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:

Regulation of apoptosis by an intrinsically disordered region of Bcl-xL

Abstract

Intrinsically disordered regions (IDRs) of proteins often regulate function upon post-translational modification (PTM) through interactions with folded domains. An IDR linking two α-helices (α1-α2) of the antiapoptotic protein Bcl-xL experiences several PTMs that reduce antiapoptotic activity. Here, we report that PTMs within the α1-α2 IDR promote its interaction with the folded core of Bcl-xL that inhibits the proapoptotic activity of two types of regulatory targets, BH3-only proteins and p53. This autoregulation utilizes an allosteric pathway whereby, in one direction, the IDR induces a direct displacement of p53 from Bcl-xL coupled to allosteric displacement of simultaneously bound BH3-only partners. This pathway operates in the opposite direction when the BH3-only protein PUMA binds to the BH3 binding groove of Bcl-xL, directly displacing other bound BH3-only proteins, and allosterically remodels the distal site, displacing p53. Our findings show how an IDR enhances functional versatility through PTM-dependent allosteric regulation of a folded protein domain.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Fig. 1: PTMs within the α1-α2 IDR of Bcl-xL downregulate its antiapoptotic function.
Fig. 2: NMR evidence of an interaction between the α1-α2 IDR and the folded core of Bcl-xL.
Fig. 3: Inhibition of the BH3-binding associated repositioning of Bcl-xL α2-α3 relative to α4-α5 by the α1-α2 IDR.
Fig. 4: Modulation of the antiapoptotic activity of Bcl-xL by mutations in the α1-α2 IDR and α2-α3.
Fig. 5: Schematic illustration of the proposed dual mechanism of allosteric regulation of Bcl-xL enabled by PTMs in the α1-α2 IDR and structural plasticity of α3.

Similar content being viewed by others

References

  1. Babu, M. M., Kriwacki, R. W. & Pappu, R. V. Structural biology. Versatility from protein disorder. Science 337, 1460–1461 (2012).

    Article  CAS  Google Scholar 

  2. Yeon, J. H., Heinkel, F., Sung, M., Na, D. & Gsponer, J. Systems-wide identification of cis-regulatory elements in proteins. Cell Syst. 2, 89–100 (2016).

    Article  CAS  Google Scholar 

  3. Showalter, S. A., Bruschweiler-Li, L., Johnson, E., Zhang, F. & Brüschweiler, R. Quantitative lid dynamics of MDM2 reveals differential ligand binding modes of the p53-binding cleft. J. Am. Chem. Soc. 130, 6472–6478 (2008).

    Article  CAS  Google Scholar 

  4. Pufall, M. A. et al. Variable control of Ets-1 DNA binding by multiple phosphates in an unstructured region. Science 309, 142–145 (2005).

    Article  CAS  Google Scholar 

  5. Youle, R. J. & Strasser, A. The BCL-2 protein family: opposing activities that mediate cell death. Nat. Rev. Mol. Cell Biol. 9, 47–59 (2008).

    Article  CAS  Google Scholar 

  6. Shamas-Din, A., Kale, J., Leber, B. & Andrews, D. W. Mechanisms of action of Bcl-2 family proteins. Cold Spring Harb. Perspect. Biol. 5, a008714 (2013).

    Article  Google Scholar 

  7. Petros, A. M. et al. Rationale for Bcl-xL/Bad peptide complex formation from structure, mutagenesis, and biophysical studies. Protein Sci. 9, 2528–2534 (2000).

    Article  CAS  Google Scholar 

  8. Follis, A. V. et al. The DNA-binding domain mediates both nuclear and cytosolic functions of p53. Nat. Struct. Mol. Biol. 21, 535–543 (2014).

    Article  CAS  Google Scholar 

  9. Follis, A. V. et al. PUMA binding induces partial unfolding within BCL-xL to disrupt p53 binding and promote apoptosis. Nat. Chem. Biol. 9, 163–168 (2013).

    Article  CAS  Google Scholar 

  10. Maundrell, K. et al. Bcl-2 undergoes phosphorylation by c-Jun N-terminal kinase/stress-activated protein kinases in the presence of the constitutively active GTP-binding protein Rac1. J. Biol. Chem. 272, 25238–25242 (1997).

    Article  CAS  Google Scholar 

  11. Clem, R. J. et al. Modulation of cell death by Bcl-XL through caspase interaction. Proc. Natl. Acad. Sci. USA 95, 554–559 (1998).

    Article  CAS  Google Scholar 

  12. Fang, G. et al. “Loop” domain is necessary for taxol-induced mobility shift and phosphorylation of Bcl-2 as well as for inhibiting taxol-induced cytosolic accumulation of cytochrome c and apoptosis. Cancer Res. 58, 3202–3208 (1998).

    CAS  Google Scholar 

  13. Fujita, N., Nagahashi, A., Nagashima, K., Rokudai, S. & Tsuruo, T. Acceleration of apoptotic cell death after the cleavage of Bcl-XL protein by caspase-3-like proteases. Oncogene 17, 1295–1304 (1998).

    Article  CAS  Google Scholar 

  14. Kirsch, D. G. et al. Caspase-3-dependent cleavage of Bcl-2 promotes release of cytochrome c. J. Biol. Chem. 274, 21155–21161 (1999).

    Article  CAS  Google Scholar 

  15. Srivastava, R. K., Mi, Q. S., Hardwick, J. M. & Longo, D. L. Deletion of the loop region of Bcl-2 completely blocks paclitaxel-induced apoptosis. Proc. Natl. Acad. Sci. USA 96, 3775–3780 (1999).

    Article  CAS  Google Scholar 

  16. Yamamoto, K., Ichijo, H. & Korsmeyer, S. J. BCL-2 is phosphorylated and inactivated by an ASK1/Jun N-terminal protein kinase pathway normally activated at G2/M. Mol. Cell. Biol. 19, 8469–8478 (1999).

    Article  CAS  Google Scholar 

  17. Kharbanda, S. et al. Translocation of SAPK/JNK to mitochondria and interaction with Bcl-xL in response to DNA damage. J. Biol. Chem. 275, 322–327 (2000).

    Article  CAS  Google Scholar 

  18. Basu, A. & Haldar, S. Identification of a novel Bcl-xL phosphorylation site regulating the sensitivity of taxol- or 2-methoxyestradiol-induced apoptosis. FEBS Lett. 538, 41–47 (2003).

    Article  CAS  Google Scholar 

  19. Du, L., Lyle, C. S. & Chambers, T. C. Characterization of vinblastine-induced Bcl-xL and Bcl-2 phosphorylation: evidence for a novel protein kinase and a coordinated phosphorylation/dephosphorylation cycle associated with apoptosis induction. Oncogene 24, 107–117 (2005).

    Article  CAS  Google Scholar 

  20. Schmitt, E., Beauchemin, M. & Bertrand, R. Nuclear colocalization and interaction between bcl-xL and cdk1(cdc2) during G2/M cell-cycle checkpoint. Oncogene 26, 5851–5865 (2007).

    Article  CAS  Google Scholar 

  21. Zhao, R. et al. DNA damage-induced Bcl-xL deamidation is mediated by NHE-1 antiport regulated intracellular pH. PLoS Biol. 5, e1 (2007).

    Article  Google Scholar 

  22. Asakura, T., Maeda, K., Omi, H., Matsudaira, H. & Ohkawa, K. The association of deamidation of Bcl-xL and translocation of Bax to the mitochondria through activation of JNK in the induction of apoptosis by treatment with GSH-conjugated DXR. Int. J. Oncol. 33, 389–395 (2008).

    CAS  Google Scholar 

  23. Upreti, M. et al. Identification of the major phosphorylation site in Bcl-xL induced by microtubule inhibitors and analysis of its functional significance. J. Biol. Chem. 283, 35517–35525 (2008).

    Article  CAS  Google Scholar 

  24. Wei, Y., Sinha, S. & Levine, B. Dual role of JNK1-mediated phosphorylation of Bcl-2 in autophagy and apoptosis regulation. Autophagy 4, 949–951 (2008).

