOR2. GFP–WDR24 expressing HeLa cells prepared as in Extended Data Fig. 4c were subsequently modified with the CRISPR–Cas9 system to create KPTN-deficient cells. These cells as well as wild-type and sgAAVS1-treated control cells were starved, or starved and restimulated with amino acids for the indicated times before processing for the detection of GFP and LAMP2 by immunofluorescence. c, The Rag GTPases localize to lysosomes regardless of SZT2 expression. Wild-type and SZT2-deficient HEK293T cells were treated as in b before processing for the detection of RAGC and LAMP2 by immunofluorescence. Quantification is shown in the bar graph (mean ± s.e.m.). Scale bars, 10 μm. Insets depict selected fields and overlays that were magnified 3.24×.
Extended Data Figure 9 Loss of KICSTOR disrupts the GATOR1–GATOR2 interaction.
a, Loss of SZT2 disrupts the interaction between GATOR1 and GATOR2. Anti-Flag immunoprecipitates were prepared from wild-type or SZT2-deficient HeLa cells that stably expressed the indicated cDNAs and starved of amino acids for 50 min or starved and restimulated with amino acids for 10 min. Immunoprecipitates and cell lysates were analysed by immunoblotting for the indicated proteins. b, KPTN is necessary for the interaction of GATOR2 with GATOR1. HEK293T cells that stably expressed the indicated sgRNAs were transfected with the indicated cDNAs and subsequently treated and analysed as in a. c, ITFG2 is also necessary for the GATOR1–GATOR2 interaction. Cells were prepared, treated and analysed as in b. d, C12orf66 is also necessary for the GATOR1–GATOR2 interaction. Cells were prepared, treated and analysed as in b.
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Wolfson, R., Chantranupong, L., Wyant, G. et al. KICSTOR recruits GATOR1 to the lysosome and is necessary for nutrients to regulate mTORC1. Nature 543, 438–442 (2017). https://doi.org/10.1038/nature21423
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DOI: https://doi.org/10.1038/nature21423
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