科技科学

A TBK1/ULK1 signalling axis couples lysosomal stress to TFEB activation

Nature作者：Alessandra Esposito2026年9月30日正文已收录本站

Data availability

Proteomic data have been deposited at the ProteomeXchange Consortium through the PRIDE partner repository with dataset identifiers PXD069600 (Fig. 2) and PXD063015 (Fig. 4). For gel source data, see Supplementary Fig. 3. Data are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank C. Settembre, J. Font-Burgada, G. Diez-Reux and A. De Matteis for critical reading of the manuscript; S. Malek for providing cell lines expressing ATP6V1B2 variants; and M. Cillo for help with generation of OCI-LY19 stable cell lines. We also thank the following TIGEM core facilities: high-content screening, MS, flow cytometry and advanced microscopy.

Funding

This work was supported by grants from the Italian Telethon Foundation (TGM22CBDM09 to G.N.), AIRC Foundation (grants MFAG-23538 and SIS−31821 to G.N.), Wereld Kanker Onderzoek Fonds (WKOF) as part of the World Cancer Research Fund International grant programme (IIG_FULL_2022_009 to G.N.), MIUR (PRIN 2022CRFNCP and PRIN P2022T4PKT to G.N.), Scuola Superiore Meridionale (SSM) postdoctoral fellowship (A.E.), University and Canton of Fribourg as part of the SKINTEGRITY.CH research network and Swiss National Science Foundation (nos. 229588 and 212187 to J.D.). The GALLIUM study (NCT01332968) and associated mutation profile analysis were sponsored by F. Hoffmann-La Roche.

Author information

Authors and Affiliations

  1. Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy

    Alessandra Esposito, Chiara Varriale, Maria Pia Cavaliere, Maria Elsa Brunetti, Rossella Agostinis, Alessia Calcagnì, Fabrizio Andreone, Jlenia Monfregola, Francesca Sacco, Andrea Ballabio & Gennaro Napolitano

  2. Scuola Superiore Meridionale (SSM), School for Advanced Studies, Genomics and Experimental Medicine program, Naples, Italy

    Alessandra Esposito, Maria Pia Cavaliere, Maria Elsa Brunetti, Andrea Ballabio & Gennaro Napolitano

  3. Department of Biology, University of Fribourg, Fribourg, Switzerland

    Ibrahim Bayramoglu & Joern Dengjel

  4. Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, USA

    Rachel M. Jansen, Dan A. Tudorica, Yongjia Duan, Martina Spacci, Roberto Zoncu & James H. Hurley

  5. California Institute for Quantitative Biosciences, University of California, Berkeley, CA, USA

    Rachel M. Jansen, Dan A. Tudorica, Yongjia Duan & James H. Hurley

  6. Medical Genetics Unit, Department of Medical and Translational Science, Federico II University, Naples, Italy

    Alessia Calcagnì, Andrea Ballabio & Gennaro Napolitano

  7. Department of Medicine III, Ludwig-Maximilians-University (LMU) Hospital, Munich, Germany

    Carolin D. Strobl, Verena Passerini & Oliver Weigert

  8. Laboratory of Experimental Leukemia and Lymphoma Research (ELLF), German Cancer Consortium (DKTK), partner site Munich (a partnership between DKFZ and LMU University Hospital), Munich, Germany

    Carolin D. Strobl, Verena Passerini & Oliver Weigert

  9. Hematopathology Section, Dpt. of Pathology, University Hospital Schleswig-Holstein (UKSH), Kiel, Germany

    Julia Richter & Wolfram Klapper

  10. Department of Biology, University of Rome Tor Vergata, Rome, Italy

    Francesca Sacco

  11. Comprehensive Cancer Center (CCC Munich LMU), Ludwig-Maximilians-Universität (LMU), Munich, Germany

    Oliver Weigert

  12. Bavarian Cancer Research Center (BZKF), LMU University Hospital, Munich, Germany

    Oliver Weigert

  13. Jan and Dan Duncan Neurological Research Institute, Texas Children’s Hospital, and Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA

    Andrea Ballabio

  14. Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, CA, USA

    James H. Hurley

Authors

  1. Alessandra Esposito
  2. Ibrahim Bayramoglu
  3. Chiara Varriale
  4. Maria Pia Cavaliere
  5. Maria Elsa Brunetti
  6. Rossella Agostinis
  7. Rachel M. Jansen
  8. Dan A. Tudorica
  9. Yongjia Duan
  10. Martina Spacci
  11. Alessia Calcagnì
  12. Carolin D. Strobl
  13. Verena Passerini
  14. Fabrizio Andreone
  15. Jlenia Monfregola
  16. Julia Richter
  17. Francesca Sacco
  18. Wolfram Klapper
  19. Oliver Weigert
  20. Andrea Ballabio
  21. Roberto Zoncu
  22. James H. Hurley
  23. Joern Dengjel
  24. Gennaro Napolitano

Contributions

A.E. and G.N. conceived the study. A.E. designed and performed most experiments. I.B. performed and analysed phosphoproteomic and in vitro kinase assays under the supervision of J.D. C.V., M.P.C., R.A., M.E.B. and A.C. performed some of the in vitro experiments. R.M.J., Y.D., D.A.T. and M.S. performed FLCN:FNIP protein purification, in vitro phosphorylation and GAP activity assays under the supervision of R.Z. and J.H.H. F.A. and J.M. generated CRISPR–Cas9 gene-edited cell lines. F.S. analysed MS data. C.D.S. generated GC B cells. J.R., V.P., W.K. and O.W. provided and analysed patient data. A.E. and G.N. wrote the original draft of the manuscript, and A.E., A.B., O.W., J.H.H., R.Z., J.D. and G.N contributed to review and editing. G.N. supervised the study.

Corresponding author

Correspondence to Gennaro Napolitano.

Ethics declarations

Competing interests

J.H.H. is a cofounder and shareholder of Casma Therapeutics and has received research funding from Genentech and Hoffmann-La Roche. A.B. is a cofounder and shareholder of Casma Therapeutics and advisory board member of Avilar Therapeutics. The other authors declare no competing interests.

Peer review

Peer review information

Nature thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.

Additional information

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 ATP6V1B2 pathogenic variants induce TFEB activation without affecting the RagA-mTORC1 axis.

HEK293T cells stably expressing TFEB-GFP and with inducible expression of either WT, Y371C, or R400Q HA-ATP6V1B2, were treated with doxycycline for 48 h and then starved (1 h) or starved and re-stimulated with amino acids (30′). Cell extracts were subjected to immunoblotting. (B) HEK293T cells with inducible expression of WT, Y371C, or R400Q HA-ATP6V1B2 were treated with doxycycline for 72 h and subjected to qRT-PCR. Relative mRNA levels of the indicated genes were normalized to ACTB levels and expressed as fold-change relative to HA-ATP6V1B2 WT expressing cells. n = 3 biological replicates; ATP6V1H: *P = 0.0135(R400Q); ATP6V1C: *P = 0.0347(R400Q)); FLCN: **P = 0.0048(R400Q); ATP6V1H: **P = 0.0081(Y371C); ATP6V1C1: **P = 0.0051(Y371C); FLCN: ***P = 0.0009(Y371C); CTSF: ***P = 0.0004(Y371C), ***P = 0.0003(R400Q). (C) Immunofluorescence analysis of mTOR lysosomal localization in HEK293T cells with inducible expression of either WT, Y371C, or R400Q HA-ATP6V1B2, starved of amino acids (-aa) for 60 min or starved and re-stimulated with amino acids (+aa) for 30 min. (D) Cells as in (C) were analyzed for mTOR-LAMP1 colocalization by calculating Manders’ colocalization coefficient. n = 5 FOV. (E) Lysates from HEK293T cells with inducible expression of either WT, Y371C, or R400Q HA-ATP6V1B2 were incubated with mTOR antibodies, followed by immunoblotting; (F) WT and NPRL3-KO HEK293T cells with inducible expression of either WT or R400Q HA-ATP6V1B2 were analyzed by immunofluorescence to assess TFEB subcellular localization; (G) Quantification of percentage of cells in (F) showing nuclear TFEB. n = 4 FOV, V1B2-WT, WT cells; n = 5, V1B2-R400Q, WT cells; n = 5, V1B2-WT, NPRL3-KO cells; n = 5, V1B2-R400Q, NPRL3-KO cells. 2way ANOVA, Dunnett’s (B) and Sidak’s (D, G) multiple comparison tests were used. ****P < 0.0001; ns, not significant. Results are mean ± SEM. Scale bars, 10 μm.

Source data

Extended Data Fig. 2 ATP6V1B2 variants promote v-ATPase assembly.

(A) Immunofluorescence analysis of HEK293T cells with inducible expression of either WT, Y371C, or R400Q HA-ATP6V1B2, co-stained with the lysosomal marker LAMP1. (B) Cells in (A) were analyzed for ATP6V1B2-LAMP1 colocalization by calculating Manders’ colocalization coefficient. n = 5 FOV; ****P < 0.0001 (one-way ANOVA Dunnett’s multiple comparison); (C) Lysates from HEK293T cells with inducible expression of either WT, Y371C, or R400Q HA-ATP6V1B2, were incubated with HA beads and analyzed by immunoblotting with the indicated antibodies; (D) Western blot analysis of cytosolic and membrane fractions from HEK293T cells with inducible expression of either WT, Y371C, or R400Q HA-ATP6V1B2; (E) Immunofluorescence analysis of HEK293T cells with inducible expression of WT HA-ATP6V1B2, either left untreated (-) or treated with 10 µM SaliPhe for 3 h. (F) Cells in (E) were analyzed for HA-ATP6V1B2-LAMP1 colocalization by calculating Manders’ colocalization coefficient. n = 5 FOV, ***P = 0.0001 (unpaired t-test). (G) U2OS cells were treated with 10 µM SaliPhe for 8 h and subjected to qRT-PCR. Relative mRNA levels of the indicated genes were normalized to HPRT levels and expressed as fold-change relative to untreated samples. n = 3 biological replicates; *P < 0.05 (FLCN: P = 0.0112); **P < 0.01 (CTSD: P = 0.0039; ATP6V0C: P = 0.0066); ****P < 0.0001; (2way ANOVA, Sidak’s multiple comparison). All data are mean ± SEM. Scale bars, 10 μm.

Source data

Extended Data Fig. 3 Lysosomal ion imbalance selectively activates TFEB.

(A) WT and NPRL3 KO HEK293T cells stably expressing TFEB-GFP, were either left untreated (−), subjected to amino acid starvation (−aa) for 1 h, or treated with 200 nM monensin (Mon), 200 nM nigericin (Nig) or 200 nM salinomycin (Sal) for 3 h. Cell extracts were analyzed by immunoblotting; (B) Confocal analysis of mTOR lysosomal localization in U2OS cells treated with 300 nM monensin (Mon), 500 nM nigericin (Nig) or 1 μM salinomycin (Sal) for 3 h. (C) Cells described in (B) were analyzed for mTOR-LAMP1 colocalization by calculating Manders’ colocalization coefficient. n = 4 FOV. (D) Immunofluorescence analysis of U2OS cells stably expressing TFEB-GFP were either left untreated or subjected to 300 nM monensin (Mon), 500 nM nigericin (Nig) or 1 μM salinomycin (Sal) treatment for 3 h, followed by 250 nM Torin1 treatment for 1 h.; (E) Cells in (D) were quantified to calculate TFEB-LAMP1 colocalization (Manders’ colocalization coefficient). n = 5 FOV. One-way ANOVA, Dunnett’s multiple comparison test was used (C, E). ****P < 0.0001; ns, not significant. Results are mean ± SEM. Scale bars, 10 µm.

Source data

Extended Data Fig. 4 Follicular lymphoma-associated ATP6V1B2 variants activate TFEB via CASM induction.

