Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2001 Oct 15;359(Pt 2):345–352. doi: 10.1042/0264-6021:3590345

Survival-factor-induced phosphorylation of Bad results in its dissociation from Bcl-x(L) but not Bcl-2.

I Hirai 1, H G Wang 1
PMCID: PMC1222152  PMID: 11583580

Abstract

The pro-apoptotic Bcl-2-family protein Bad heterodimerizes with Bcl-2 and Bcl-x(L) in the outer mitochondrial membranes, nullifying their anti-apoptotic activities and promoting cell death. We report that interleukin-3 (IL-3) stimulation induces Bad phosphorylation and triggers its translocation from mitochondria to cytoplasm in cells expressing Bcl-x(L) but not Bcl-2. Overexpression of Bad sensitized Bcl-x(L)-expressing FL5.12 cells to apoptosis induced by IL-3 deprivation, but had no effect on the viability of cells expressing Bcl-2. IL-3 stimulation induced Bad phosphorylation at Ser-112, impairing its binding to Bcl-x(L) and resulting in its association with 14-3-3 proteins in the cytosol. However, Ser-112 phosphorylation could not trigger Bad dissociation from mitochondria in FL5.12 cells expressing Bcl-2. In 293T cells expressing Bcl-x(L), Bad was phosphorylated at three serines, 112, 136 and 155, and was largely localized in the cytosolic fraction. In contrast, overexpression of Bcl-2 prevented phosphorylation of Bad at Ser-136 and Ser-155, sequestering this protein in the mitochondrial membranes. When the N-terminal regions of Bcl-2 and Bcl-x(L) were swapped with each other, the Bcl-x(L)(N)-Bcl-2 chimaeric protein (containing the N-terminal region of Bcl-x(L)) failed to prevent Bad phosphorylation in cells and was unable to block the cytosolic distribution of this pro-apoptotic protein. Additional experiments with the Bcl-2(N)-Bcl-x(L) chimaeric protein (containing the N-terminal region of Bcl-2) indicated that, although the N-terminal region of Bcl-2 is necessary, it is not sufficient for sequestering Bad in the mitochondrial membranes. These observations suggest that growth-factor-mediated phosphorylation of Bad contributes to the cytoprotective function of Bcl-x(L) but not Bcl-2.

Full Text

The Full Text of this article is available as a PDF (311.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ayllón V., Martínez-A C., García A., Cayla X., Rebollo A. Protein phosphatase 1alpha is a Ras-activated Bad phosphatase that regulates interleukin-2 deprivation-induced apoptosis. EMBO J. 2000 May 15;19(10):2237–2246. doi: 10.1093/emboj/19.10.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blume-Jensen P., Janknecht R., Hunter T. The kit receptor promotes cell survival via activation of PI 3-kinase and subsequent Akt-mediated phosphorylation of Bad on Ser136. Curr Biol. 1998 Jun 18;8(13):779–782. doi: 10.1016/s0960-9822(98)70302-1. [DOI] [PubMed] [Google Scholar]
  3. Bonni A., Brunet A., West A. E., Datta S. R., Takasu M. A., Greenberg M. E. Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and -independent mechanisms. Science. 1999 Nov 12;286(5443):1358–1362. doi: 10.1126/science.286.5443.1358. [DOI] [PubMed] [Google Scholar]
  4. Breitschopf K., Haendeler J., Malchow P., Zeiher A. M., Dimmeler S. Posttranslational modification of Bcl-2 facilitates its proteasome-dependent degradation: molecular characterization of the involved signaling pathway. Mol Cell Biol. 2000 Mar;20(5):1886–1896. doi: 10.1128/mcb.20.5.1886-1896.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chang B. S., Minn A. J., Muchmore S. W., Fesik S. W., Thompson C. B. Identification of a novel regulatory domain in Bcl-X(L) and Bcl-2. EMBO J. 1997 Mar 3;16(5):968–977. doi: 10.1093/emboj/16.5.968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cheng E. H., Kirsch D. G., Clem R. J., Ravi R., Kastan M. B., Bedi A., Ueno K., Hardwick J. M. Conversion of Bcl-2 to a Bax-like death effector by caspases. Science. 1997 Dec 12;278(5345):1966–1968. doi: 10.1126/science.278.5345.1966. [DOI] [PubMed] [Google Scholar]
  7. Clem R. J., Cheng E. H., Karp C. L., Kirsch D. G., Ueno K., Takahashi A., Kastan M. B., Griffin D. E., Earnshaw W. C., Veliuona M. A. Modulation of cell death by Bcl-XL through caspase interaction. Proc Natl Acad Sci U S A. 1998 Jan 20;95(2):554–559. doi: 10.1073/pnas.95.2.554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Datta S. R., Brunet A., Greenberg M. E. Cellular survival: a play in three Akts. Genes Dev. 1999 Nov 15;13(22):2905–2927. doi: 10.1101/gad.13.22.2905. [DOI] [PubMed] [Google Scholar]
  9. Datta S. R., Dudek H., Tao X., Masters S., Fu H., Gotoh Y., Greenberg M. E. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell. 1997 Oct 17;91(2):231–241. doi: 10.1016/s0092-8674(00)80405-5. [DOI] [PubMed] [Google Scholar]
  10. Datta S. R., Katsov A., Hu L., Petros A., Fesik S. W., Yaffe M. B., Greenberg M. E. 14-3-3 proteins and survival kinases cooperate to inactivate BAD by BH3 domain phosphorylation. Mol Cell. 2000 Jul;6(1):41–51. [PubMed] [Google Scholar]
  11. Fuhlbrigge R. C., Fine S. M., Unanue E. R., Chaplin D. D. Expression of membrane interleukin 1 by fibroblasts transfected with murine pro-interleukin 1 alpha cDNA. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5649–5653. doi: 10.1073/pnas.85.15.5649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Harada H., Becknell B., Wilm M., Mann M., Huang L. J., Taylor S. S., Scott J. D., Korsmeyer S. J. Phosphorylation and inactivation of BAD by mitochondria-anchored protein kinase A. Mol Cell. 1999 Apr;3(4):413–422. doi: 10.1016/s1097-2765(00)80469-4. [DOI] [PubMed] [Google Scholar]
  13. Hsu Y. T., Youle R. J. Nonionic detergents induce dimerization among members of the Bcl-2 family. J Biol Chem. 1997 May 23;272(21):13829–13834. doi: 10.1074/jbc.272.21.13829. [DOI] [PubMed] [Google Scholar]
  14. Komatsu K., Miyashita T., Hang H., Hopkins K. M., Zheng W., Cuddeback S., Yamada M., Lieberman H. B., Wang H. G. Human homologue of S. pombe Rad9 interacts with BCL-2/BCL-xL and promotes apoptosis. Nat Cell Biol. 2000 Jan;2(1):1–6. doi: 10.1038/71316. [DOI] [PubMed] [Google Scholar]
  15. Lizcano J. M., Morrice N., Cohen P. Regulation of BAD by cAMP-dependent protein kinase is mediated via phosphorylation of a novel site, Ser155. Biochem J. 2000 Jul 15;349(Pt 2):547–557. doi: 10.1042/0264-6021:3490547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Muchmore S. W., Sattler M., Liang H., Meadows R. P., Harlan J. E., Yoon H. S., Nettesheim D., Chang B. S., Thompson C. B., Wong S. L. X-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death. Nature. 1996 May 23;381(6580):335–341. doi: 10.1038/381335a0. [DOI] [PubMed] [Google Scholar]
