A substrate-specific mTORC1 pathway underlies Birt–Hogg–Dubé syndrome

Abstract

The mechanistic target of rapamycin complex 1 (mTORC1) is a key metabolic hub that controls the cellular response to environmental cues by exerting its kinase activity on multiple substrates1,2,3. However, whether mTORC1 responds to diverse stimuli by differentially phosphorylating specific substrates is poorly understood. Here we show that transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy4,5, is phosphorylated by mTORC1 via a substrate-specific mechanism that is mediated by Rag GTPases. Owing to this mechanism, the phosphorylation of TFEB—unlike other substrates of mTORC1, such as S6K and 4E-BP1— is strictly dependent on the amino-acid-mediated activation of RagC and RagD GTPases, but is insensitive to RHEB activity induced by growth factors. This mechanism has a crucial role in Birt–Hogg–Dubé syndrome, a disorder that is caused by mutations in the RagC and RagD activator folliculin (FLCN) and is characterized by benign skin tumours, lung and kidney cysts and renal cell carcinoma6,7. We found that constitutive activation of TFEB is the main driver of the kidney abnormalities and mTORC1 hyperactivity in a mouse model of Birt–Hogg–Dubé syndrome. Accordingly, depletion of TFEB in kidneys of these mice fully rescued the disease phenotype and associated lethality, and normalized mTORC1 activity. Our findings identify a mechanism that enables differential phosphorylation of mTORC1 substrates, the dysregulation of which leads to kidney cysts and cancer.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: TFEB phosphorylation is insensitive to the RHEB–TSC axis.
Fig. 2: Unconventional recruitment of an mTORC1 substrate by Rag GTPases.
Fig. 3: Activation of RagC has a differential effect on mTORC1 substrates.
Fig. 4: TFEB depletion rescues renal pathology and lethality in FLCN-knockout mice.

Data availability

Full scans for all western blots as well as raw data for all the graphs are provided with this manuscript. No datasets were generated or analysed during the current study. All other data are available from the corresponding author on reasonable request. Source data are provided with this paper.

References

  1. 1.

    Liu, G. Y. & Sabatini, D. M. mTOR at the nexus of nutrition, growth, ageing and disease. Nat. Rev. Mol. Cell Biol. 21, 183–203 (2020).

    CAS  PubMed  Google Scholar 

  2. 2.

    Ben-Sahra, I. & Manning, B. D. mTORC1 signaling and the metabolic control of cell growth. Curr. Opin. Cell Biol. 45, 72–82 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    González, A. & Hall, M. N. Nutrient sensing and TOR signaling in yeast and mammals. EMBO J. 36, 397–408 (2017).

    PubMed  PubMed Central  Google Scholar 

  4. 4.

    Sardiello, M. et al. A gene network regulating lysosomal biogenesis and function. Science 325, 473–477 (2009).

    ADS  CAS  PubMed  Google Scholar 

  5. 5.

    Settembre, C. et al. TFEB links autophagy to lysosomal biogenesis. Science 332, 1429–1433 (2011).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Schmidt, L. S. & Linehan, W. M. FLCN: the causative gene for Birt–Hogg–Dubé syndrome. Gene 640, 28–42 (2018).

    CAS  PubMed  Google Scholar 

  7. 7.

    Schmidt, L. S. & Linehan, W. M. Molecular genetics and clinical features of Birt–Hogg–Dubé syndrome. Nat. Rev. Urol. 12, 558–569 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Inoki, K., Li, Y., Xu, T. & Guan, K. L. Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling. Genes Dev. 17, 1829–1834 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Long, X., Lin, Y., Ortiz-Vega, S., Yonezawa, K. & Avruch, J. Rheb binds and regulates the mTOR kinase. Curr. Biol. 15, 702–713 (2005).

    CAS  PubMed  Google Scholar 

  10. 10.

    Tee, A. R., Manning, B. D., Roux, P. P., Cantley, L. C. & Blenis, J. Tuberous sclerosis complex gene products, tuberin and hamartin, control mTOR signaling by acting as a GTPase-activating protein complex toward Rheb. Curr. Biol. 13, 1259–1268 (2003).

    CAS  PubMed  Google Scholar 

  11. 11.

    Yang, H. et al. Mechanisms of mTORC1 activation by RHEB and inhibition by PRAS40. Nature 552, 368–373 (2017).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Sancak, Y. et al. The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science 320, 1496–1501 (2008).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Kim, E., Goraksha-Hicks, P., Li, L., Neufeld, T. P. & Guan, K. L. Regulation of TORC1 by Rag GTPases in nutrient response. Nat. Cell Biol. 10, 935–945 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    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).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    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).

    CAS  PubMed  Google Scholar 

  17. 17.

    Lawrence, R. E. et al. Structural mechanism of a Rag GTPase activation checkpoint by the lysosomal folliculin complex. Science 366, 971–977 (2019).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Shen, K. et al. Cryo-EM structure of the human FLCN–FNIP2–Rag–Ragulator complex. Cell 179, 1319–1329.e8 (2019).

    CAS  PubMed  Google Scholar 

  19. 19.

    Settembre, C. et al. A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB. EMBO J. 31, 1095–1108 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Martina, J. A., Chen, Y., Gucek, M. & Puertollano, R. MTORC1 functions as a transcriptional regulator of autophagy by preventing nuclear transport of TFEB. Autophagy 8, 903–914 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Roczniak-Ferguson, A. et al. The transcription factor TFEB links mTORC1 signaling to transcriptional control of lysosome homeostasis. Sci. Signal. 5, ra42 (2012).

    PubMed  PubMed Central  Google Scholar 

  22. 22.

    Napolitano, G. et al. mTOR-dependent phosphorylation controls TFEB nuclear export. Nat. Commun. 9, 3312 (2018).

    ADS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Medina, D. L. et al. Lysosomal calcium signalling regulates autophagy through calcineurin and TFEB. Nat. Cell Biol. 17, 288–299 (2015).

    PubMed  PubMed Central  Google Scholar 

  24. 24.

    Ballabio, A. & Bonifacino, J. S. Lysosomes as dynamic regulators of cell and organismal homeostasis. Nat. Rev. Mol. Cell Biol. 21, 101–118 (2019).

    PubMed  Google Scholar 

  25. 25.

    Di Malta, C. et al. Transcriptional activation of RagD GTPase controls mTORC1 and promotes cancer growth. Science 356, 1188–1192 (2017).

    PubMed  PubMed Central  Google Scholar 

  26. 26.

    Martina, J. A. & Puertollano, R. Rag GTPases mediate amino acid-dependent recruitment of TFEB and MITF to lysosomes. J. Cell Biol. 200, 475–491 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Schalm, S. S. & Blenis, J. Identification of a conserved motif required for mTOR signaling. Curr. Biol. 12, 632–639 (2002).

    CAS  PubMed  Google Scholar 

  28. 28.

    Schalm, S. S., Fingar, D. C., Sabatini, D. M. & Blenis, J. TOS motif-mediated raptor binding regulates 4E-BP1 multisite phosphorylation and function. Curr. Biol. 13, 797–806 (2003).

    CAS  PubMed  Google Scholar 

  29. 29.

    Lawrence, R. E. et al. A nutrient-induced affinity switch controls mTORC1 activation by its Rag GTPase–Ragulator lysosomal scaffold. Nat. Cell Biol. 20, 1052–1063 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Baba, M. et al. Kidney-targeted Birt–Hogg–Dubé gene inactivation in a mouse model: Erk1/2 and Akt–mTOR activation, cell hyperproliferation, and polycystic kidneys. J. Natl. Cancer Inst. 100, 140–154 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Chen, J. et al. Deficiency of FLCN in mouse kidney led to development of polycystic kidneys and renal neoplasia. PLoS ONE 3, e3581 (2008).

    ADS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Palmieri, M. et al. Characterization of the CLEAR network reveals an integrated control of cellular clearance pathways. Hum. Mol. Genet. 20, 3852–3866 (2011).

    CAS  PubMed  Google Scholar 

  33. 33.

    Wada, S. et al. The tumor suppressor FLCN mediates an alternate mTOR pathway to regulate browning of adipose tissue. Genes Dev. 30, 2551–2564 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Anandapadamanaban, M. et al. Architecture of human Rag GTPase heterodimers and their complex with mTORC1. Science 366, 203–210 (2019).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Rogala, K. B. et al. Structural basis for the docking of mTORC1 on the lysosomal surface. Science 366, 468–475 (2019).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Kauffman, E. C. et al. Molecular genetics and cellular features of TFE3 and TFEB fusion kidney cancers. Nat. Rev. Urol. 11, 465–475 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Calcagnì, A. et al. Modelling TFE renal cell carcinoma in mice reveals a critical role of WNT signaling. eLife 5, e17047 (2016).

    PubMed  PubMed Central  Google Scholar 

  38. 38.

    Perera, R. M., Di Malta, C. & Ballabio, A. MiT/TFE family of transcription factors, lysosomes, and cancer. Annu Rev Cancer Biol 3, 203–222 (2019).

    PubMed  Google Scholar 

  39. 39.

    Baba, M. et al. Folliculin encoded by the BHD gene interacts with a binding protein, FNIP1, and AMPK, and is involved in AMPK and mTOR signaling. Proc. Natl Acad. Sci. USA 103, 15552–15557 (2006).

    ADS  CAS  PubMed  Google Scholar 

  40. 40.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Mansueto, G. et al. Transcription factor EB controls metabolic flexibility during exercise. Cell Metab. 25, 182–196 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Endoh, M. et al. A FLCN–TFE3 feedback loop prevents excessive glycogenesis and phagocyte activation by regulating lysosome activity. Cell Rep. 30, 1823–1834 (2020).

    CAS  PubMed  Google Scholar 

  43. 43.

    Possik, E. et al. FLCN and AMPK confer resistance to hyperosmotic stress via remodeling of glycogen stores. PLoS Genet. 11, e1005520 (2015).

    PubMed  PubMed Central  Google Scholar 

  44. 44.

    Perera, R. M. et al. Transcriptional control of autophagy–lysosome function drives pancreatic cancer metabolism. Nature 524, 361–365 (2015).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Kawakami, K. Tol2: a versatile gene transfer vector in vertebrates. Genome Biol. 8, S7 (2007).

    PubMed  PubMed Central  Google Scholar 

  46. 46.

    de Araujo, M. E. G. et al. Crystal structure of the human lysosomal mTORC1 scaffold complex and its impact on signaling. Science 358, 377–381 (2017).

    ADS  PubMed  Google Scholar 

  47. 47.

    Napolitano, G. et al. Impairment of chaperone-mediated autophagy leads to selective lysosomal degradation defects in the lysosomal storage disease cystinosis. EMBO Mol. Med. 7, 158–174 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Shao, X., Johnson, J. E., Richardson, J. A., Hiesberger, T. & Igarashi, P. A minimal Ksp-cadherin promoter linked to a green fluorescent protein reporter gene exhibits tissue-specific expression in the developing kidney and genitourinary tract. J. Am. Soc. Nephrol. 13, 1824–1836 (2002).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank M. A. De Matteis, G. Diez-Roux, L. Murphy and C. Settembre for the critical reading of the manuscript; A. Iuliano for statistical analyisis; C. Soldati for software analysis of TFEB subcellular distribution; L. D’Orsi and N. Zampelli for technical help; and M. Mea for help in drawing the model figure. This work was supported by grants from the Italian Telethon Foundation ‘TGM16CB6’ (A.B.); MIUR ‘PRIN 2017E5L5P3’ (A.B) and ‘PRIN 2017YF9FBS’ (G.N.); European Research Council H2020 AdG ‘LYSOSOMICS 694282’ (A.B.); European Union’s Horizon 2020 MSCA ‘REBuILD 661271’ (G.N.); US National Institutes of Health ‘R01-NS078072’ (A.B.); Huffington Foundation (A.B.); European Regional Development Fund - POR Campania FESR 2014/2020 (A.B); Associazione Italiana per la Ricerca sul Cancro A.I.R.C. ‘IG-22103’ and ‘5x1000-21051’ (A.B.), ‘MFAG-23538’ (G.N.) and ‘IG-18988’ (P.P.D.F.); University of Naples ‘Federico II’ ‘STAR L1 2018’ (G.N.); MCO 10000 (P.P.D.F.); and Italian Ministry of Health ‘RF-2016-02361540’ (P.P.D.F.).