    Article  CAS  Google Scholar 

  25. Tamura, Y. et al. Polo-like kinase 1 phosphorylates and regulates Bcl-xL during pironetin-induced apoptosis. Oncogene 28, 107–116 (2009).

    Article  CAS  Google Scholar 

  26. Terrano, D. T., Upreti, M. & Chambers, T. C. Cyclin-dependent kinase 1-mediated Bcl-xL/Bcl-2 phosphorylation acts as a functional link coupling mitotic arrest and apoptosis. Mol. Cell. Biol. 30, 640–656 (2010).

    Article  CAS  Google Scholar 

  27. Dho, S. H. et al. Control of cellular Bcl-xL levels by deamidation-regulated degradation. PLoS Biol. 11, e1001588 (2013).

    Article  CAS  Google Scholar 

  28. Bah, N. et al. Bcl-xL controls a switch between cell death modes during mitotic arrest. Cell Death Dis. 5, e1291 (2014).

    Article  CAS  Google Scholar 

  29. Kim, S. Y., Song, X., Zhang, L., Bartlett, D. L. & Lee, Y. J. Role of Bcl-xL/Beclin-1 in interplay between apoptosis and autophagy in oxaliplatin and bortezomib-induced cell death. Biochem. Pharmacol. 88, 178–188 (2014).

    Article  CAS  Google Scholar 

  30. Maiuri, M. C. et al. Functional and physical interaction between Bcl-XL and a BH3-like domain in Beclin-1. EMBO J. 26, 2527–2539 (2007).

    Article  CAS  Google Scholar 

  31. Tyler-Cross, R. & Schirch, V. Effects of amino acid sequence, buffers, and ionic strength on the rate and mechanism of deamidation of asparagine residues in small peptides. J. Biol. Chem. 266, 22549–22556 (1991).

    CAS  Google Scholar 

  32. Muchmore, S. W. et al. X-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death. Nature 381, 335–341 (1996).

    Article  CAS  Google Scholar 

  33. Sattler, M. et al. Structure of Bcl-xL-Bak peptide complex: recognition between regulators of apoptosis. Science 275, 983–986 (1997).

    Article  CAS  Google Scholar 

  34. Lee, E. F. et al. Crystal structure of ABT-737 complexed with Bcl-xL: implications for selectivity of antagonists of the Bcl-2 family. Cell Death Differ. 14, 1711–1713 (2007).

    Article  CAS  Google Scholar 

  35. Yao, Y. et al. Conformation of BCL-XL upon Membrane Integration. J. Mol. Biol. 427, 2262–2270 (2015).

    Article  CAS  Google Scholar 

  36. Yao, Y., Bobkov, A. A., Plesniak, L. A. & Marassi, F. M. Mapping the interaction of pro-apoptotic tBID with pro-survival BCL-XL. Biochemistry 48, 8704–8711 (2009).

    Article  CAS  Google Scholar 

  37. Wysoczanski, P. et al. NMR solution structure of a photoswitchable apoptosis activating Bak peptide bound to Bcl-xL. J. Am. Chem. Soc. 134, 7644–7647 (2012).

    Article  CAS  Google Scholar 

  38. Hagn, F., Klein, C., Demmer, O., Marchenko, N., Vaseva, A., Moll, U. M. & Kessler, H. BclxL changes conformation upon binding to wild-type but not mutant p53 DNA binding domain. J. Biol. Chem. 285, 3439–3450 (2010).

    Article  CAS  Google Scholar 

  39. Basu, A., DuBois, G. & Haldar, S. Posttranslational modifications of Bcl2 family members–a potential therapeutic target for human malignancy. Front. Biosci. 11, 1508–1521 (2006).

    Article  CAS  Google Scholar 

  40. Kutuk, O. & Letai, A. Regulation of Bcl-2 family proteins by posttranslational modifications. Curr. Mol. Med. 8, 102–118 (2008).

    Article  CAS  Google Scholar 

  41. Vermes, I., Haanen, C., Steffens-Nakken, H. & Reutelingsperger, C. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J. Immunol. Methods 184, 39–51 (1995).

    Article  CAS  Google Scholar 

  42. Czabotar, P. E. et al. Mutation to Bax beyond the BH3 domain disrupts interactions with pro-survival proteins and promotes apoptosis. J. Biol. Chem. 286, 7123–7131 (2011).

    Article  CAS  Google Scholar 

  43. Domina, A. M., Vrana, J. A., Gregory, M. A., Hann, S. R. & Craig, R. W. MCL1 is phosphorylated in the PEST region and stabilized upon ERK activation in viable cells, and at additional sites with cytotoxic okadaic acid or taxol. Oncogene 23, 5301–5315 (2004).

    Article  CAS  Google Scholar 

  44. Maurer, U., Charvet, C., Wagman, A. S., Dejardin, E. & Green, D. R. Glycogen synthase kinase-3 regulates mitochondrial outer membrane permeabilization and apoptosis by destabilization of MCL-1. Mol. Cell 21, 749–760 (2006).

    Article  CAS  Google Scholar 

  45. 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  Google Scholar 

  46. Follis, A. V., Llambi, F., Merritt, P., Chipuk, J. E., Green, D. R. & Kriwacki, R. W. Pin1-induced proline ssomerization in cytosolic p53 mediates BAX activation and apoptosis. Mol. Cell 59, 677–684 (2015).

    Article  CAS  Google Scholar 

  47. Chipuk, J. E. et al. Mechanism of apoptosis induction by inhibition of the anti-apoptotic BCL-2 proteins. Proc. Natl. Acad. Sci. USA 105, 20327–20332 (2008).

    Article  CAS  Google Scholar 

  48. Neidhardt, F. C., Bloch, P. L. & Smith, D. F. Culture medium for enterobacteria. J. Bacteriol. 119, 736–747 (1974).

    CAS  Google Scholar 

  49. Güntert, P. Automated NMR structure calculation with CYANA. Methods Mol. Biol. 278, 353–378 (2004).

    Google Scholar 

  50. Shen, Y., Delaglio, F., Cornilescu, G. & Bax, A. TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts. J. Biomol. NMR 44, 213–223 (2009).

    Article  CAS  Google Scholar 

  51. Brünger, A. T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921 (1998).

    Article  Google Scholar 

  52. Bhattacharya, A., Tejero, R. & Montelione, G. T. Evaluating protein structures determined by structural genomics consortia. Proteins 66, 778–795 (2007).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors acknowledge C.R. Grace (St. Jude Children’s Research Hospital, SJCRH) for assistance with NMR experiments and B. Cassell and P. Rodrigues (SJCRH) for peptide synthesis. This work was supported by NIH R01CA082491 and 1R01GM083159 (to R.W.K.); R01GM96208 (to D.R.G.); R01 CA179087 and R35 GM118186 (to F.M.M.); National Cancer Institute Cancer Center Support grants P30CA21765 (to SJCRH) and P30CA030199 (to S.B.P.); and ALSAC (to SJCRH). A.V.F. was the recipient of the Neoma Boadway Fellowship from SJCRH.

Author information

Authors and Affiliations

Authors

Contributions

A.V.F. designed experiments, performed experiments, analyzed data and wrote the manuscript; F.L. designed and performed cell experiments; H.K. designed experiments, performed experiments, and analyzed data; Y.Y. designed experiments, performed experiments, and analyzed data; A.H.P. designed experiments, performed experiments, and analyzed data; C.-G.P. and F.M.M. designed experiments and analyzed data; D.R.G. designed experiments and wrote the manuscript; and R.W.K. designed experiments, analyzed data and wrote the manuscript. All authors reviewed and edited the manuscript.

Corresponding author

Correspondence to Richard W. Kriwacki.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1–2, Supplementary Figures 1–10

Life Sciences Reporting Summary

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Follis, A.V., Llambi, F., Kalkavan, H. et al. Regulation of apoptosis by an intrinsically disordered region of Bcl-xL. Nat Chem Biol 14, 458–465 (2018). https://doi.org/10.1038/s41589-018-0011-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-018-0011-x

This article is cited by

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