  17. Muslin A. J., Tanner J. W., Allen P. M., Shaw A. S. Interaction of 14-3-3 with signaling proteins is mediated by the recognition of phosphoserine. Cell. 1996 Mar 22;84(6):889–897. doi: 10.1016/s0092-8674(00)81067-3. [DOI] [PubMed] [Google Scholar]
  18. Neshat M. S., Raitano A. B., Wang H. G., Reed J. C., Sawyers C. L. The survival function of the Bcr-Abl oncogene is mediated by Bad-dependent and -independent pathways: roles for phosphatidylinositol 3-kinase and Raf. Mol Cell Biol. 2000 Feb;20(4):1179–1186. doi: 10.1128/mcb.20.4.1179-1186.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Oltvai Z. N., Milliman C. L., Korsmeyer S. J. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell. 1993 Aug 27;74(4):609–619. doi: 10.1016/0092-8674(93)90509-o. [DOI] [PubMed] [Google Scholar]
  20. Raff M. C. Social controls on cell survival and cell death. Nature. 1992 Apr 2;356(6368):397–400. doi: 10.1038/356397a0. [DOI] [PubMed] [Google Scholar]
  21. Salomoni P., Wasik M. A., Riedel R. F., Reiss K., Choi J. K., Skorski T., Calabretta B. Expression of constitutively active Raf-1 in the mitochondria restores antiapoptotic and leukemogenic potential of a transformation-deficient BCR/ABL mutant. J Exp Med. 1998 Jun 15;187(12):1995–2007. doi: 10.1084/jem.187.12.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sattler M., Liang H., Nettesheim D., Meadows R. P., Harlan J. E., Eberstadt M., Yoon H. S., Shuker S. B., Chang B. S., Minn A. J. Structure of Bcl-xL-Bak peptide complex: recognition between regulators of apoptosis. Science. 1997 Feb 14;275(5302):983–986. doi: 10.1126/science.275.5302.983. [DOI] [PubMed] [Google Scholar]
  23. Schürmann A., Mooney A. F., Sanders L. C., Sells M. A., Wang H. G., Reed J. C., Bokoch G. M. p21-activated kinase 1 phosphorylates the death agonist bad and protects cells from apoptosis. Mol Cell Biol. 2000 Jan;20(2):453–461. doi: 10.1128/mcb.20.2.453-461.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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 U S A. 1999 Mar 30;96(7):3775–3780. doi: 10.1073/pnas.96.7.3775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Vaux D. L., Korsmeyer S. J. Cell death in development. Cell. 1999 Jan 22;96(2):245–254. doi: 10.1016/s0092-8674(00)80564-4. [DOI] [PubMed] [Google Scholar]
  26. Virdee K., Parone P. A., Tolkovsky A. M. Phosphorylation of the pro-apoptotic protein BAD on serine 155, a novel site, contributes to cell survival. Curr Biol. 2000 Sep 21;10(18):1151–1154. doi: 10.1016/s0960-9822(00)00702-8. [DOI] [PubMed] [Google Scholar]
  27. Wang H. G., Millan J. A., Cox A. D., Der C. J., Rapp U. R., Beck T., Zha H., Reed J. C. R-Ras promotes apoptosis caused by growth factor deprivation via a Bcl-2 suppressible mechanism. J Cell Biol. 1995 May;129(4):1103–1114. doi: 10.1083/jcb.129.4.1103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wang H. G., Pathan N., Ethell I. M., Krajewski S., Yamaguchi Y., Shibasaki F., McKeon F., Bobo T., Franke T. F., Reed J. C. Ca2+-induced apoptosis through calcineurin dephosphorylation of BAD. Science. 1999 Apr 9;284(5412):339–343. doi: 10.1126/science.284.5412.339. [DOI] [PubMed] [Google Scholar]
  29. Wang H. G., Rapp U. R., Reed J. C. Bcl-2 targets the protein kinase Raf-1 to mitochondria. Cell. 1996 Nov 15;87(4):629–638. doi: 10.1016/s0092-8674(00)81383-5. [DOI] [PubMed] [Google Scholar]
  30. Wang H. G., Reed J. C. Mechanisms of Bcl-2 protein function. Histol Histopathol. 1998 Apr;13(2):521–530. doi: 10.14670/HH-13.521. [DOI] [PubMed] [Google Scholar]
  31. 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 G(2)/M. Mol Cell Biol. 1999 Dec;19(12):8469–8478. doi: 10.1128/mcb.19.12.8469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Yang E., Zha J., Jockel J., Boise L. H., Thompson C. B., Korsmeyer S. J. Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death. Cell. 1995 Jan 27;80(2):285–291. doi: 10.1016/0092-8674(95)90411-5. [DOI] [PubMed] [Google Scholar]
  33. Zha J., Harada H., Yang E., Jockel J., Korsmeyer S. J. Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-X(L) Cell. 1996 Nov 15;87(4):619–628. doi: 10.1016/s0092-8674(00)81382-3. [DOI] [PubMed] [Google Scholar]
  34. Zhou X. M., Liu Y., Payne G., Lutz R. J., Chittenden T. Growth factors inactivate the cell death promoter BAD by phosphorylation of its BH3 domain on Ser155. J Biol Chem. 2000 Aug 11;275(32):25046–25051. doi: 10.1074/jbc.M002526200. [DOI] [PubMed] [Google Scholar]
  35. del Peso L., González-García M., Page C., Herrera R., Nuñez G. Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science. 1997 Oct 24;278(5338):687–689. doi: 10.1126/science.278.5338.687. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES