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TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling

Abstract

Tuberous sclerosis (TSC) is an autosomal dominant disorder characterized by the formation of hamartomas in a wide range of human tissues. Mutation in either the TSC1 or TSC2 tumour suppressor gene is responsible for both the familial and sporadic forms of this disease. TSC1 and TSC2 proteins form a physical and functional complex in vivo. Here, we show that TSC1–TSC2 inhibits the p70 ribosomal protein S6 kinase 1 (an activator of translation) and activates the eukaryotic initiation factor 4E binding protein 1 (4E-BP1, an inhibitor of translational initiation). These functions of TSC1–TSC2 are mediated by inhibition of the mammalian target of rapamycin (mTOR). Furthermore, TSC2 is directly phosphorylated by Akt, which is involved in stimulating cell growth and is activated by growth stimulating signals, such as insulin. TSC2 is inactivated by Akt-dependent phosphorylation, which destabilizes TSC2 and disrupts its interaction with TSC1. Our data indicate a molecular mechanism for TSC2 in insulin signalling, tumour suppressor functions and in the inhibition of cell growth.

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Figure 1: Inhibition of S6K by TSC1–TSC2.
Figure 2: Effects of endogenous TSC2 and disease-derived mutations on phosphorylation of S6K.
Figure 3: Phosphorylation of TSC2 by Akt.
Figure 4: Determination of Akt-dependent phosphorylation sites in TSC2.
Figure 5: Mutation of Akt phosphorylation sites alters TSC2 activity.
Figure 6: TSC1–TSC2 inhibits nutrient-stimulated phosphorylation of S6K and 4E-BP1.
Figure 7: TSC1–TSC2 functions through mTOR to inhibit S6K.

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References

  1. Young, J. & Povey, S. The genetic basis of tuberous sclerosis. Mol. Med. Today 4, 313–319 (1998).

    Article  CAS  PubMed  Google Scholar 

  2. Gomez, M. R. Phenotypes of the tuberous sclerosis complex with a revision of diagnostic criteria. Ann. NY Acad. Sci. 615, 1–7 (1991).

    Article  CAS  PubMed  Google Scholar 

  3. The European Chromosome 16 Tuberous Sclerosis Consortium. Identification and characterization of the tuberous sclerosis gene on chromosome 16. Cell 75, 1305–1315 (1993).

  4. Wienecke, R., Konig, A. & DeClue, J. E. Identification of tuberin, the tuberous sclerosis-2 product. Tuberin possesses specific Rap1GAP activity. J. Biol. Chem. 270, 16409–16414 (1995).

    Article  CAS  PubMed  Google Scholar 

  5. Xiao, G. H., Shoarinejad, F., Jin, F., Golemis, E. A. & Yeung, R. S. The tuberous sclerosis 2 gene product, tuberin, functions as a Rab5 GTPase activating protein (GAP) in modulating endocytosis. J. Biol. Chem. 272, 6097–6100 (1997).

    Article  CAS  PubMed  Google Scholar 

  6. Onda, H., Lueck, A., Marks, P. W., Warren, H. B. & Kwiatkowski, D. J. Tsc2(+/−) mice develop tumors in multiple sites that express gelsolin and are influenced by genetic background. J. Clin. Invest. 104, 687–695 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Au, K. S. et al. Germ-line mutational analysis of the TSC2 gene in 90 tuberous-sclerosis patients. Am. J. Hum. Genet. 62, 286–294 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Kobayashi, T. et al. A germ-line Tsc1 mutation causes tumor development and embryonic lethality that are similar, but not identical to, those caused by Tsc2 mutation in mice. Proc. Natl Acad. Sci. USA 98, 8762–8767 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Gao, X. & Pan, D. TSC1 and TSC2 tumor suppressors antagonize insulin signaling in cell growth. Genes Dev. 15, 1383–1392 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Ito, N. & Rubin, G. M. gigas, a Drosophila homolog of tuberous sclerosis gene product-2, regulates the cell cycle. Cell 96, 529–539 (1999).

    Article  CAS  PubMed  Google Scholar 

  11. Potter, C. J., Huang, H. & Xu, T. Drosophila Tsc1 functions with Tsc2 to antagonize insulin signaling in regulating cell growth, cell proliferation, and organ size. Cell 105, 357–368 (2001).

    Article  CAS  PubMed  Google Scholar 

  12. Tapon, N., Ito, N., Dickson, B. J., Treisman, J. E. & Hariharan, I. K. The Drosophila tuberous sclerosis complex gene homologs restrict cell growth and cell proliferation. Cell 105, 345–355 (2001).

    Article  CAS  PubMed  Google Scholar 

  13. Kozma, S. C. & Thomas, G. Regulation of cell size in growth, development and human disease: PI3K, PKB and S6K. Bioessays 24, 65–71 (2002).

    Article  CAS  PubMed  Google Scholar 

  14. Stocker, H. & Hafen, E. Genetic control of cell size. Curr. Opin. Genet. Dev. 10, 529–535 (2000).

    Article  CAS  PubMed  Google Scholar 

  15. Weinkove, D. & Leevers, S. J. The genetic control of organ growth: insights from Drosophila. Curr. Opin. Genet. Dev. 10, 75–80 (2000).

    Article  CAS  PubMed  Google Scholar 

  16. Shioi, T. et al. The conserved phosphoinositide 3-kinase pathway determines heart size in mice. EMBO J. 19, 2537–2548 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Shioi, T. et al. Akt/Protein kinase B promotes organ growth in transgenic mice. Mol. Cell. Biol. 22, 2799–2809 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. DeChiara, T. M., Efstratiadis, A. & Robertson, E. J. A growth-deficiency phenotype in heterozygous mice carrying an insulin-like growth factor II gene disrupted by targeting. Nature 345, 78–80 (1990).

    Article  CAS  PubMed  Google Scholar 

  19. Liu, J. P., Baker, J., Perkins, A. S., Robertson, E. J. & Efstratiadis, A. Mice carrying null mutations of the genes encoding insulin-like growth factor I (Igf-1) and type 1 IGF receptor (Igf1r). Cell 75, 59–72 (1993).

    CAS  PubMed  Google Scholar 

  20. Tamemoto, H. et al. Insulin resistance and growth retardation in mice lacking insulin receptor substrate-1. Nature 372, 182–186 (1994).

    Article  CAS  PubMed  Google Scholar 

  21. Shima, H. et al. Disruption of the p70(s6k)/p85(s6k) gene reveals a small mouse phenotype and a new functional S6 kinase. EMBO J. 17, 6649–6659 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Vogt, P. K. PI 3-kinase, mTOR, protein synthesis and cancer. Trends Mol. Med. 7, 482–484 (2001).

    Article  CAS  PubMed  Google Scholar 

  23. Neshat, M. S. et al. Enhanced sensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR. Proc. Natl Acad. Sci. USA 98, 10314–10319 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Schmelzle, T. & Hall, M. N. TOR, a central controller of cell growth. Cell 103, 253–262 (2000).

    Article  CAS  PubMed  Google Scholar 

  25. Shah, O. J., Anthony, J. C., Kimball, S. R. & Jefferson, L. S. 4E-BP1 and S6K1: translational integration sites for nutritional and hormonal information in muscle. Am. J. Physiol. Endocrinol. Metab. 279, E715–E729 (2000).

    Article  CAS  PubMed  Google Scholar 

  26. Sonenberg, N. & Gingras, A. C. The mRNA 5′ cap-binding protein eIF4E and control of cell growth. Curr. Opin. Cell Biol. 10, 268–275 (1998).

    Article  CAS  PubMed  Google Scholar 

  27. Podsypanina, K. et al. An inhibitor of mTOR reduces neoplasia and normalizes p70/S6 kinase activity in Pten+/− mice. Proc. Natl Acad. Sci. USA 98, 10320–10325 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Dufner, A. & Thomas, G. Ribosomal S6 kinase signaling and the control of translation. Exp. Cell Res. 253, 100–109 (1999).

    Article  CAS  PubMed  Google Scholar 

  29. Pearson, R. B. et al. The principal target of rapamycin-induced p70s6k inactivation is a novel phosphorylation site within a conserved hydrophobic domain. EMBO J. 14, 5279–5287 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Dabora, S. L. et al. Mutational analysis in a cohort of 224 tuberous sclerosis patients indicates increased severity of TSC2, compared with TSC1, disease in multiple organs. Am. J. Hum. Genet. 68, 64–80 (2001).

    Article  CAS  PubMed  Google Scholar 

  31. Jones, A. C. et al. Comprehensive mutation analysis of TSC1 and TSC2 and phenotypic correlations in 150 families with tuberous sclerosis. Am. J. Hum. Genet. 64, 1305–1315 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Datta, S. R., Brunet, A. & Greenberg, M. E. Cellular survival: a play in three Akts. Genes Dev. 13, 2905–2927 (1999).

    Article  CAS  PubMed  Google Scholar 

  33. Miron, M. et al. The translational inhibitor 4E-BP is an effector of PI(3)K/Akt signalling and cell growth in Drosophila. Nature Cell Biol. 3, 596–601 (2001).

    Article  CAS  PubMed  Google Scholar 

  34. Benvenuto, G. et al. The tuberous sclerosis-1 (TSC1) gene product hamartin suppresses cell growth and augments the expression of the TSC2 product tuberin by inhibiting its ubiquitination. Oncogene 19, 6306–6316 (2000).

    Article  CAS  PubMed  Google Scholar 

  35. Hara, K. et al. Amino acid sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a common effector mechanism. J. Biol. Chem. 273, 14484–14494 (1998).

    Article  CAS  PubMed  Google Scholar 

  36. Gingras, A. C. et al. Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism. Genes Dev. 13, 1422–1437 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Dennis, P. B. et al. Mammalian TOR: a homeostatic ATP sensor. Science 294, 1102–1105 (2001).

    Article  CAS  PubMed  Google Scholar 

  38. Weng, Q. P., Andrabi, K., Kozlowski, M. T., Grove, J. R. & Avruch, J. Multiple independent inputs are required for activation of the p70 S6 kinase. Mol. Cell. Biol. 15, 2333–2340 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Schalm, S. S. & Blenis, J. Identification of a Conserved Motif Required for mTOR Signaling. Curr. Biol. 12, 632–639 (2002).

    Article  CAS  PubMed  Google Scholar 

  40. Nave, B. T., Ouwens, M., Withers, D. J., Alessi, D. R. & Shepherd, P. R. Mammalian target of rapamycin is a direct target for protein kinase B: identification of a convergence point for opposing effects of insulin and amino-acid deficiency on protein translation. Biochem J. 344, 427–431 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Scott, P. H., Brunn, G. J., Kohn, A. D., Roth, R. A. & Lawrence, J. C. Jr. Evidence of insulin-stimulated phosphorylation and activation of the mammalian target of rapamycin mediated by a protein kinase B signaling pathway. Proc. Natl Acad. Sci. USA 95, 7772–7777 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Sekulic, A. et al. A direct linkage between the phosphoinositide 3-kinase-AKT signaling pathway and the mammalian target of rapamycin in mitogen-stimulated and transformed cells. Cancer Res. 60, 3504–3513 (2000).

    CAS  PubMed  Google Scholar 

  43. Aoki, M., Blazek, E. & Vogt, P. K. A role of the kinase mTOR in cellular transformation induced by the oncoproteins P3k and Akt. Proc. Natl Acad. Sci. USA 98, 136–141 (2001).

    Article  CAS  PubMed  Google Scholar 

  44. Burgering, B. M. & Coffer, P. J. Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature 376, 599–602 (1995).

    Article  CAS  PubMed  Google Scholar 

  45. Kwiatkowski, D. J. et al. A mouse model of TSC1 reveals sex-dependent lethality from liver hemangiomas, and up-regulation of p70S6 kinase activity in Tsc1 null cells. Hum. Mol. Genet. 11, 525–534 (2002).

    Article  CAS  PubMed  Google Scholar 

  46. Radimerski, T. et al. dS6K-regulated cell growth is dPKB/dPI(3)K-independent, but requires dPDK1. Nature Cell Biol. 4, 251–255 (2002).

    Article  CAS  PubMed  Google Scholar 

  47. Tuttle, R. L. et al. Regulation of pancreatic β-cell growth and survival by the serine/threonine protein kinase Akt1/PKBα. Nature Med. 7, 1133–1137 (2001).

    Article  CAS  PubMed  Google Scholar 

  48. Reynolds, I. T., Bodine, S. C. & Lawrence, J. C. Jr. Control of Ser 2448 phosphorylation in the mammalian target of rapamycin by insulin and skeletal muscle load. J. Biol. Chem. 277, 17657–17662 (2002).

    Article  CAS  PubMed  Google Scholar 

  49. Peterson, R. T., Desai, B. N., Hardwick, J. S. & Schreiber, S. L. Protein phosphatase 2A interacts with the 70-kDa S6 kinase and is activated by inhibition of FKBP12-rapamycin-associated protein. Proc. Natl Acad. Sci. USA 96, 4438–4442 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Elbashir, S. M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498 (2001).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We are indebted to J. Blenis, Y. Xiong, and S. Schreiber for providing cDNAs. We thank T. Xu for personal communication. We also thank E. Tang and H.G. Vikis for critical reading of the manuscript, and B. Chia for construction of TSC2 fragment 1 and fragment 2 plasmids. This work was supported by grants from the National Institutes of Health and the Walther Cancer Institute.

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Correspondence to Kun-Liang Guan.

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Inoki, K., Li, Y., Zhu, T. et al. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat Cell Biol 4, 648–657 (2002). https://doi.org/10.1038/ncb839

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