Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Inhibition of caspase-9 through phosphorylation at Thr 125 by ERK MAPK

Nature Cell Biology volume 5, pages 647–654 (2003)Cite this article

Abstract

Many pro-apoptotic signals activate caspase-9, an initiator protease that activates caspase-3 and downstream caspases to initiate cellular destruction1. However, survival signals can impinge on this pathway and suppress apoptosis. Activation of the Ras–Raf–MEK–ERK mitogen-activated protein kinase (MAPK) pathway is associated with protection of cells from apoptosis and inhibition of caspase-3 activation2,3,4,5, although the targets are unknown. Here, we show that the ERK MAPK pathway inhibits caspase-9 activity by direct phosphorylation. In mammalian cell extracts, cytochrome c-induced activation of caspases-9 and -3 requires okadaic-acid-sensitive protein phosphatase activity. The opposing protein kinase activity is overcome by treatment with the broad-specificity kinase inhibitor staurosporine or with inhibitors of MEK1/2. Caspase-9 is phosphorylated at Thr 125, a conserved MAPK consensus site targeted by ERK2 in vitro, in a MEK-dependent manner in cells stimulated with epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA). Phosphorylation at Thr 125 is sufficient to block caspase-9 processing and subsequent caspase-3 activation. We suggest that phosphorylation and inhibition of caspase-9 by ERK promotes cell survival during development and tissue homeostasis. This mechanism may also contribute to tumorigenesis when the ERK MAPK pathway is constitutively activated.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Inhibition of caspase-9 activation in cell extracts.
Figure 2: Caspase-9 is phosphorylated at Thr 125.
Figure 3: Phosphorylation of Thr 125 is sufficient to inhibit caspase-9 activation and function.
Figure 4: Phosphorylation of caspase-9 at Thr 125 in vivo.
Figure 5: EGF- and TPA-stimulated phosphorylation of endogenous caspase-9 at Thr 125.

Similar content being viewed by others

References

  1. Budihardjo, I., Oliver, H., Lutter, M., Luo, X. & Wang, X. Biochemical pathways of caspase activation during apoptosis. Annu. Rev. Cell Dev. Biol. 15, 269–290 (1999).

    Article  CAS  Google Scholar 

  2. Xia, Z., Dickens, M., Raingeaud, J., Davis, R.J. & Greenberg, M.E. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270, 1326–1331 (1995).

    Article  CAS  Google Scholar 

  3. Erhardt, P., Schremser, E.J. & Cooper, G.M. B-Raf inhibits programmed cell death downstream of cytochrome c release from mitochondria by activating the MEK/Erk pathway. Mol. Cell. Biol. 19, 5308–5315 (1999).

    Article  CAS  Google Scholar 

  4. Le Gall, M. et al. The p42/p44 MAP kinase pathway prevents apoptosis induced by anchorage and serum removal. Mol. Biol. Cell 11, 1103–1112 (2000).

    Article  CAS  Google Scholar 

  5. von Gise, A. et al. Apoptosis suppression by Raf-1 and MEK1 requires MEK- and phosphatidylinositol 3-kinase-dependent signals. Mol. Cell. Biol. 21, 2324–2336 (2001).

    Article  CAS  Google Scholar 

  6. Jacobson, M.D., Weil, M. & Raff, M.C. Programmed cell death in animal development. Cell 88, 347–354 (1997).

    Article  CAS  Google Scholar 

  7. Zou, H., Henzel, W.J., Liu, X., Lutschg, A. & Wang, X. Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 90, 405–413 (1997).

    Article  CAS  Google Scholar 

  8. Acehan, D. et al. Three-dimensional structure of the apoptosome: implications for assembly, procaspase-9 binding, and activation. Mol. Cell 9, 423–432 (2002).

    Article  CAS  Google Scholar 

  9. Cecconi, F., Alvarez-Bolado, G., Meyer, B.I., Roth, K.A. & Gruss, P. Apaf1 (CED-4 homolog) regulates programmed cell death in mammalian development. Cell 94, 727–737 (1998).

    Article  CAS  Google Scholar 

  10. Yoshida, H. et al. Apaf1 is required for mitochondrial pathways of apoptosis and brain development. Cell 94, 739–750 (1998).

    Article  CAS  Google Scholar 

  11. Hakem, R. et al. Differential requirement for caspase 9 in apoptotic pathways in vivo. Cell 94, 339–352 (1998).

    Article  CAS  Google Scholar 

  12. Kuida, K. et al. Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9. Cell 94, 325–337 (1998).

    Article  CAS  Google Scholar 

  13. Soengas, M.S. et al. Apaf-1 and caspase-9 in p53-dependent apoptosis and tumor inhibition. Science 284, 156–159 (1999).

    Article  CAS  Google Scholar 

  14. Soengas, M.S. et al. Inactivation of the apoptosis effector Apaf-1 in malignant melanoma. Nature 409, 207–211 (2001).

    Article  CAS  Google Scholar 

  15. Wang, X., Martindale, J.L., Liu, Y. & Holbrook, N.J. The cellular response to oxidative stress: influences of mitogen-activated protein kinase signalling pathways on cell survival. Biochem. J. 333, 291–300 (1998).

    Article  CAS  Google Scholar 

  16. Rytömaa, M., Lehmann, K. & Downward, J. Matrix detachment induces caspase-dependent cytochrome c release from mitochondria: inhibition by PKB/Akt but not Raf signalling. Oncogene 19, 4461–4468 (2000).

    Article  Google Scholar 

  17. MacKeigan, J.P., Collins, T.S. & Ting, J.P. MEK inhibition enhances paclitaxel-induced tumor apoptosis. J. Biol. Chem. 275, 38953–38956 (2000).

    Article  CAS  Google Scholar 

  18. Tashker, J.S., Olson, M. & Kornbluth, S. Post-cytochrome c protection from apoptosis conferred by a MAPK pathway in Xenopus egg extracts. Mol. Biol. Cell 13, 393–401 (2002).

    Article  CAS  Google Scholar 

  19. Li, P. et al. Cytochrome c and dATP-dependent formation of Apaf-1–caspase-9 complex initiates an apoptotic protease cascade. Cell 91, 479–489 (1997).

    Article  CAS  Google Scholar 

  20. Clarke, P.R. in Apoptosis: The Molecular Biology of Programmed Cell Death (eds Jacobson, M.D. & McCarthy, N.) 176–199 (Oxford University Press, Oxford, 2002).

    Google Scholar 

  21. Davies, S.P., Reddy, H., Caivano, M. & Cohen, P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem. J. 351, 95–105 (2000).

    Article  CAS  Google Scholar 

  22. Jiang, X. & Wang, X. Cytochrome c promotes caspase-9 activation by inducing nucleotide binding to Apaf-1. J. Biol. Chem. 275, 31199–31203 (2000).

    Article  CAS  Google Scholar 

  23. Hu, Y., Ding, L., Spencer, D.M. & Nunez, G. WD-40 repeat region regulates Apaf-1 self-association and procaspase-9 activation. J. Biol. Chem. 273, 33489–33494 (1998).

    Article  CAS  Google Scholar 

  24. Srinivasula, S.M., Ahmad, M., Fernandes-Alnemri, T. & Alnemri, E.S. Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization. Mol. Cell 1, 949–957 (1998).

    Article  CAS  Google Scholar 

  25. Cardone, M.H. et al. Regulation of cell death protease caspase-9 by phosphorylation. Science 282, 1318–1321 (1998).

    Article  CAS  Google Scholar 

  26. Mody, N., Leitch, J., Armstrong, C., Dixon, J. & Cohen, P. Effects of MAP kinase cascade inhibitors on the MKK5/ERK5 pathway. FEBS Lett. 502, 21–24 (2001).

    Article  CAS  Google Scholar 

  27. Bergmann, A., Tugentman, M., Shilo, B.Z. & Steller, H. Regulation of cell number by MAPK-dependent control of apoptosis: a mechanism for trophic survival signaling. Dev. Cell 2, 159–170 (2002).

    Article  CAS  Google Scholar 

  28. Bergmann, A., Agapite, J., McCall, K. & Steller, H. The Drosophila gene hid is a direct molecular target of Ras-dependent survival signaling. Cell 95, 331–341 (1998).

    Article  CAS  Google Scholar 

  29. Kurada, P. & White, K. Ras promotes cell survival in Drosophila by downregulating hid expression. Cell 95, 319–329 (1998).

    Article  CAS  Google Scholar 

  30. Sapkota, G.P. et al. Phosphorylation of the protein kinase mutated in Peutz-Jeghers cancer syndrome, LKB1/STK11, at Ser431 by p90(RSK) and cAMP-dependent protein kinase, but not its farnesylation at Cys(433), is essential for LKB1 to suppress cell growth. J. Biol. Chem. 276, 19469–19482 (2001).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We are grateful to D. Alessi for reagents and discussion, and S. Keyse for critical reading of the manuscript. Initial studies on the inhibition of caspase activation by okadaic acid were performed by S. Cosulich and P. Savory. This work was supported by Cancer Research UK, the Medical Research Council and the Association for International Cancer Research.

Author information

Authors and Affiliations

  1. Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY, Scotland, UK

    Lindsey A. Allan, Suzanne Brady, Gareth Magee, Shalini Pathak & Paul R. Clarke

  2. Medical Research Council Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dundee, DD1 5EH, Scotland, UK

    Nick Morrice

Authors
  1. Lindsey A. Allan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nick Morrice
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Suzanne Brady
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gareth Magee
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Shalini Pathak
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Paul R. Clarke
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Paul R. Clarke.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

41556_2003_BFncb1005_MOESM1_ESM.pdf

Figure S1. Inhibition of protein phosphatase activity in cell extracts blocks caspase-9 activation in a MEK-dependent manner. (PDF 204 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Allan, L., Morrice, N., Brady, S. et al. Inhibition of caspase-9 through phosphorylation at Thr 125 by ERK MAPK. Nat Cell Biol 5, 647–654 (2003). https://doi.org/10.1038/ncb1005

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb1005

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing