Article | Published:

Caspase-activated phosphoinositide binding by CNT-1 promotes apoptosis by inhibiting the AKT pathway

Nature Structural & Molecular Biology volume 21, pages 10821090 (2014) | Download Citation

Abstract

Inactivation of cell-survival factors is a crucial step in apoptosis. The phosphoinositide 3-kinase (PI3K)-AKT signaling pathway promotes cell growth, proliferation and survival, and its deregulation causes cancer. How this pathway is suppressed to promote apoptosis is poorly understood. Here we report the identification of a CED-3 caspase substrate in Caenorhabditis elegans, CNT-1, that is cleaved during apoptosis to generate an N-terminal phosphoinositide-binding fragment (tCNT-1). tCNT-1 translocates from the cytoplasm to the plasma membrane and blocks AKT binding to phosphatidylinositol (3,4,5)-trisphosphate, thereby disabling AKT activation and its prosurvival activity. Our findings reveal a new mechanism that negatively regulates AKT cell signaling to promote apoptosis and that may restrict cell growth and proliferation in normal cells.

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References

  1. 1.

    Mechanisms and genes of cellular suicide. Science 267, 1445–1449 (1995).

  2. 2.

    & Caspase substrates and cellular remodeling. Annu. Rev. Biochem. 80, 1055–1087 (2011).

  3. 3.

    & Membrane and morphological changes in apoptotic cells regulated by caspase-mediated activation of PAK2. Science 276, 1571–1574 (1997).

  4. 4.

    , & Genetics of programmed cell death in C. elegans: past, present and future. Trends Genet. 14, 410–416 (1998).

  5. 5.

    , , , & Caspase-dependent conversion of Dicer ribonuclease into a death-promoting deoxyribonuclease. Science 328, 327–334 (2010).

  6. 6.

    et al. Caenorhabditis elegans drp-1 and fis-2 regulate distinct cell-death execution pathways downstream of ced-3 and independent of ced-9. Mol. Cell 31, 586–597 (2008).

  7. 7.

    , , , & Caspase-mediated activation of Caenorhabditis elegans CED-8 promotes apoptosis and phosphatidylserine externalization. Nat. Commun. 4, 2726 (2013).

  8. 8.

    , , & Beyond PTEN mutations: the PI3K pathway as an integrator of multiple inputs during tumorigenesis. Nat. Rev. Cancer 6, 184–192 (2006).

  9. 9.

    , & Targeting the PI3K-Akt pathway in human cancer: rationale and promise. Cancer Cell 4, 257–262 (2003).

  10. 10.

    , & Isolation of transforming murine leukemia viruses from mice with a high incidence of spontaneous lymphoma. Proc. Natl. Acad. Sci. USA 74, 3065–3067 (1977).

  11. 11.

    et al. PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science 275, 1943–1947 (1997).

  12. 12.

    & The phosphatidylinositol 3-Kinase–AKT pathway in human cancer. Nat. Rev. Cancer 2, 489–501 (2002).

  13. 13.

    et al. Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the protein kinase Akt2 (PKBβ). Science 292, 1728–1731 (2001).

  14. 14.

    , & Akt signalling in health and disease. Cell. Signal. 23, 1515–1527 (2011).

  15. 15.

    & The genetics of aging. Annu. Rev. Genomics Hum. Genet. 2, 435–462 (2001).

  16. 16.

    The plasticity of aging: insights from long-lived mutants. Cell 120, 449–460 (2005).

  17. 17.

    & The trifecta of aging in Caenorhabditis elegans. Exp. Gerontol. 41, 894–903 (2006).

  18. 18.

    , , & daf-2, an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans. Science 277, 942–946 (1997).

  19. 19.

    , & A phosphatidylinositol-3-OH kinase family member regulating longevity and diapause in Caenorhabditis elegans. Nature 382, 536–539 (1996).

  20. 20.

    , , & Insulin receptor substrate and p55 orthologous adaptor proteins function in the Caenorhabditis elegans daf-2/insulin-like signaling pathway. J. Biol. Chem. 277, 49591–49597 (2002).

  21. 21.

    , , , & PDK1 homolog is necessary and sufficient to transduce AGE-1 PI3 kinase signals that regulate diapause in Caenorhabditis elegans. Genes Dev. 13, 1438–1452 (1999).

