Skip to main content
SpringerLink
Log in

Phosphorylation of Bad is not essential for PKB-mediated survival signaling in hemopoietic cells

  • Published:
Apoptosis Aims and scope Submit manuscript

Abstract

This study was designed to investigate Bad phosphorylation at several of its key regulatory Ser residues in cytokine-dependent hemopoietic cells. These studies were initiated in light of numerous studies that have reported a key role for phosphorylated Bad in preventing apoptosis. One key question is whether the survival signaling effect of the PI 3-kinase pathway is mediated by PKB phosphorylation of Bad. We confirm previous reports that if Bad is overexpressed or if active PKB is overexpressed, then the increased phosphorylation of Bad at Ser136 is apparent. However, we were unable to detect phosphorylation of endogenous Bad at Ser136 in the MC/9 mast cell line or in murine bone marrow-derived macrophages. On the other hand, phosphorylation of Bad at Ser112 and Ser155 was observed in response to IL-3 or GM-CSF, which activate the MEK/erk pathway, but not with IL-4, which activates the PI 3-kinase, but not the MEK/erk pathway, and also promotes cell survival. In contrast to previous reports, we found that ceramide had no effect on the phosphorylation status of Bad. In summary, our results suggest that Bad phosphorylation at any of the three major sites is not a required event for cytokine-dependent cell survival, and in particular, the activation of PI 3-kinase/PKB pathway can be dissociated from phosphorylation of Bad at Ser136.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Park JR. Cytokine regulation of apoptosis in hematopoietic precursor cells. Curr. Opinion in Hematology 1996; 3: 191.

    Article  CAS  Google Scholar 

  2. Lotem, J, Sachs L. Control of apoptosis in hematopoiesis and leukemia by cytokines, tumor suppressor and oncogenes. Leukemia 1996; 10: 925.

    CAS  PubMed  Google Scholar 

  3. Brach MA, deVos S, Gruss HJ, Herrmann F. Prolongation of survival of human polymorphonuclear neutrophils by granulocyte-macrophage colony-stimulating factor is caused by inhibition of programmed cell death. Blood 1992; 80: 2920.

    CAS  PubMed  Google Scholar 

  4. Scheid MP, Lauener RW, Duronio V. Role of phosphatidylinositol 3-OH-kinase activity in the inhibition of apoptosis in haemopoietic cells: Phosphatidylinositol 3-OH-kinase inhibitors reveal a difference in signalling between interleukin-3 and granulocyte-macrophage colony stimulating factor. Biochem J 1995; 312: 159.

    CAS  PubMed  Google Scholar 

  5. Webb PR, Wang, KQ, Scheel-Toellner D, Pongracz J, Salmon M, Lord JM. Regulation of neutrophil apoptosis: A role for protein kinase C and phosphatidylinositol-3-kinase. Apoptosis 2000; 5: 451.

    CAS  PubMed  Google Scholar 

  6. Minshall C, Arkins S, Freund GG, Kelley KW. Requirement for phosphatidylinositol 3′-kinase to protect hemopoietic progenitors against apoptosis depends upon the extracellular survival factor. J Immunol 1996; 156: 939.

    CAS  PubMed  Google Scholar 

  7. Dijkers PF, Birkenkamp KU, Lam EW, et al. FKHR-L1 can act as a critical effector of cell death induced by cytokine withdrawal: Protein kinase B-enhanced cell survival through maintenance of mitochondrial integrity. J Cell Biol 2002; 156: 531.

    CAS  PubMed  Google Scholar 

  8. Reed JC, Jurgensmeier JM, Matsuyama S. Bcl-2 family proteins and mitochondria. Biochim Biophys Acta 1998; 1366: 127.

    CAS  PubMed  Google Scholar 

  9. Korsmeyer SJ. BCL-2 gene family and the regulation of programmed cell death. Cancer Res 1999; 59: 1693.

    Google Scholar 

  10. Wei MC, Zong WX, Cheng EH, et al. Proapoptotic BAX and BAK: A requisite gateway to mitochondrial dysfunction and death. Science 2001; 292: 727.

    Article  CAS  PubMed  Google Scholar 

  11. Zong, WX, Lindsten T, Ross AJ, MacGregor GR, Thompson CB. BH3-only proteins that bind pro-survival Bcl-2 family members fail to induce apoptosis in the absence of Bax and Bak. Genes & Development 2001; 15: 1481.

    CAS  Google Scholar 

  12. Huang DCS, Strasser A. BH3-Only proteins—Essential initiators of apoptotic cell death. Cell 2000; 103: 839.

    CAS  PubMed  Google Scholar 

  13. Zha J, Harada H, Yang E, Jockel J, Korsmeyer SJ. Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-X(L). Cell 1996; 87: 619.

    CAS  PubMed  Google Scholar 

  14. del Peso L, Gonzalez-Garcia M, Page C, Herrera R, Nunez G. Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science 1997; 278: 687.

    CAS  PubMed  Google Scholar 

  15. Datta SR, Dudek H, Tao X, et al. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 1997; 91: 231.

    CAS  PubMed  Google Scholar 

  16. 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; 8: 779.

    CAS  PubMed  Google Scholar 

  17. Scheid MP, Schubert KM, Duronio V. Regulation of Bad phosphorylation and association with Bcl-xL by the MAPK/Erk kinase. J Biol Chem 1999; 274: 31108.

    CAS  PubMed  Google Scholar 

  18. Fang XJ, Yu SX, Eder A, et al. Regulation of BAD phosphorylation at serine 112 by the Ras-mitogen-activated protein kinase pathway. Oncogene 1999; 18: 6635.

    CAS  PubMed  Google Scholar 

  19. Shimamura A, Ballif BA, Richards SA, Blenis J. Rsk1 mediates a MEK-MAP kinase cell survival signal. Curr Biol 2000; 10: 127.

    CAS  PubMed  Google Scholar 

  20. Bonni A, Brunet A., West AE, Datta SR, Takasu MA, Greenberg ME. Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and -independent mechanisms. Science 1999; 286: 1358.

    CAS  PubMed  Google Scholar 

  21. Bertolotto C, Maulon L, Filippa N, Baier G, Auberger P. Protein kinase C theta and epsilon promote T-cell survival by a rsk-dependent phosphorylation and inactivation of BAD. J Biol Chem 2000; 275: 37246.

    CAS  PubMed  Google Scholar 

  22. Datta SR, Katsov A, Hu L, et al. 14-3-3 proteins and survival kinases cooperate to inactivate BAD by BH3 domain phosphorylation. Mol Cell 2000; 6: 41.

    CAS  PubMed  Google Scholar 

  23. Masters SC, Yang H, Datta SR, Greenberg ME, Fu H. 14-3-3 inhibits Bad-induced cell death through interaction with serine-136. Mol Pharmacol 2001; 60: 1325.

    CAS  PubMed  Google Scholar 

  24. Tan Y, Demeter MR, Ruan H, Comb MJ. BAD ser-155 phosphorylation regulates BAD/Bcl-XL interaction and cell survival. J Biol Chem 2000; 275: 25865.

    CAS  PubMed  Google Scholar 

  25. Zhou XM, Liu YM, Payne G, Lutz RJ, Chittenden T. Growth factors inactivate the cell death promoter BAD by phosphorylation of its BH3 domain on Ser(155). J Biol Chem 2000; 275: 25046.

    CAS  PubMed  Google Scholar 

  26. Lizcano JM, Morrice N, Cohen P. Regulation of BAD by cAMP-dependent protein kinase is mediated via phosphorylation of a novel site, Ser(155). Biochem J 2000; 349: 547.

    CAS  PubMed  Google Scholar 

  27. Valks DM, Cook SA, Pham FH, Morrison PR, Clerk A, Sugden PH. Phenylephrine promotes phosphorylation of Bad in cardiac myocytes through the extracellular signal-regulated kinases 1/2 and protein kinase A. J Mol Cell Cardiol 2002; 34: 749.

    CAS  PubMed  Google Scholar 

  28. Virdee K, Parone PA, Tolkovsky AM. Phosphorylation of the pro-apoptotic protein BAD on serine 155, a novel site, contributes to cell survival (vol. 10, p. 1151, 2000). Curr Biol 2000; 10: 1151.

    CAS  PubMed  Google Scholar 

  29. Virdee K, Parone PA, Tolkovsky AM. Phosphorylation of the pro-apoptotic protein BAD on serine 155, a novel site, contributes to cell survival. Curr Biol 2000; 10: 1151.

    CAS  PubMed  Google Scholar 

  30. Liu Y. Hepatocyte growth factor promotes renal epithelial cell survival by dual mechanisms. Am J Physiol 1999; 277: F624.

    CAS  PubMed  Google Scholar 

  31. Yano S, Tokumitsu H, Soderling TR. Calcium promotes cell survival through CaM-K kinase activation of the protein-kinase-B pathway. Nature 1998; 396: 584.

    CAS  PubMed  Google Scholar 

  32. Li WQ, Jiang Q, Khaled AR, Keller JR, Durum SK. Interleukin-7 inactivates the pro-apoptotic protein Bad promoting T cell survival. J Biol Chem 2004; 279: 29160.

    Article  CAS  PubMed  Google Scholar 

  33. Scheid MP, Duronio V. Dissociation of cytokine-induced phosphorylation of Bad and activation of PKB/akt: Involvement of MEK upstream of Bad phosphorylation. Proc Nat Acad Sci USA 1998; 95: 7439.

    CAS  PubMed  Google Scholar 

  34. Hermann C, Assmus B, Urbich C, Zeiher AM, Dimmeler S. Insulin-mediated stimulation of protein kinase Akt: A potent survival signaling cascade for endothelial cells. Arterioscler Thromb Vasc Biol 2000; 20: 402.

    CAS  PubMed  Google Scholar 

  35. Hinton HJ, Welham MJ. Cytokine-induced protein kinase B activation and bad phosphorylation do not correlate with cell survival of hemopoietic cells. J Immunol 1999; 162: 7002.

    CAS  PubMed  Google Scholar 

  36. Mabuchi S, Ohmichi M, Kimura A, et al. Inhibition of phosphorylation of BAD and Raf-1 by Akt sensitizes human ovarian cancer cells to paclitaxel. J Biol Chem 2002; 277: 33490.

    CAS  PubMed  Google Scholar 

  37. Tran NL, Adams DG, Vaillancourt RR, Heimark RL. Signal transduction from N-cadherin increases Bcl-2. Regulation of the phosphatidylinositol 3-kinase/Akt pathway by homophilic adhesion and actin cytoskeletal organization. J Biol Chem 2002; 277: 32905.

    CAS  PubMed  Google Scholar 

  38. Jin Z, Gao F, Flagg T, Deng X. Nicotine induces multi-site phosphorylation of Bad in association with suppression of apoptosis. J Biol Chem 2004; 279: 23837.

    CAS  PubMed  Google Scholar 

  39. Wang HG, Pathan N, Ethell IM, et al. Ca2 +-induced apoptosis through calcineurin dephosphorylation of BAD. Science 1999; 284: 339.

    CAS  PubMed  Google Scholar 

  40. Ayllon V, Martinez AC, Garcia A, Cayla X, Rebollo A. Protein phosphatase 1alpha is a Ras-activated Bad phosphatase that regulates interleukin-2 deprivation-induced apoptosis. EMBO J 2000; 19: 2237.

    CAS  PubMed  Google Scholar 

  41. Chiang CW, Harris G, Ellig C, et al. Protein phosphatase 2A activates the proapoptotic function of BAD in interleukin- 3-dependent lymphoid cells by a mechanism requiring 14-3-3 dissociation. Blood 2001; 97: 1289.

    CAS  PubMed  Google Scholar 

  42. Kolesnick RN, Kronke M. Regulation of ceramide production and apoptosis. Ann Rev.0 Physiol 1998; 60: 643.

    CAS  Google Scholar 

  43. Pettus BJ, Chalfant CE, Hannun YA. Ceramide in apoptosis: an overview and current perspectives. Biochim Biophys Acta 2002; 1585: 114.

    CAS  PubMed  Google Scholar 

  44. Schubert KM, Scheid MP, Duronio V. Ceramide inhibits protein kinase B/Akt by promoting dephosphorylation of serine 473. J Biol Chem 2000; 275: 13330.

    CAS  PubMed  Google Scholar 

  45. Basu S, Bayoumy S, Zhang Y, Lozano J, Kolesnick R. BAD enables ceramide to signal apoptosis via Ras and Raf-1. J Biol Chem 1998; 273: 30419.

    CAS  PubMed  Google Scholar 

  46. Stoica BA, Movsesyan VA, Lea PMT, Faden AI. Ceramide-induced neuronal apoptosis is associated with dephosphorylation of Akt, BAD, FKHR, GSK-3beta, and induction of the mitochondrial-dependent intrinsic caspase pathway. Mol Cell Neurosci 2003; 22: 365.

    CAS  PubMed  Google Scholar 

  47. Dramsi S, Scheid MP, Maiti A, et al. Identification of a novel phosphorylation site, Ser-170, as a regulator of bad pro-apoptotic activity. J Biol Chem 2002; 277: 6399.

    CAS  PubMed  Google Scholar 

  48. Kohn AD, Barthel A, Kovacina KS, et al. Construction and characterization of a conditionally active version of the serine/threonine kinase Akt. J Biol Chem 1998; 273: 11937.

    CAS  PubMed  Google Scholar 

  49. Hundal RS, Salh BS, Schrader JW, Gomez-Munoz A, Duronio V, Steinbrecher UP. Oxidized low density lipoprotein inhibits macrophage apoptosis through activation of the PI 3-kinase/PKB pathway. J Lipid Res 2001; 42: 1483.

    CAS  PubMed  Google Scholar 

  50. Duronio V, Clark-Lewis I, Federsppiel B, Wieler JS, Schrader JW. Tyrosine phosphorylation of receptor beta subunits and common substrates in response to interleukin-3 and granulocyte-macrophage colony-stimulating factor. J Biol Chem 1992; 267: 21856.

    CAS  PubMed  Google Scholar 

  51. Welham MJ, Duronio V, Leslie KB, Bowtell D, Schrader JW. Multiple hemopoietins, with the exception of interleukin-4, induce modification of Shc and mSos1, but not their translocation. J Biol Chem 1994; 269: 21165.

    CAS  PubMed  Google Scholar 

  52. Welham MJ, Duronio V, Sanghera JS, Pelech SL, Schrader JW. Multiple hemopoietic growth factors stimulate activation of mitogen-activated protein kinase family members. J Immunol 1992; 149: 1683.

    CAS  PubMed  Google Scholar 

  53. Gold MR, Duronio V, Saxena SP, Schrader JW, Aebersold R. Multiple cytokines activate phosphatidylinositol 3-kinase in hemopoietic cells. Association of the enzyme with various tyrosine-phosphorylated proteins. J Biol Chem 1994; 269: 5403.

    CAS  PubMed  Google Scholar 

  54. Scheid MP, Foltz IN, Young PR, Schrader JW, Duronio V. Ceramide and cyclic adenosine monophosphate (cAMP) induce cAMP response element binding protein phosphorylation via distinct signaling pathways while having opposite effects on myeloid cell survival. Blood 1999; 93: 217.

    CAS  PubMed  Google Scholar 

  55. Hundal RS, Gomez-Munoz A, Kong JY, et al. Oxidized low density lipoprotein inhibits macrophage apoptosis by blocking ceramide generation, thereby maintaining protein kinase B activation and Bcl-XL levels. J Biol Chem 2003; 278: 24399.

    CAS  PubMed  Google Scholar 

  56. Datta SR, Ranger AM, Lin MZ, et al. Survival factor-mediated BAD phosphorylation raises the mitochondrial threshold for apoptosis. Dev Cell 2002; 3: 631.

    CAS  PubMed  Google Scholar 

  57. Ranger AM, Zha J, Harada H, et al. Bad-deficient mice develop diffuse large B cell lymphoma. Proc Natl Acad Sci USA 2003; 100: 9324.

    PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Medicine, University of British Columbia and Vancouver Coastal Health Research Institute, Jack Bell Research Centre, 2660 Oak St., Vancouver, BC, Canada, V6H 3Z6

    S. W. Wang, T. A. Denny, U. P. Steinbrecher & V. Duronio

  2. Jack Bell Research Centre, 2660 Oak St., Room 255, Vancouver, BC, Canada, V6H 3Z6

    V. Duronio

Authors
  1. S. W. Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. T. A. Denny
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. U. P. Steinbrecher
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. V. Duronio
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to V. Duronio.

Additional information

Supported by grants from the Cancer Research Society Inc. and the Heart and Stroke Foundation of Canada.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, S.W., Denny, T.A., Steinbrecher, U.P. et al. Phosphorylation of Bad is not essential for PKB-mediated survival signaling in hemopoietic cells. Apoptosis 10, 341–348 (2005). https://doi.org/10.1007/s10495-005-0808-4

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10495-005-0808-4

Keywords

Search

Navigation