Author information

Affiliations

Authors

Contributions

G.N., C.D.M. and A.B. conceived the study. G.N., C.D.M. and A.E. designed the experiments. G.N. and M.M. performed the majority of co-immunoprecipitation and the in vitro experiments involving RagC. A.E. performed experiments involving FBS, RHEB and TSC2, TFEB chimaera and Lys- and Mit-RAPTOR. C.D.M. and A.Z. performed the experiments on FLCN-knockout HeLa cells, and generated and characterized the mouse lines described in the study. D.S. was involved in the characterization of mouse lines and analysis of RagA-knockout cells. C.V. was involved in some experiments on FLCN-knockout cells. E.N. helped with mouse handling. V.B. and A.V. performed some of the experiments involving Lys–RAPTOR. M.C. was involved in virus and cell line preparation. J.M. generated CRISPR–Cas9 gene-edited cell lines and performed some microscopy experiments. A.C. was involved in some of the experiments related to RHEB depletion. M.E.G.d.A., T.S. and L.A.H. performed and analysed biochemistry experiments. S.P., G.B. and P.P.D.F. performed and analysed histology experiments. G.N., C.D.M. and A.B. wrote the manuscript. A.B. supervised the study.

Corresponding author

Correspondence to Andrea Ballabio.

Ethics declarations

Competing interests

A.B. is cofounder of CASMA Therapeutics, Inc.

Additional information

Peer review information Nature thanks Kun-Liang Guan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 TFEB phosphorylation is insensitive to serum starvation.

a, HeLa cells stably expressing GFP–TFEB were starved of serum (FBS) for 2 h in the presence or absence of 250 nM torin and analysed by immunoblotting with the indicated antibodies (replicated twice). bf, HeLa (b, c), U2OS (d, e) or primary MEFs (f) were starved of amino acids or serum for 2 h and then analysed by immunoblotting for the indicated proteins (b, d, f) or by immunofluorescence to assess TFEB subcellular localization (c, e) (replicated twice). Scale bars, 10 μm. g, HeLa cells stably expressing GFP–TFEB were either starved of amino acids (−aa) or serum (−FBS) for 8 h and subjected to qRT–PCR (replicated twice). Relative mRNA levels of the indicated genes were normalized to levels of HPRT1 and expressed as fold change relative to control (fed) samples. Results are mean ± s.e.m.; n = 3; *P < 0.05 (GBA, −aa P = 0.0322); ***P < 0.001 (FLCN, −aa P < 0.0001; RRAGC, −aa P < 0.0001; RRAGD, −aa P < 0.0001; ATP6V1H, −aa P = 0.0004; CTNS, −aa P < 0.0001; SQSTM1, −aa P < 0.0001; NPC1, −aa P < 0.0001; MCOLN1, −aa P < 0.0001); NS, non-significant (FLCN, −FBS P = 0.8619; RRAGC, −FBS P = 0.99; RRAGD, −FBS P = 0.2558; ATP6V1H, −FBS P = 0.9890; CTNS, −FBS P > 0.9999; SQSTM1, −FBS P = 0.9270; GBA, −FBS P = 0.9359; NPC1, −FBS P = 0.1933; MCOLN1, −FBS P = 0.7916). Dunnett’s multiple comparisons test. Source data

Extended Data Fig. 2 TFEB phosphorylation is insensitive to the RHEB–TSC axis.

