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.

  • Article
  • Published:

Hierarchical regulation of mitochondrion-dependent apoptosis by BCL-2 subfamilies

Nature Cell Biology volume 8, pages 1348–1358 (2006)Cite this article

Abstract

Although the BCL-2 family constitutes a crucial checkpoint in apoptosis, the intricate interplay between these family members remains elusive. Here, we demonstrate that BIM and PUMA, similar to truncated BID (tBID), directly activate BAX–BAK to release cytochrome c. Conversely, anti-apoptotic BCL-2–BCL-XL–MCL-1 sequesters these 'activator' BH3-only molecules into stable complexes, thus preventing the activation of BAX–BAK. Extensive mutagenesis of BAX–BAK indicates that their activity is not kept in check by BCL-2–BCL-XL–MCL-1. Anti-apoptotic BCL-2 members are differentially inactivated by the remaining 'inactivator' BH3-only molecules including BAD, NOXA, BMF, BIK/BLK and HRK/DP5. BAD displaces tBID, BIM or PUMA from BCL-2–BCL-XL to activate BAX–BAK, whereas NOXA specifically antagonizes MCL-1. Coexpression of BAD and NOXA killed wild-type but not Bax, Bak doubly deficient cells or Puma deficient cells with Bim knockdown, indicating that activator BH3-only molecules function downstream of inactivator BH3-only molecules to activate BAX–BAK. Our data establish a hierarchical regulation of mitochondrion-dependent apoptosis by various BCL-2 subfamilies.

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: BID, BIM and PUMA directly activate BAX and BAK to induce cytochrome c release from mitochondria.
Figure 2: BAK is kept in check by VDAC2 but not by BCL-XL or MCL-1.
Figure 3: BAX is not kept in check by BCL-XL or MCL-1.
Figure 4: Anti-apoptotic BCL-2 proteins inhibit apoptosis by sequestering activator BH3-only molecules, preventing them from activating BAX–BAK.
Figure 5: Activator BH3-only molecules sequestered by anti-apoptotic BCL- members can be displaced by inactivator BH3-only molecules to activate BAX–BAK-dependent mitochondrial apoptosis.
Figure 6: BAD and NOXA synergize to trigger apoptosis by displacing BIM and PUMA from anti-apoptotic BCL-2 members to activate BAX and BAK.
Figure 7: The anti-apoptotic activity of BCL-2, BCL-XL and MCL-1 against various death stimuli is differentially regulated by specific inactivator BH3-only molecules.

Similar content being viewed by others

References

  1. Horvitz, H.R. Worms, life, and death. Chembiochem 4, 697–711 (2003).

    Article  CAS  PubMed  Google Scholar 

  2. Danial, N.N. & Korsmeyer, S.J. Cell death: critical control points. Cell 116, 205–219 (2004).

    Article  CAS  PubMed  Google Scholar 

  3. Degterev, A., Boyce, M. & Yuan, J. A decade of caspases. Oncogene 22, 8543–8567 (2003).

    Article  CAS  PubMed  Google Scholar 

  4. Green, D.R. & Reed, J.C. Mitochondria and apoptosis. Science 281, 1309–1312 (1998).

    Article  CAS  PubMed  Google Scholar 

  5. Wang, X. The expanding role of mitochondria in apoptosis. Genes Dev. 15, 2922–2933 (2001).

    CAS  PubMed  Google Scholar 

  6. Cory, S. & Adams, J.M. The Bcl2 family: regulators of the cellular life-or-death switch. Nature Rev. Cancer 2, 647–656 (2002).

    Article  CAS  Google Scholar 

  7. Lindsten, T. et al. The combined functions of proapoptotic Bcl-2 family members Bak and Bax are essential for normal development of multiple tissues. Mol. Cell 6, 1389–1399 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Wei, M.C. et al. Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science 292, 727–730 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Hsu, Y.T., Wolter K.G. & Youle, R.J. Cytosol-to-membrane redistribution of Bax and Bcl-X(L) during apoptosis. Proc. Natl Acad. Sci. USA 94, 3668–3672 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Wei, M.C. et al. tBID, a membrane-targeted death ligand, oligomerizes BAK to release cytochrome c. Genes Dev. 14, 2060–2071 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Cheng, E.H., Sheiko T.V., Fisher, J.K., Craigen, W.J. & Korsmeyer, S.J. VDAC2 inhibits BAK activation and mitochondrial apoptosis. Science 301, 513–517 (2003).

    Article  CAS  PubMed  Google Scholar 

  12. Antonsson, B., Montessuit, S., Sanchez, B. & Martinou, J.C. Bax is present as a high molecular weight oligomer/complex in the mitochondrial membrane of apoptotic cells. J. Biol. Chem. 276, 11615–11623 (2001).

    Article  CAS  PubMed  Google Scholar 

  13. Cheng, E.H. et al. BCL-2, BCL-X(L) sequester BH3 domain-only molecules preventing BAX- and BAK-mediated mitochondrial apoptosis. Mol. Cell 8, 705–711 (2001).

    Article  CAS  PubMed  Google Scholar 

  14. Zong, W.X., Lindsten, T., Ross, A.J., MacGregor, G.R . & Thompson, C.B. BH3-only proteins that bind pro-survival Bcl-2 family members fail to induce apoptosis in the absence of Bax and Bak. Genes Dev. 15, 1481–1486 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Letai, A. et al. Distinct BH3 domains either sensitize or activate mitochondrial apoptosis, serving as prototype cancer therapeutics. Cancer Cell 2, 183–192 (2002).

    Article  CAS  PubMed  Google Scholar 

  16. Kuwana, T. et al. BH3 domains of BH3-only proteins differentially regulate Bax-mediated mitochondrial membrane permeabilization both directly and indirectly. Mol. Cell 17, 525–535 (2005).

    Article  CAS  PubMed  Google Scholar 

  17. Chen, L. et al. Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function. Mol. Cell 17, 393–403 (2005).

    Article  CAS  PubMed  Google Scholar 

  18. Li, H., Zhu, H., Xu, C.J. & Yuan, J. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell 94, 491–501 (1998).

    Article  CAS  PubMed  Google Scholar 

  19. Luo, X., Budihardjo, I., Zou, H., Slaughter, C. & Wang, X. Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell 94, 481–490 (1998).

    Article  CAS  PubMed  Google Scholar 

  20. Cuconati, A., Mukherjee, C., Perez, D. & White, E. DNA damage response and MCL-1 destruction initiate apoptosis in adenovirus-infected cells. Genes Dev. 17, 2922–2932 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Willis, S.N. et al. Proapoptotic Bak is sequestered by Mcl-1 and Bcl-xL, but not Bcl-2, until displaced by BH3-only proteins. Genes Dev. 19, 1294–1305 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Nijhawan, D. et al. Elimination of Mcl-1 is required for the initiation of apoptosis following ultraviolet irradiation. Genes Dev. 17, 1475–1486 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Opferman, J.T. et al. Development and maintenance of B and T lymphocytes requires antiapoptotic MCL-1. Nature 426, 671–676 (2003).

    Article  CAS  PubMed  Google Scholar 

  24. Inohara, N., Ding, L., Chen, S. & Nunez, G. harakiri, a novel regulator of cell death, encodes a protein that activates apoptosis and interacts selectively with survival-promoting proteins Bcl-2 and Bcl-X(L). EMBO J. 16, 1686–1694 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Puthalakath, H. et al. Bmf: a proapoptotic BH3-only protein regulated by interaction with the myosin V actin motor complex, activated by anoikis. Science 293, 1829–1832 (2001).

    Article  CAS  PubMed  Google Scholar 

  26. Terradillos, O., Montessuit, S., Huang, D.C. & Martinou, J.C. Direct addition of BimL to mitochondria does not lead to cytochrome c release. FEBS Lett. 522, 29–34 (2002).

  27. Cartron, P.F. et al. The first α-helix of Bax plays a necessary role in its ligand-induced activation by the BH3-only proteins Bid and PUMA. Mol. Cell 16, 807–818 (2004).

    Article  CAS  PubMed  Google Scholar 

  28. Chipuk, J.E., Bouchier-Hayes, L., Kuwana, T., Newmeyer, D.D. & Green, D.R. PUMA couples the nuclear and cytoplasmic proapoptotic function of p53. Science 309, 1732–1735 (2005).

    Article  CAS  PubMed  Google Scholar 

  29. Mihara, M. et al. p53 has a direct apoptogenic role at the mitochondria. Mol. Cell 11, 577–590 (2003).

    Article  CAS  PubMed  Google Scholar 

  30. Nakano, K. & Vousden, K.H. PUMA, a novel proapoptotic gene, is induced by p53. Mol. Cell 7, 683–694 (2001).

    Article  CAS  PubMed  Google Scholar 

  31. Li, C. et al. Bcl-X(L) affects Ca2+ homeostasis by altering expression of inositol 1,4,5-trisphosphate receptors. Proc. Natl Acad. Sci. USA 99, 9830–9835 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Scorrano, L. et al. BAX and BAK regulation of endoplasmic reticulum Ca2+: a control point for apoptosis. Science 300, 135–139 (2003).

    Article  CAS  PubMed  Google Scholar 

  33. Oda, E. et al. Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis. Science 288, 1053–1058 (2000).

    Article  CAS  PubMed  Google Scholar 

  34. Yu, J., Zhang, L., Hwang, P.M., Kinzler, K.W. & Vogelstein, B. PUMA induces the rapid apoptosis of colorectal cancer cells. Mol. Cell 7, 673–682 (2001).

    Article  CAS  PubMed  Google Scholar 

  35. Imaizumi, K. et al. Molecular cloning of a novel polypeptide, DP5, induced during programmed neuronal death. J. Biol. Chem. 272, 18842–18848 (1997).

    Article  CAS  PubMed  Google Scholar 

  36. Putcha, G.V. et al. Induction of BIM, a proapoptotic BH3-only BCL-2 family member, is critical for neuronal apoptosis. Neuron 29, 615–628 (2001).

