Cellular and Molecular Life Sciences

, Volume 76, Issue 19, pp 3843–3859 | Cite as

Bcl-2 and IP3 compete for the ligand-binding domain of IP3Rs modulating Ca2+ signaling output

  • Hristina Ivanova
  • Larry E. WagnerII
  • Akihiko Tanimura
  • Elien Vandermarliere
  • Tomas Luyten
  • Kirsten Welkenhuyzen
  • Kamil J. Alzayady
  • Liwei Wang
  • Kozo Hamada
  • Katsuhiko Mikoshiba
  • Humbert De Smedt
  • Lennart Martens
  • David I. Yule
  • Jan B. Parys
  • Geert BultynckEmail author
Original Article


Bcl-2 proteins have emerged as critical regulators of intracellular Ca2+ dynamics by directly targeting and inhibiting the IP3 receptor (IP3R), a major intracellular Ca2+-release channel. Here, we demonstrate that such inhibition occurs under conditions of basal, but not high IP3R activity, since overexpressed and purified Bcl-2 (or its BH4 domain) can inhibit IP3R function provoked by low concentration of agonist or IP3, while fails to attenuate against high concentration of agonist or IP3. Surprisingly, Bcl-2 remained capable of inhibiting IP3R1 channels lacking the residues encompassing the previously identified Bcl-2-binding site (a.a. 1380–1408) located in the ARM2 domain, part of the modulatory region. Using a plethora of computational, biochemical and biophysical methods, we demonstrate that Bcl-2 and more particularly its BH4 domain bind to the ligand-binding domain (LBD) of IP3R1. In line with this finding, the interaction between the LBD and Bcl-2 (or its BH4 domain) was sensitive to IP3 and adenophostin A, ligands of the IP3R. Vice versa, the BH4 domain of Bcl-2 counteracted the binding of IP3 to the LBD. Collectively, our work reveals a novel mechanism by which Bcl-2 influences IP3R activity at the level of the LBD. This allows for exquisite modulation of Bcl-2’s inhibitory properties on IP3Rs that is tunable to the level of IP3 signaling in cells.


Inositol 1,4,5-trisphosphate receptor Calcium channels Protein binding Ligand–receptor interaction Ligand-binding domain Inhibition Mechanism of interaction 



The authors thank Anja Florizoone and Marina Crabbé for the excellent technical help. The authors are very grateful to Dr. Colin W. Taylor and Dr. Vera Konieczny (Department of Pharmacology, University of Cambridge, England, UK) and to Dr. Llewelyn Roderick (Department of Cardiovascular Sciences, KU Leuven, Belgium) for their assistance in preliminary work. The authors thank all members of the Lab. Molecular and Cellular Signaling in Leuven and Clark W. Distelhorst (Case Western Reserve University, Cleveland OH) for fruitful discussions. The work was supported by Grants from the Research Foundation-Flanders (FWO Grants 6.057.12, G.0819.13, G.0C91.14, G.0A34.16, G.0901.18), by the Research Council of the KU Leuven (OT Grant 14/101) and by the Interuniversity Attraction Poles Program (Belgian Science Policy; IAP-P7/13). GB, JBP and DIY are partners of the FWO Scientific Research Network (CaSign W0.019.17N). HI and EV are recipients of post-doctoral fellowships of the FWO. HI was supported by a mobility Grant from the FWO for a stay in the team of Dr. Yule (Rochester University, NY).

Supplementary material

18_2019_3091_MOESM1_ESM.docx (305 kb)
Supplementary material 1 (DOCX 304 kb)


  1. 1.
    Tsujimoto Y et al (1985) Involvement of the bcl-2 gene in human follicular lymphoma. Science 228(4706):1440–1443CrossRefGoogle Scholar
  2. 2.
    Vaux DL, Cory S, Adams JM (1988) Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature 335(6189):440–442CrossRefPubMedGoogle Scholar
  3. 3.
    Cuende E et al (1993) Programmed cell death by bcl-2-dependent and independent mechanisms in B lymphoma cells. EMBO J 12(4):1555–1560CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Czabotar PE et al (2014) Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy. Nat Rev Mol Cell Biol 15(1):49–63CrossRefGoogle Scholar
  5. 5.
    Brunelle JK, Letai A (2009) Control of mitochondrial apoptosis by the Bcl-2 family. J Cell Sci 122(Pt 4):437–441CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Cory S, Adams JM (2002) The Bcl2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer 2(9):647–656CrossRefPubMedGoogle Scholar
  7. 7.
    Gross A, McDonnell JM, Korsmeyer SJ (1999) BCL-2 family members and the mitochondria in apoptosis. Genes Dev 13(15):1899–1911CrossRefPubMedGoogle Scholar
  8. 8.
    Antonsson B et al (1997) Inhibition of Bax channel-forming activity by Bcl-2. Science 277(5324):370–372CrossRefPubMedGoogle Scholar
  9. 9.
    Barclay LA et al (2015) Inhibition of Pro-apoptotic BAX by a noncanonical interaction mechanism. Mol Cell 57(5):873–886CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Baffy G et al (1993) Apoptosis induced by withdrawal of interleukin-3 (IL-3) from an IL-3-dependent hematopoietic cell line is associated with repartitioning of intracellular calcium and is blocked by enforced Bcl-2 oncoprotein production. J Biol Chem 268(9):6511–6519PubMedGoogle Scholar
  11. 11.
    Vervliet T, Parys JB, Bultynck G (2016) Bcl-2 proteins and calcium signaling: complexity beneath the surface. Oncogene 35(39):5079–5092CrossRefPubMedGoogle Scholar
  12. 12.
    Pinton P, Rizzuto R (2006) Bcl-2 and Ca2+ homeostasis in the endoplasmic reticulum. Cell Death Differ 13(8):1409–1418CrossRefPubMedGoogle Scholar
  13. 13.
    Rong Y, Distelhorst CW (2008) Bcl-2 protein family members: versatile regulators of calcium signaling in cell survival and apoptosis. Annu Rev Physiol 70:73–91CrossRefPubMedGoogle Scholar
  14. 14.
    Chen R et al (2004) Bcl-2 functionally interacts with inositol 1,4,5-trisphosphate receptors to regulate calcium release from the ER in response to inositol 1,4,5-trisphosphate. J Cell Biol 166(2):193–203CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Hanson CJ et al (2008) Bcl-2 suppresses Ca2+ release through inositol 1,4,5-trisphosphate receptors and inhibits Ca2+ uptake by mitochondria without affecting ER calcium store content. Cell Calcium 44(3):324–338CrossRefPubMedGoogle Scholar
  16. 16.
    Rong YP et al (2008) Targeting Bcl-2-IP3 receptor interaction to reverse Bcl-2’s inhibition of apoptotic calcium signals. Mol Cell 31(2):255–265CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Monaco G et al (2012) Profiling of the Bcl-2/Bcl-XL-binding sites on type 1 IP3 receptor. Biochem Biophys Res Commun 428(1):31–35CrossRefPubMedGoogle Scholar
  18. 18.
    Monaco G et al (2012) Selective regulation of IP3-receptor-mediated Ca2+ signaling and apoptosis by the BH4 domain of Bcl-2 versus Bcl-Xl. Cell Death Differ 19(2):295–309CrossRefPubMedGoogle Scholar
  19. 19.
    Yoshikawa F et al (1999) Trypsinized cerebellar inositol 1,4,5-trisphosphate receptor. Structural and functional coupling of cleaved ligand binding and channel domains. J Biol Chem 274(1):316–327CrossRefPubMedGoogle Scholar
  20. 20.
    Maes K et al (2001) Mapping of the ATP-binding sites on inositol 1,4,5-trisphosphate receptor type 1 and type 3 homotetramers by controlled proteolysis and photoaffinity labeling. J Biol Chem 276(5):3492–3497CrossRefPubMedGoogle Scholar
  21. 21.
    Fan G et al (2015) Gating machinery of InsP3R channels revealed by electron cryomicroscopy. Nature 527(7578):336–341CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Eckenrode EF et al (2010) Apoptosis protection by Mcl-1 and Bcl-2 modulation of inositol 1,4,5-trisphosphate receptor-dependent Ca2+ signaling. J Biol Chem 285(18):13678–13684CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Rong YP et al (2009) The BH4 domain of Bcl-2 inhibits ER calcium release and apoptosis by binding the regulatory and coupling domain of the IP3 receptor. Proc Natl Acad Sci USA 106(34):14397–14402CrossRefPubMedGoogle Scholar
  24. 24.
    Rong YP et al (2009) Targeting Bcl-2 based on the interaction of its BH4 domain with the inositol 1,4,5-trisphosphate receptor. Biochim Biophys Acta 1793(6):971–978CrossRefPubMedGoogle Scholar
  25. 25.
    Ivanova H et al (2016) The trans-membrane domain of Bcl-2α, but not its hydrophobic cleft, is a critical determinant for efficient IP3 receptor inhibition. Oncotarget 7:55704–55720CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Fan G et al (2018) Cryo-EM reveals ligand induced allostery underlying InsP3R channel gating. Cell Res 28(12):1158–1170CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Zhong F et al (2006) Bcl-2 differentially regulates Ca2+ signals according to the strength of T cell receptor activation. J Cell Biol 172(1):127–137CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Suzuki J et al (2014) Imaging intraorganellar Ca2+ at subcellular resolution using CEPIA. Nat Commun 5:4153CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Vervliet T et al (2015) Regulation of the ryanodine receptor by anti-apoptotic Bcl-2 is independent of its BH3-domain-binding properties. Biochem Biophys Res Commun 463(3):174–179CrossRefPubMedGoogle Scholar
  30. 30.
    Bultynck G et al (2004) Thimerosal stimulates Ca2+ flux through inositol 1,4,5-trisphosphate receptor type 1, but not type 3, via modulation of an isoform-specific Ca2+-dependent intramolecular interaction. Biochem J 381(Pt 1):87–96CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Wagner LE, Yule DI (2012) Differential regulation of the InsP3 receptor type-1 and -2 single channel properties by InsP3, Ca2+ and ATP. J Physiol 590(Pt 14):3245–3259CrossRefPubMedGoogle Scholar
  32. 32.
    Alzayady KJ et al (2016) Defining the stoichiometry of inositol 1,4,5-trisphosphate binding required to initiate Ca2+ release. Sci Signal 9(422):ra35CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Missiaen L et al (2014) Measurement of intracellular Ca2+ release in intact and permeabilized cells using 45Ca2+. Cold Spring Harb Protoc 2014(3):263–270CrossRefPubMedGoogle Scholar
  34. 34.
    Pierce BG et al (2014) ZDOCK server: interactive docking prediction of protein–protein complexes and symmetric multimers. Bioinformatics 30(12):1771–1773CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Seo MD et al (2012) Structural and functional conservation of key domains in InsP3 and ryanodine receptors. Nature 483(7387):108–112CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Souers AJ et al (2013) ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets. Nat Med 19(2):202–208CrossRefPubMedGoogle Scholar
  37. 37.
    Mashiach E, Nussinov R, Wolfson HJ (2010) FiberDock: a web server for flexible induced-fit backbone refinement in molecular docking. Nucleic Acids Res 38(suppl_2):W457–W461CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Lin CC, Baek K, Lu Z (2011) Apo and InsP3-bound crystal structures of the ligand-binding domain of an InsP3 receptor. Nat Struct Mol Biol 18(10):1172–1174CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Monaco G et al (2013) Alpha-helical destabilization of the Bcl-2-BH4-domain peptide abolishes its ability to inhibit the IP3 receptor. PLoS One 8(8):e73386CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Fouque A et al (2016) The apoptotic members CD95, Bcl-Xl, and Bcl-2 cooperate to promote cell migration by inducing Ca2+ flux from the endoplasmic reticulum to mitochondria. Cell Death Differ 23(10):1702–1716CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Ivanova H et al (2017) The BH4 domain of Bcl-2 orthologues from different classes of vertebrates can act as an evolutionary conserved inhibitor of IP3 receptor channels. Cell Calcium 62:41–46CrossRefPubMedGoogle Scholar
  42. 42.
    Vervliet T et al (2014) Bcl-2 binds to and inhibits ryanodine receptors. J Cell Sci 127(Pt 12):2782–2792CrossRefPubMedGoogle Scholar
  43. 43.
    Takahashi S, Kinoshita T, Takahashi M (1994) Adenophostins A and B: potent agonists of inositol-1,4,5-trisphosphate receptor produced by Penicillium brevicompactum structure elucidation. J Antibiot (Tokyo) 47(1):95–100CrossRefGoogle Scholar
  44. 44.
    Sims CE, Allbritton NL (1998) Metabolism of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate by the oocytes of Xenopus laevis. J Biol Chem 273(7):4052–4058CrossRefPubMedGoogle Scholar
  45. 45.
    Yoshikawa F et al (1996) Mutational analysis of the ligand binding site of the inositol 1,4,5-trisphosphate receptor. J Biol Chem 271(30):18277–18284CrossRefPubMedGoogle Scholar
  46. 46.
    Oura T et al (2016) Highly sensitive measurement of inositol 1,4,5-trisphosphate by using a new fluorescent ligand and ligand binding domain combination. ChemBioChem 17(16):1509–1512CrossRefPubMedGoogle Scholar
  47. 47.
    Greenberg EF et al (2015) Synergistic killing of human small cell lung cancer cells by the Bcl-2-inositol 1,4,5-trisphosphate receptor disruptor BIRD-2 and the BH3-mimetic ABT-263. Cell Death Dis 6:e2034CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Greenberg EF, Lavik AR, Distelhorst CW (2014) Bcl-2 regulation of the inositol 1,4,5-trisphosphate receptor and calcium signaling in normal and malignant lymphocytes: potential new target for cancer treatment. Biochim Biophys Acta 1843(10):2205–2210CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Hamada K et al (2017) IP3-mediated gating mechanism of the IP3 receptor revealed by mutagenesis and X-ray crystallography. Proc Natl Acad Sci USA 114(18):4661–4666CrossRefPubMedGoogle Scholar
  50. 50.
    Bonneau B et al (2016) IRBIT controls apoptosis by interacting with the Bcl-2 homolog, Bcl2l10, and by promoting ER-mitochondria contact. Elife 5:e19896CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Bonneau B et al (2014) The Bcl-2 homolog Nrz inhibits binding of IP3 to its receptor to control calcium signaling during zebrafish epiboly. Sci Signal 7(312):ra14CrossRefPubMedGoogle Scholar
  52. 52.
    Nougarede A et al (2018) Breast cancer targeting through inhibition of the endoplasmic reticulum-based apoptosis regulator Nrh/BCL2L10. Cancer Res 78(6):1404–1417CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular MedicineLeuven Cancer Institute (LKI), KU LeuvenLeuvenBelgium
  2. 2.University of Rochester Medical Center School of Medicine and DentistryRochesterUSA
  3. 3.Department of Pharmacology, School of DentistryHealth Sciences University of HokkaidoHokkaidoJapan
  4. 4.Center for Medical Biotechnology, VIB-UGentGhentBelgium
  5. 5.Department of BiochemistryGhent UniversityGhentBelgium
  6. 6.Bioinformatics Institute GhentGhent UniversityGhentBelgium
  7. 7.Lab Developmental NeurobiologyRIKEN Brain Science InstituteSaitamaJapan
  8. 8.SIAIS (Shanghai Institute for Advanced Immunochemical Studies)ShanghaiTech UniversityShanghaiChina

Personalised recommendations