(A) HEK293T cells stably expressing TFEB-GFP and with inducible expression of either WT, Y371C, or R400Q HA-ATP6V1B2, were treated with doxycycline for 48 h and then starved (1 h) or starved and re-stimulated with amino acids (30’). Cell extracts were subjected to immunoblotting. n = 3 biological replicates. ****P < 0.0001, ns, not significant (LC3I/GAPDH, LC3II/GAPDH: 2way ANOVA, Dunnett’s multiple comparison; LC3II/LC3I:2way ANOVA, Sidak’s multiple comparison). (B) HEK293T cells with inducible expression of WT or R400Q HA-ATP6V1B2 were transfected with siRNA targeting either ATG9 (siATG9), ATG16L1 (siATG16L1), or control siRNA (siCtrl). After 24 h, cells were treated with doxycycline for 48 h and analyzed by confocal microscopy. (C) Cells in (B) were quantified to calculate the number of LC3 spots per cell. n = 3 FOV, ***P = 0.0001, ns, not significant (2way ANOVA, Dunnett’s multiple comparison); (D) Cells treated as in (B) were analyzed by immunofluorescence to assess TFEB subcellular localization. White arrows indicate HA-positive cells. (E) Cells in (D) were quantified to calculate the percentage of cells with nuclear TFEB,****P < 0.0001, ns, not significant (2way ANOVA, Dunnett’s multiple comparison). (F) Cells as in (B) were analyzed by immunoblotting with the indicated antibodies. All results are mean ± SEM. Scale bars, 10 μm.

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Extended Data Fig. 5 TBK1 functions redundantly with ULK1 to control TFEB subcellular localization.

(A) HEK293T cells expressing TMEM192-3xHA and with inducible expression of WT or R400Q FLAG-ATP6V1B2 were subjected to Lyso-IP and analyzed by immunoblotting; (B) U2OS cells were treated with increasing concentrations of SaliPhe (5, 10, and 20 µM) for 3 h and analyzed by immunoblotting; (C) Immunoblotting analysis of U2OS cells treated with 20 µM MG132 for either 4 h or 5 h; (D) Immunofluorescence analysis showing TFEB subcellular localization in WT and TBK1 KO U2OS cells treated with 300 nM or 600 nM monensin for 3 h. Scale bar, 10 μm; (E) Cells in (D) were quantified to calculate the percentage of cells with nuclear TFEB. Bars are mean ± SEM. n = 3 fields of view (FOV). (F-G) Immunofluorescence analysis (F) and quantification (G) of TFEB nuclear localization in TBK1 KO U2OS cells reconstituted with GFP-TBK1 and either left untreated or treated with monensin for 3 h; Scale bar, 10 μm. Results are mean ± SEM. n = 5 FOV. ****P < 0.0001 (unpaired t-test); (H) WT and TBK1 KO U2OS cells were transfected for 72 h with siRNA targeting either LRRK1, LRRK2, ULK1, ULK2, IKKε, or control siRNA, treated with 600 nM monensin for 3 h and analyzed by immunofluorescence. Scale bar, 10 μm; (I) Cells in (H) were analyzed to calculate the percentage of cells with nuclear TFEB. Bars are mean ± SEM. n = 5 FOV. ****P < 0.0001, ns, not significant (2way ANOVA, Sidak’s multiple comparison).

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Extended Data Fig. 6 TBK1 and ULK1 are required for TFEB activation in response to lysosomal stress.

(A) WT and TBK1-KO cells stably expressing TFEB-GFP, transfected for 72 h with either control or ULK1-targeting siRNA (siULK1), respectively, were either left untreated (−) or treated with 600 nM monensin (mon), 1 µM nigericin (nig), or 2 µM salinomycin (sal) for 3 h, and analyzed by confocal microscopy. Scale bar, 10 μm; (B) High content imaging analysis of WT and TBK1-KO cells stably expressing TFEB-GFP, transfected for 72 h with either ULK1-targeting (siULK1) or control (siCtrl) siRNA, plated in 96-well plates and either left untreated or treated with the indicated concentrations of monensin, nigericin and salinomycin (3 h). Cells were analyzed to calculate the nucleo-cytoplasmic ratio of TFEB fluorescence intensity, using a dedicated script. Ratios were analyzed from hundreds of cells from n = 3 different wells. (C) WT and TBK1 KO cells stably expressing TFEB-GFP, transfected for 72 h with either ULK1-targeting (siULK1) or control (siCtrl) siRNA, were treated with monensin at the indicated concentrations for 3 h and analyzed by immunoblotting. n = 2 biologically independent experiments, (D) Immunofluorescence analysis of WT and TBK1-KO cells, transfected for 72 h with ULK1-targeting (siULK1) or control (siCtrl) siRNA, left untreated or treated with 20 µM MG132 for 4 h, 5 h, or 6 h, as indicated; (E) Cells in (D) were analyzed to calculate the percentage of cells with nuclear TFEB. n = 5 FOV. (F) High content imaging analysis of WT and TBK1 KO cells transfected for 72 h with either ULK1-targeting (siULK1) or control (siCtrl) siRNA, as indicated, plated in 96-well plates and either left untreated or treated with 20 µM MG132 for 4 h, 5 h, or 6 h. Cells were analyzed to calculate the nuclear-cytoplasmic ratio of TFEB fluorescence intensity, using a dedicated script. Ratios were analyzed from hundreds of cells from n = 3 different wells. *P = 0.0362 (4 h); ***P = 0.0003 (5 h). (G) Immunofluorescence analysis of WT and TBK1 KO cells stably expressing a lysosomal version of TAX1BP1 (Lyso-GFP-TAX1BP1), transfected for 72 h with ULK1-targeting or control siRNA. (H) Cells in (G) were analyzed to calculate the percentage of cells with nuclear TFEB. n = 3 biological replicates. (I) Cells as in (G) were analyzed by western blotting; (J) WT and TBK1 KO HEK 293 T cells with inducible expression of either WT or R400Q HA-ATP6V1B2 were transfected with control or ULK-targeting siRNA for 24 h, then treated with doxycycline for 48 h, and analyzed by immunofluorescence. (K) Quantification of percentage of cells in (J) showing nuclear TFEB. n = 4 FOV, V1B2-WT, WT cells; n = 5, V1B2-WT, TBK1 KO siULK1 cells; n = 4, V1B2-R400Q, WT cells; n = 4, V1B2- R400Q, TBK1 KO siULK1 cells. Unpaired t-test (H), 2way ANOVA, Dunnett’s (B), or 2way ANOVA, Sidak’s (E, F, K) multiple comparison tests were used. All bars are mean ± SEM. ****P < 0.0001; ns, not significant. Scale bars, 10 μm.

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Extended Data Fig. 7 TBK1 and ULK1 lysosomal tethering promotes TFEB nuclear translocation.

(A) Immunofluorescence analysis of WT cells stably expressing a lysosomal version of either GFP alone (Lyso-GFP), GFP-JNK (Lyso-GFP-JNK), GFP-TBK1 (Lyso-GFP-TBK1) or GFP-ULK1 (Lyso-GFP-ULK1). Scale bar, 10 μm; (B) Cells in (A) were analyzed to calculate the percentage of cells with nuclear TFEB. Bars represent mean ± SEM. n = 10 fields of view (FOV). ****P < 0.0001, ns, not significant (one-way ANOVA, Dunnett’s multiple comparison); (C-D) Immunofluorescence analysis (C) and quantification (D) of TFEB nuclear localization in TBK1 KO U2OS cells reconstituted with either Lyso-GFP-TBK1-WT or Lyso-GFP-TBK1(D135N) kinase dead mutant; Scale bar, 10 μm. Results are mean ± SEM. n = 10 FOV. ****P < 0.0001 (unpaired t-test); (E-F) Immunofluorescence analysis (E) and quantification (F) of TFEB nuclear localization in U2OS cells expressing either Lyso-GFP-ULK1-WT or Lyso-GFP-ULK1(D165A) kinase dead mutant. Scale bar, 10 μm. Results are mean ± SEM. n = 10 FOV. ****P < 0.0001 (unpaired t-test).

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Extended Data Fig. 8 TBK1 and ULK1 control TFEB subcellular localization independently of nutrient availability.

(A-B) Immunofluorescence analysis (A) and quantification (B) of TFEB nuclear translocation in WT and TBK1-KO cells transfected with the indicated siRNA for 72 h and subjected to amino acid starvation for 60 min or to amino acid starvation/re-feeding for 30 min. Bars are mean ± SEM; n = 4 FOV. Scale bar, 10 μM (C) Cells as in (A) were analyzed by immunoblotting with the indicated antibodies; (D) WT cells transfected with control siRNA (siCtrl) and TBK1-KO U2OS cells transfected with ULK1-targeting siRNA (siULK1), stably expressing TFEB-GFP, were either left untreated or treated with 600 nM monensin, 1 μM nigericin or 2 μM salinomycin for 3 h, followed by Torin1 treatment (250 nM) for 1 h to induce TFEB lysosomal localization. Cells were stained and analyzed by confocal microscopy. Scale bar, 10 μm. (E) Cells in (D) were analyzed for TFEB-LAMP1 colocalization by calculating Manders’ colocalization coefficient. Results are mean ± SEM. n = 5 FOV; ****P < 0.0001, ns, not significant (2way ANOVA, Sidak’s multiple comparison).

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Extended Data Fig. 9 TBK1-mediated activation of TFEB is separable from IRF3 activation.

(A-B) Immunofluorescence analysis (A) and quantification (B) of TFEB and IRF3 nuclear localization in U2OS cells either left untreated (−), treated with Mon 600 nM, or treated with 2 µM Poly I:C for 3 h. Results are mean ± SEM; n = 5 FOV. ****P < 0.0001, ns, not significant (2way ANOVA, Sidak’s multiple comparison). Scale bar, 10 μm. (C) Cells as in (A) were analyzed by immunoblotting with the indicated antibodies.

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Extended Data Fig. 10 Phosphorylation of FNIP1 Ser296 promotes TFEB nuclear translocation.

(A) Clustal W alignment of FNIP1 and FNIP2 sequences surrounding S296 and S277, respectively, with well-established phosphorylation sites of TBK1. Residues belonging to the same amino acid category are highlighted; (B) U2OS cells transfected with either WT or S296A HA-tagged FNIP1 were left untreated or treated with 600 nM monensin for 3 h, followed by incubation of cell lysates with FLAG-beads and immunoblotting analysis; (C) U2OS cells co-transfected with TFEB-GFP and either WT or S296A HA-tagged FNIP1 were left untreated or treated with 300 nM monensin for 3 h, followed by incubation of cell lysates with GFP-beads and immunoblotting analysis; (D) WT and TBK1-KO U2OS cells were transfected with control siRNA (siCtrl) and ULK1-targeting siRNA (siULK1), respectively. After 48 h, cells were co-transfected with TFEB-GFP and either WT or S296D HA-tagged FNIP1, followed by GFP immunoprecipitation and immunoblotting analysis; (E) WT and TBK1-KO U2OS cells transfected with HA-tagged S153D, S280D, S296D, S714D or WT FNIP1 were analyzed by immunofluorescence. Scale bar, 10 μm; (F) Cells in (E) were quantified to calculate the percentage of cells showing TFEB nuclear localization. Results are mean ± SEM. n = 3 biological replicates. ***P = 0.0007, ns, not significant (one-way ANOVA, Dunnett’s multiple comparison); (G) PhosTag gel of FLCN:FNIP1 prior or after incubation with recombinant TBK1; (H) HEK293T cells were left untreated or treated with either 200 nM monensin, 200 nM nigericin, or 200 nM salinomycin for 3 h, or subjected to glucose starvation for 2 h and analyzed by immunoblotting.

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Esposito, A., Bayramoglu, I., Varriale, C. et al. A TBK1/ULK1 signalling axis couples lysosomal stress to TFEB activation. Nature (2026). https://doi.org/10.1038/s41586-026-11093-3

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