ac, HeLa (a), HEK293T (b) or ARPE19 (c) cells were transfected with either siRNA targeting both RHEB and RHEBL1 or with scramble siRNA. Seventy-two hours after transfection cells were either starved of amino acids for 60 min or starved and restimulated with amino acids for 30 min, in the presence or absence of 250 nM torin, and analysed by immunoblotting with the indicated antibodies (replicated twice). d, HeLa cells stably expressing GFP–TFEB were transfected with siRNA targeting either RHEB and RHEBL1 (siRHEB/L1), MTOR (simTOR), or control siRNA (siCtrl) for 72 h, and subjected to qRT–PCR (replicated three times). Relative mRNA levels of the indicated genes were normalized to levels of HPRT1 and expressed as fold change relative to control (siCtrl) samples. Results are mean ± s.e.m.; n = 3; *P < 0.05 (MCOLN1, siRHEB P = 0.0212); ***P < 0.001 (FLCN, siMTOR P < 0.0001; RRAGC, siMTOR P < 0.0001; ATP6V1H, siMTOR P < 0.0001; NEU1, siRHEB P < 0.0001; NEU1, siMTOR P < 0.0001; GBA, siMTOR P < 0.0001; NPC1, siMTOR P < 0.0001; MCOLN1, siMTOR P < 0.0001); ns, non-significant (FLCN, siRHEB P = 0.9908; RRAGC, siRHEB P = 0.6937; ATP6V1H, siRHEB P = 0.0714; SQSTM1, siRHEB P = 0.2846; SQSTM1, siMTOR P = 0.0528; GBA, siRHEB P = 0.0597; NPC1, siRHEB P = 0.7753). Dunnett’s multiple comparisons test. e, HEK293A cells stably expressing GFP–TFEB, transfected with either empty vector or with increasing amounts of Flag–RHEB, were either left untreated or starved of amino acids for 60 min and analysed by immunoblotting (replicated three times). Source data

Extended Data Fig. 3 Rag GTPases are required for TFEB phosphorylation.

a, HeLa cells stably expressing GFP–TFEB were transfected with siRNAs targeting both RRAGC and RRAGD or with a control siRNA (siCtrl). Seventy-two hours after transfection, cells were either starved of amino acids for 60 min or starved and restimulated with amino acids for 30 min in the presence or absence of torin and analysed by immunoblotting using the indicated antibodies (replicated three times). b, Immunofluorescence analysis of TFEB localization in HeLa cells stably expressing GFP–TFEB and transfected with RRAGC- and RRAGD-targeting siRNA or with control siRNA (siCtrl) and after 48 h with empty vector (left), HA–RagC or HA–RagD (right). Cells were either starved for amino acids for 60 min (−aa) or starved and then restimulated with amino acids for 30 min (+aa) (replicated three times). Scale bar, 10 μm.

Extended Data Fig. 4 Rag GTPases are required for TFEB phosphorylation regardless of mTORC1 activation status.

a, b, GFP immunoprecipitates were prepared from HEK293A cells stably expressing GFP–TFEB (a) or GFP–S6K (b) and analysed by immunoblotting for the indicated proteins (replicated three times). c, d, HEK293T cells (c) or HeLa cells stably expressing GFP–TFEB (d) were transduced with lentiviruses expressing Lys–RAPTOR or with control lentiviruses and transfected with siRNA targeting both RRAGC and RRAGD (siRagC/D) or with scramble siRNA (siCtrl). Seventy-two hours after transfection, cells were either starved of amino acids for 60 min or starved and restimulated with amino acids for 30 min and analysed by immunoblotting using the indicated antibodies (replicated three times). e, f, HEK293T cells (e) or HeLa cells stably expressing GFP–TFEB (f) transiently expressing the indicated combinations of mitochondria-targeted RAPTOR (Mit-Raptor: Flag–RAPTOR–OMP25) and RHEB (Mit-Rheb: Myc–RHEB–OMP25) were starved of amino acids for 60 min or starved and re-stimulated with amino acids for 30 min and analysed by immunoblotting using the indicated antibodies (replicated three times). g, HeLa cells stably expressing GFP–TFEB were transfected and treated as in f and analysed by immunofluorescence for the indicated proteins. GFP–TFEB was pseudocoloured to magenta to allow better visualization of mTOR and RAPTOR–OMP25 staining (replicated three times). Scale bar, 10 μm.

Extended Data Fig. 5 The mTORC1 substrate-recruitment mechanism of TFEB is determined by its N-terminal region.

HeLa cells were transiently transfected with plasmids expressing either GFP alone or GFP-tagged versions of the following proteins: TFEB (GFP–WT–TFEB), a TFEB deletion mutant that lacks the first 30 amino acids (GFP–Δ30TFEB), a chimeric protein in which the first 30 amino acids of S6K (containing the TOS motif) were fused to the D30TFEB mutant (GFP-TOS-Δ30TFEB), or the TOS-D30TFEB chimeric protein in which a key phenylalanine residue (F5) of the TOS motif was mutagenized to alanine (GFP-F5A-Δ30TFEB). Twenty-four hours after transfection cell lysates were incubated with GFP beads and subjected to immunoblotting using the indicated antibodies (replicated three times).

Extended Data Fig. 6 Addition of a TOS motif to a Rag-binding-deficient TFEB mutant rescues its phosphorylation and subcellular localization.

a, HeLa cells transiently expressing the cDNAs described in Extended Data Fig. 5 were starved of amino acids for 60 min and restimulated with amino acids for 30 min, in the presence or absence of 250 nM torin, and analysed by immunoblotting using the indicated antibodies (replicated three times). b, Cells described in a were either starved of amino acids for 60 min or starved and restimulated with amino acids for 30 min, in the presence or absence of torin, and analysed for TFEB subcellular localization by immunofluorescence (replicated twice). c, Representative immunoblotting and quantification (mean ± s.e.m.; n = 3) of HeLa cells stably expressing GFP–TFEB or GFP–TOS-D30TFEB. Cells were either kept fed, starved of amino acids (−aa) or serum (−FBS) for 2 h. d, Cells described and treated as in c were analysed by immunofluorescence to assess TFEB subcellular localization (replicated three times). Scale bar, 10 μm. e, Analysis of TFEB localization performed using a dedicated script (Columbus software; Perkin-Elmer) that calculates the ratio value resulting from the average intensity of nuclear TFEB–GFP fluorescence divided by the average of the cytosolic intensity of TFEB–GFP fluorescence. Results are mean ± s.e.m. P values were calculated on the basis of mean values from 3 or 4 independent fields (Sidak’s multiple comparisons test). ***P < 0.0001; NS, non-significant (fed, P = 0.9989; −aa, P = 0.0946). Source data

Extended Data Fig. 7 Activation of RagA is essential for mTOR lysosomal recruitment and TFEB cytosolic localization.

a, b, Representative immunofluorescence images of endogenous mTOR (a) and endogenous TFEB (b) upon transfection of a construct encoding active (RagA(Q66L)) or inactive (RagA(T21L)) HA-tagged RagA or an empty vector in RagA-knockout HeLa cells. Cells were deprived of amino acids for 50 min and then stimulated with amino acids for 15 min. Scale bars, 10 μm.

Extended Data Fig. 8 TFEB phosphorylation and cytosolic retention requires active RagC/D.

a, RagC-knockout HeLa cells were transfected with RRAGD-targeting siRNA (siRagD) for 72 h, then either starved of amino acids for 60 min or starved and restimulated with amino acids for 30 min and analysed by immunoblotting using the indicated antibodies (replicated three times). b, FLCN-knockout and control HeLa cells overexpressing the TFEB–GFP construct were either starved of amino acids for 60 min, or starved and restimulated with amino acids for 30 min in the presence or absence of 250 nM torin, and then analysed by immunoblotting with the indicated antibodies (replicated three times). c, Immunofluorescence analysis representative of triplicate experiments of TFEB in FLCN-knockout and control HeLa cells kept in amino acid deprived medium (−aa) or restimulated in amino-acid-containing medium (+aa). Scale bars, 10 μm. d, FLCN-knockout HeLa cells transfected with empty vector or with constitutively active RagD (RagD(S75L)) were either starved of amino acids for 60 min, or starved and restimulated with amino acids for 30 min, and analysed by immunoblotting with the indicated antibodies (replicated three times). e, f, FLCN-knockout Hela cells transfected with empty vector or constitutively active RagC (RagC(S75L)) (d) or constitutively active RagD (RagD(S77L)) (e) and kept in basal medium were immunostained with the indicated antibodies (replicated three times). Scale bars, 10 μm.

Extended Data Fig. 9 Genomic and mRNA analysis of transgenic mouse lines.

a, PCR analysis of genotypes from kidney samples of Flcnflox/flox;Ksp-cre+ (Flcn-KO) mice and Flcnflox/flox;Tfebflox/flox;Ksp-cre+ (Flcn-Tfeb-DKO) mice and corresponding controls (replicated three times). In detail, genotypes of Ctrl-Flcn mice were the following: Flcnflox/+- Flcnflox/flox - Flcnflox/+;Ksp-cre+. Genotypes of Ctrl-Flcn-Tfeb mice were the following: Flcnflox/+;Tfebflox/+-Flcnflox/flox;Tfebflox/flox-Flcnflox/+;Tfebflox/flox. b, mRNA levels of the indicated genes in Tfebflox/flox;Ksp-cre+ (Tfeb-KO), Flcnflox/flox;Ksp-cre+ (Flcn-KO), Flcnflox/flox;Tfebflox/flox;Ksp-cre+(Flcn-Tfeb-DKO) and corresponding control mice at p2. Bars represent mean ± s.e.m. for each group and are expressed as fold change compared with control mice, normalized to cyclophilin gene expression. **P < 0.01, ***P < 0.001, two-sided Student t-test. S16 expression is shown as a control, unrelated gene (n = 4 Ctrl-Tfeb; n = 3 Tfeb-KO; n = 3 Ctrl-Flcn; n = 4 Flcn-KO; n = 3 Ctrl-Flcn-Tfeb; n = 4 Flcn-Tfeb-DKO). Source data

Extended Data Fig. 10 TFEB is constitutively nuclear and active in FLCN-knockout kidneys, and its depletion rescues mTORC1 hyperactivation.

a, Representative images from three independent histopathological analyses of FLCN-knockout kidney tissues showing magnifications of areas with tubular papillary atypical hyperplasia (arrowheads, top), hyperplasia with considerable alterations of the tubular morphology (marked by asterisks in the bottom left panel) and atypical hyperplasia with multiple mitoses (represented in boxed areas and magnified in indents in bottom right panel). Scale bar, 100 μm. b, Representative immunofluorescence analysis of triplicate experiments of TFEB in kidney sections from mice of the indicated genotypes. Insets show higher magnification of the boxed area. Scale bars, 100 μm. c, Immunoblotting analysis of the indicated proteins in cytosolic and nuclear fractions of kidneys from Flcnflox/flox (Ctrl) and Flcnflox/flox;Ksp-cre+ (Flcn-KO) mice (replicated three times). d, Immunoblotting analysis of the indicated proteins in kidneys from Flcnflox/flox;Ksp-cre+ (Flcn-KO) mice and Flcnflox/flox;Tfebflox/flox;Ksp-cre+ (Flcn-Tfeb-DKO) mice and corresponding controls (replicated three times). e, mRNA levels of several TFEB target genes were analysed in kidney samples from FLCN-knockout mice relative to control mice. Bars represent mean ± s.e.m. for n = 5 mice for each group and are expressed as fold change compared with control mice, normalized to cyclophilin gene expression. *P < 0.05, **P < 0.01, ***P < 0.001, two-sided Student t-test. S16 expression is shown as a control, unrelated gene. f, Immunohistochemical analysis of LAMP1 in kidney sections from FLCN-knockout mice and control mice (replicated three times). Insets show higher magnification of the boxed area. Scale bars, 50 μm. In af, analysis was performed on 21-day-old mice. Source data

Supplementary information

Supplementary Figure 1

This file contains the western blot source data.

Reporting Summary

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Napolitano, G., Di Malta, C., Esposito, A. et al. A substrate-specific mTORC1 pathway underlies Birt–Hogg–Dubé syndrome. Nature (2020). https://doi.org/10.1038/s41586-020-2444-0

Download citation

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.