    Article  CAS  PubMed  Google Scholar 

  37. Dijkers, P.F., Medema, R.H., Lammers, J.W., Koenderman, L. & Coffer, P.J. Expression of the pro-apoptotic Bcl-2 family member Bim is regulated by the forkhead transcription factor FKHR-L1. Curr. Biol. 10, 1201–1204 (2000).

    Article  CAS  PubMed  Google Scholar 

  38. Morrison, D.J., English, M.A. & Licht, J.D. WT1 induces apoptosis through transcriptional regulation of the proapoptotic Bcl-2 family member Bak. Cancer Res. 65, 8174–8182 (2005).

    Article  CAS  PubMed  Google Scholar 

  39. Yin, X.M. et al. Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis. Nature 400, 886–891 (1999).

    Article  CAS  PubMed  Google Scholar 

  40. Bouillet, P. et al. Proapoptotic Bcl-2 relative Bim required for certain apoptotic responses, leukocyte homeostasis, and to preclude autoimmunity. Science 286, 1735–1738 (1999).

    Article  CAS  PubMed  Google Scholar 

  41. Imaizumi, K. et al. Critical role for DP5/Harakiri, a Bcl-2 homology domain 3-only Bcl-2 family member, in axotomy-induced neuronal cell death. J. Neurosci. 24, 3721–3725 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Jeffers, J.R. et al. Puma is an essential mediator of p53-dependent and -independent apoptotic pathways. Cancer Cell 4, 321–328 (2003).

    Article  CAS  PubMed  Google Scholar 

  43. Villunger, A. et al. p53- and drug-induced apoptotic responses mediated by BH3-only proteins Puma and Noxa. Science 302, 1036–1038 (2003).

    Article  CAS  PubMed  Google Scholar 

  44. Shibue, T. et al. Integral role of Noxa in p53-mediated apoptotic response. Genes Dev. 17, 2233–2238 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Coultas, L. et al. Proapoptotic BH3-only Bcl-2 family member Bik/Blk/Nbk is expressed in hemopoietic and endothelial cells but is redundant for their programmed death. Mol. Cell Biol. 24, 1570–1581 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Ranger, A.M. et al. Bad-deficient mice develop diffuse large B cell lymphoma. Proc. Natl Acad. Sci. USA 100, 9324–9329 (2003).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Bouillet, P., Cory, S., Zhang, L.C., Strasser, A. & Adams, J.M. Degenerative disorders caused by Bcl-2 deficiency prevented by loss of its BH3-only antagonist Bim. Dev. Cell 1, 645–653 (2001).

    Article  CAS  PubMed  Google Scholar 

  48. Walensky, L.D. et al. Activation of apoptosis in vivo by a hydrocarbon-stapled BH3 helix. Science 305, 1466–1470 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Oltersdorf, T. et al. An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature 435, 677–681 (2005).

    Article  CAS  PubMed  Google Scholar 

  50. Harada, H., Quearry, B., Ruiz-Vela, A. & Korsmeyer, S.J. Survival factor-induced extracellular signal-regulated kinase phosphorylates BIM, inhibiting its association with BAX and proapoptotic activity. Proc. Natl Acad. Sci. USA 101, 15313–15317 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We would like to thank N. Inohara, E. Johnson and H. Harada for generously providing Hrk, Dp5 and Bim knockdown constructs. E.H-Y.C. is supported by the National Cancer Institute (NCI) Howard Temin Award, Searle Scholars Program and Edward Mallinckrodt, Jr. Foundation. G.P.Z. is supported by a National Institutes of Health (NIH) grant (CA63230).

Author information

Authors and Affiliations

  1. Department of Medicine, Molecular Oncology, Washington University School of Medicine, St. Louis, 63110, MO, USA

    Hyungjin Kim, Mubina Rafiuddin-Shah, Ho-Chou Tu, James J.-D. Hsieh & Emily H.-Y. Cheng

  2. Siteman Cancer Center, Washington University School of Medicine, St. Louis, 63110, MO, USA

    James J.-D. Hsieh & Emily H.-Y. Cheng

  3. Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, 63110, MO, USA

    Emily H.-Y. Cheng

  4. St. Jude Children's Research Hospital, Memphis, 38105, TN, USA

    John R. Jeffers & Gerard P. Zambetti

Authors
  1. Hyungjin Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mubina Rafiuddin-Shah
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ho-Chou Tu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. John R. Jeffers
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Gerard P. Zambetti
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. James J.-D. Hsieh
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Emily H.-Y. Cheng
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Emily H.-Y. Cheng.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary figures S1, S2, S3 and S4 (PDF 551 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kim, H., Rafiuddin-Shah, M., Tu, HC. et al. Hierarchical regulation of mitochondrion-dependent apoptosis by BCL-2 subfamilies. Nat Cell Biol 8, 1348–1358 (2006). https://doi.org/10.1038/ncb1499

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb1499

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