  22. 22.

    & Caenorhabditis elegans Akt/PKB transduces insulin receptor-like signals from AGE-1 PI3 kinase to the DAF-16 transcription factor. Genes Dev. 12, 2488–2498 (1998).

  23. 23.

    , & C. elegans SGK-1 is the critical component in the Akt/PKB kinase complex to control stress response and life span. Dev. Cell 6, 577–588 (2004).

  24. 24.

    , , & daf-16: an HNF-3/forkhead family member that can function to double the life-span of Caenorhabditis elegans. Science 278, 1319–1322 (1997).

  25. 25.

    et al. The Fork head transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C. elegans. Nature 389, 994–999 (1997).

  26. 26.

    , & Regulation of C. elegans DAF-16 and its human ortholog FKHRL1 by the daf-2 insulin-like signaling pathway. Curr. Biol. 11, 1950–1957 (2001).

  27. 27.

    , , & Regulation of the Caenorhabditis elegans longevity protein DAF-16 by insulin/IGF-1 and germline signaling. Nat. Genet. 28, 139–145 (2001).

  28. 28.

    , , , & Regulation of the insulin-like developmental pathway of Caenorhabditis elegans by a homolog of the PTEN tumor suppressor gene. Proc. Natl. Acad. Sci. USA 96, 2925–2930 (1999).

  29. 29.

    , , , & The PTEN tumor suppressor homolog in Caenorhabditis elegans regulates longevity and dauer formation in an insulin receptor-like signaling pathway. Proc. Natl. Acad. Sci. USA 96, 7427–7432 (1999).

  30. 30.

    & The C. elegans PTEN homolog, DAF-18, acts in the insulin receptor-like metabolic signaling pathway. Mol. Cell 2, 887–893 (1998).

  31. 31.

    et al. Regulation of dauer larva development in Caenorhabditis elegans by daf-18, a homologue of the tumour suppressor PTEN. Curr. Biol. 9, 329–332 (1999).

  32. 32.

    , , & The age-1 and daf-2 genes function in a common pathway to control the lifespan of Caenorhabditis elegans. Genetics 141, 1399–1406 (1995).

  33. 33.

    , & Genes that regulate both development and longevity in Caenorhabditis elegans. Genetics 139, 1567–1583 (1995).

  34. 34.

    , & AKT-1 regulates DNA-damage-induced germline apoptosis in C. elegans. Curr. Biol. 17, 286–292 (2007).

  35. 35.

    et al. Mitochondrial endonuclease G is important for apoptosis in C. elegans. Nature 412, 90–94 (2001).

  36. 36.

    & Functional genomic analysis of apoptotic DNA degradation in C. elegans. Mol. Cell 11, 987–996 (2003).

  37. 37.

    & The ced-8 gene controls the timing of programmed cell deaths in C. elegans. Mol. Cell 5, 423–433 (2000).

  38. 38.

    , , & The nongenotoxic carcinogens naphthalene and para-dichlorobenzene suppress apoptosis in Caenorhabditis elegans. Nat. Chem. Biol. 2, 338–345 (2006).

  39. 39.

    & The C. elegans protein EGL-1 is required for programmed cell death and interacts with the Bcl-2-like protein CED-9. Cell 93, 519–529 (1998).

  40. 40.

    , & The Caenorhabditis elegans cell-death protein CED-3 is a cysteine protease with substrate specificities similar to those of the human CPP32 protease. Genes Dev. 10, 1073–1083 (1996).

  41. 41.

    et al. Comprehensive identification of PIP3-regulated PH domains from C. elegans to H. sapiens by model prediction and live imaging. Mol. Cell 30, 381–392 (2008).

  42. 42.

    et al. Direct inhibition of the longevity-promoting factor SKN-1 by insulin-like signaling in C. elegans. Cell 132, 1025–1038 (2008).

  43. 43.

    , & Cellular survival: a play in three Akts. Genes Dev. 13, 2905–2927 (1999).

  44. 44.

    et al. Identification and analysis of PH domain-containing targets of phosphatidylinositol 3-kinase using a novel in vivo assay in yeast. EMBO J. 17, 5374–5387 (1998).

  45. 45.

    , , , & G protein signaling events are activated at the leading edge of chemotactic cells. Cell 95, 81–91 (1998).

  46. 46.

    et al. Chemoattractant-mediated transient activation and membrane localization of Akt/PKB is required for efficient chemotaxis to cAMP in Dictyostelium. EMBO J. 18, 2092–2105 (1999).

  47. 47.

    et al. Polarization of chemoattractant receptor signaling during neutrophil chemotaxis. Science 287, 1037–1040 (2000).

  48. 48.

    , , & Protein kinase B/Akt at a glance. J. Cell Sci. 118, 5675–5678 (2005).

  49. 49.

    et al. Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN. Cell 95, 29–39 (1998).

  50. 50.

    , , , & The PTEN/MMAC1 tumor suppressor phosphatase functions as a negative regulator of the phosphoinositide 3-kinase/Akt pathway. Proc. Natl. Acad. Sci. USA 95, 15587–15591 (1998).

  51. 51.

    et al. Regulation of phosphorylation of Thr-308 of Akt, cell proliferation, and survival by the B55α regulatory subunit targeting of the protein phosphatase 2A holoenzyme to Akt. J. Biol. Chem. 283, 1882–1892 (2008).

  52. 52.

    et al. A PP2A regulatory subunit regulates C. elegans insulin/IGF-1 signaling by modulating AKT-1 phosphorylation. Cell 136, 939–951 (2009).

  53. 53.

    , & PHLPP: a phosphatase that directly dephosphorylates Akt, promotes apoptosis, and suppresses tumor growth. Mol. Cell 18, 13–24 (2005).

  54. 54.

    , , & PHLPP and a second isoform, PHLPP2, differentially attenuate the amplitude of Akt signaling by regulating distinct Akt isoforms. Mol. Cell 25, 917–931 (2007).

  55. 55.

    et al. Advances of AKT pathway in human oncogenesis and as a target for anti-cancer drug discovery. Curr. Cancer Drug Targets 8, 2–6 (2008).

  56. 56.

    The genetics of Caenorhabditis elegans. Genetics 77, 71–94 (1974).

  57. 57.

    et al. C. elegans mitochondrial factor WAH-1 promotes phosphatidylserine externalization in apoptotic cells through phospholipid scramblase SCRM-1. Nat. Cell Biol. 9, 541–549 (2007).

  58. 58.

    et al. Noncanonical control of C. elegans germline apoptosis by the insulin/IGF-1 and Ras/MAPK signaling pathways. Cell Death Differ. 20, 97–107 (2013).

  59. 59.

    , , & Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 10, 3959–3970 (1991).

  60. 60.

    , & Caenorhabditis elegans SUR-5, a novel but conserved protein, negatively regulates LET-60 Ras activity during vulval induction. Mol. Cell. Biol. 18, 4556–4564 (1998).

  61. 61.

    et al. The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell 99, 123–132 (1999).

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Acknowledgements

We thank B. Derry (Hospital for Sick Children) for anti–AKT-1 antibodies, M. Han (University of Colorado) for some RNAi clones, Y. Kohara (Japan National Institute of Genetics) for akt-1 cDNA, M. Valencia and Y. Shi for making some of the constructs, S. Mitani (Tokyo Women's Medical University) and G. Ruvkun (Massachusetts General Hospital) for strains, R.R. Skeen-Gaar for assistance in generating transgenic strains and B.L. Harry, T. Blumenthal, B. Olwin, and J.M. Espinosa for comments on the manuscript. Some of the worm strains used in this study were kindly provided by the Caenorhabditis Genetics Center, which is funded by the US National Institutes of Health. This work was supported by US National Institutes of Health (grants R01 GM59083, R01 GM79097 and R01 GM088241 to D.X.).

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Affiliations

  1. Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Boulder, Colorado, USA.

    • Akihisa Nakagawa
    • , Kelly D Sullivan
    •  & Ding Xue

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Contributions

A.N. and D.X. conceived the research and designed experiments. A.N. carried out and analyzed experiments. K.D.S. assisted in some experiments. A.N. and D.X. wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Ding Xue.

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https://doi.org/10.1038/nsmb.2915

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