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A tuberous sclerosis complex signalling node at the peroxisome regulates mTORC1 and autophagy in response to ROS

Nature Cell Biology volume 15, pages 1186–1196 (2013)Cite this article

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Abstract

Subcellular localization is emerging as an important mechanism for mTORC1 regulation. We report that the tuberous sclerosis complex (TSC) signalling node, TSC1, TSC2 and Rheb, localizes to peroxisomes, where it regulates mTORC1 in response to reactive oxygen species (ROS). TSC1 and TSC2 were bound by peroxisomal biogenesis factors 19 and 5 (PEX19 and PEX5), respectively, and peroxisome-localized TSC functioned as a Rheb GTPase-activating protein (GAP) to suppress mTORC1 and induce autophagy. Naturally occurring pathogenic mutations in TSC2 decreased PEX5 binding, and abrogated peroxisome localization, Rheb GAP activity and suppression of mTORC1 by ROS. Cells lacking peroxisomes were deficient in mTORC1 repression by ROS, and peroxisome-localization-deficient TSC2 mutants caused polarity defects and formation of multiple axons in neurons. These data identify a role for the TSC in responding to ROS at the peroxisome, and identify the peroxisome as a signalling organelle involved in regulation of mTORC1.

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Figure 1: TSC1 and TSC2 localization to peroxisomes.
Figure 2: Cell fractionation demonstrating the TSC signalling node at the peroxisome.
Figure 3: Active TSC signalling node resident at peroxisome membrane.
Figure 4: The TSC signalling node at the peroxisome induces autophagy in response to ROS.
Figure 5: The ability of TSC to suppress mTORC1 is abrogated in peroxisome-deficient Zellweger cells.
Figure 6: TSC1 and TSC2 interact with PEX19 and PEX5.
Figure 7: TSC2 functions at the peroxisome to repress mTORC1.

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Acknowledgements

We thank G. Mills and Y. Lu (University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA) for the MCF-7 cell line stably expressing GFP–LC3, and RIKEN BRC for providing the ATG5+/+ MEFs and ATG5−/− MEFs. We are also grateful for the assistance of K. Dunner in electron microscopy image acquisition and analysis and T. Berry, X. Tong and S. Hensley for technical assistance. This work was supported by National Institutes of Health (NIH) Grant R01 CA143811 to C.L.W., NIH R01CA157216 to M.B.K., and NIH R01NS058956, the John Merck Fund, and the Children’s Hospital Boston Translational Research Program to M.S. A.R.T. was supported by the Association for International Cancer Research Career Development Fellowship (No. 06-914/915).

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  1. Jiangwei Zhang and Jinhee Kim: These authors contributed equally to this work

Authors and Affiliations

  1. Center for Translational Cancer Research, Institute for Biosciences and Technology, Texas A&M Health Science Center, Houston, Texas 77030, USA

    Jiangwei Zhang, Angela Alexander, Shengli Cai, Durga Nand Tripathi, Ruhee Dere & Cheryl Lyn Walker

  2. Korea Institute of Oriental Medicine, Dajeon 305-811, South Korea

    Jinhee Kim

  3. Institute of Medical Genetics, Cardiff University, Cardiff CF14 4XN, UK

    Andrew R. Tee, Elaine A. Dunlop & Kayleigh M. Dodd

  4. Departments of Pediatrics, Pharmacology and Cancer Biology, Duke Cancer Institute, Duke University Medical Center, Durham, North Carolina 27710, USA

    Jacqueline Tait-Mulder & Michael B. Kastan

  5. Department of Neurology, The F.M. Kirby Neurobiology Center, Children’s Hospital Boston, Harvard Medical School, Boston, Massachusetts 02115, USA

    Alessia Di Nardo, Juliette M. Han, Erica Kwiatkowski & Mustafa Sahin

  6. Department of Pathology (Neuropathology), Brigham and Women’s Hospital, Children’s Hospital Boston, Harvard Medical School, Boston, Massachusetts 02115, USA

    Rebecca D. Folkerth

  7. Department of Pathology and Cell Biology, Columbia University, New York, New York 10032, USA

    Phyllis L. Faust

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Contributions

J.Z., J.K. and C.L.W. designed research; J.Z., J.K., A.A., S.C., D.N.T., R.D., A.R.T., J.T-M., A.D.N., J.M.H., E.K., E.A.D. and K.M.D. performed research; J.Z., J.K., A.R.T., R.D.F., P.L.F., M.B.K., M.S. and C.L.W. analysed data; J.Z., J.K. and C.L.W. wrote the paper.

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Supplementary Figure 1 TSC2 Localization at the peroxisome.

(a) Representative images of FAO cell showing endogenous TSC2 (green) and MTC02 (mitochondria marker) or EEA1 (endosome marker) (red). (Scale bar - 10 μm). (b) Representative images of TSC2+/+ and TSC2−/− MEFs showing endogenous TSC2 (green) and PMP70 (red). (Scale bar -10 μm). (c and d) Subcellular fractionation of HEK 293 (c) or HeLa (d) cells to separate nuclear (N), cytosolic (C), membrane (M), and peroxisome (P) fractions. Immuno-blot analyses were performed using antibodies to TSC2, TSC1, Rheb, TBC1D7 (HEK 293, Supplementary Fig. S1c), AKT, catalase, PMP70 and lamin A/C. The degree of enrichment for peroxisomes in fractionated lysates was evaluated by assessing markers for endosomes (EEA1), lysosomes (LAMP1) and mitochondria (VDAC). WCE– whole cell extracts. Uncropped images of western blots are shown in Supplementary Fig. S6.

Supplementary Figure 2 Induction of autophagy in response to ROS.

(a) Western analyses of MCF-7 stably expressing GFP-LC3 cells treated with 0.4 mM H2O2 for the indicated time with markers for autophagy (p62 and LC3), mTORC1 signaling (pS6K (T389), S6K, pS6 (S235/236) and S6). (b) Quantitation of the ratio of LC3 II/Actin from Fig. S2a. (±s.e.m., n = 3 independent experiments). *p<0.05,***p<0.001, NS, not significant. (c) Western analysis of GFP-LC3 MCF7 cells pre-incubated in the presence or absence 100 nM Bafilomycin A1 (BafA1) for 1hr before treatement with 0.4 mM H2O2 for 7hr using anti-p62 and LC3 antibodies. (d) Quantitation of the ratio of LC3 II/Actin and p62/Actin from Supplementary Fig. S2c. (±s.e.m., n = 3 independent experiments). *p<0.05,**p<0.01, NS, not significant. (e) Western analysis of Atg5+/+ MEFs and Atg5−/− MEFs treated with 0.4 mM H2O2 for 24hr using anti-p62 and Atg5 antibodies. Uncropped images of western blots are shown in Supplementary Fig. S6. Source data of statistical analysis are shown in Supplementary Table S1.

Supplementary Figure 3 mTORC1 signaling and autophagy in Zellweger cells with ROS and amino acids.

(a) Western analysis of human fibroblasts obtained from Zellweger (GM13269) or corresponding control patient with Ehlers-Danlos syndre (GM13427) treated with indicated doses of H2O2 for 1 hr. mTORC1 signaling monitored by western analysis for pS6K (T389), S6K, pS6 (S235/236), S6, p4E-BP1 (T37/46), 4E-BP1, pATM (S1981), ATM, pAMPK (T172), AMPK, p62 and LC3. (b) Western analysis of Zellweger cells (GM13267) or control fibroblasts (GM15871) cells pre-incubated in the presence or absence of 100 nM Bafilomycin A1 (BafA1) for 1hr before treatment with 0.4 mM H2O2 for 1hr using anti-p62 and LC3 antibodies. (c) Quantitation of the ratio of LC3 II/Actin and p62/Actin from Supplementary Fig. S3b (±s.e.m., n = 3 independent experiments). *p<0.05,**p<0.01,***p<0.001, NS, not significant. (d) Representative western analysis using cell extracts from human fibroblasts obtained from a Zellweger patient (GM13269) or control fibroblasts (GM13427) treated with amino acid free media for 60 min, and stimulated with amino acid containing media for 10 min. mTORC1 signaling was monitored using anti-pS6K (T389), S6K, pS6 (S235/236), S6, p4EBP1(T37/46) and 4EBP1. Uncropped images of western blots are shown in Supplementary Fig. S6. Source data of statistical analysis are shown in Supplementary Table S1.

Supplementary Figure 4 Localization of TSC2 PEX5 binding mutants.

(a) Representative images using HeLa cells transfected with Myc-TSC1 and Flag-TSC2 wild type (WT) and or the Flag-TSC2 PxBS mutants (RQ, RG, RW) stained with Flag (red) and PMP70 (peroxisome marker, green). (Scale bar - 10 μm). (b) Representative images using HeLa cells transfected with Myc-TSC1 and Flag-TSC2 mutants (RQ, RG and RW) stained with Flag (green) and LAMP1 (marker for lysosome, red). As a control, the cells were stained by anti-mTOR (green) and anti-LAMP1 (red). (Scale bar - 10 μm). (c) Representative images using HeLa cells transfected with Myc-TSC1 and Flag-TSC2 mutant (RQ) stained with Flag (red) and either calnexin (marker for endoplasmic reticulum, green) or VDAC (marker for mitochondria, green). (Scale bar - 10 μm). (d) HEK 293 cells were transfected with Myc-TSC1 and Flag-TSC2 wild type (WT) or the Flag-TSC2 mutants (RQ, RG, RW) or Flag-TSC2 L1624P (GAP mutant) or Flag-TSC2 G294E (TSC1 binding mutant). The lysates were immunoprecipitated using anti-Myc and control IgG, and samples were analyzed using anti-Flag and anti-myc antibodies. (e) Functional assays were performed using HEK 293 cells expressing Flag–TSC1, myc–Rheb, and Flag–TSC2 wild type (WT) or Flag-TSC2 mutant (RQ). mTOR signaling was monitored by measuring the ratio of pS6K (T389) to S6K level. Graph represents densitometric quantitation of the ratio of phospho-S6K to total S6K (±s.e.m., n = 3 independent experiments). *p<0.05, **p<0.01. Uncropped images of western blots are shown in Supplementary Fig. S6. Source data of statistical analysis are shown in Supplementary Table S1.

Supplementary Figure 5 Model for TSC complex localization on peroxisomal membranes, and activation of the TSC1-TSC2-Rheb signaling node by peroxisomal ROS to repress mTORC1 and induce autophagy, or inactivation by AKT phosphorylation of TSC2, with subsequent binding of TSC2 by 14-3-3 and sequestration in cytosol.

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Zhang, J., Kim, J., Alexander, A. et al. A tuberous sclerosis complex signalling node at the peroxisome regulates mTORC1 and autophagy in response to ROS. Nat Cell Biol 15, 1186–1196 (2013). https://doi.org/10.1038/ncb2822

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