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

I Hirai1, H G Wang

  • 1Drug Discovery Program, H. Lee Moffitt Cancer Center and Research Institute, Department of Interdisciplinary Oncology, University of South Florida College of Medicine, 12902 Magnolia Drive, Tampa FL 33612, USA.

The Biochemical Journal
|October 5, 2001
PubMed

Insights

Interleukin-3 (IL-3) stimulation causes phosphorylation of the pro-apoptotic protein Bad, moving it to the cytoplasm in cells with Bcl-x(L) but not Bcl-2. This phosphorylation is key to Bcl-x(L)

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Apoptosis Research

Background:

  • The Bcl-2 protein family regulates apoptosis, with anti-apoptotic members like Bcl-2 and Bcl-x(L) inhibiting cell death.
  • The pro-apoptotic protein Bad heterodimerizes with Bcl-2 and Bcl-x(L), neutralizing their function and promoting apoptosis.
  • Interleukin-3 (IL-3) is a growth factor that promotes cell survival, particularly in hematopoietic cells.

Purpose of the Study:

  • To investigate the differential effects of Bcl-2 and Bcl-x(L) on the regulation of the pro-apoptotic protein Bad.
  • To elucidate the role of Bad phosphorylation and subcellular localization in mediating the cytoprotective effects of growth factors like IL-3.
  • To determine how the interaction between Bad and Bcl-x(L) versus Bcl-2 is modulated by IL-3 stimulation.

Main Methods:

  • Cell culture experiments using FL5.12 and 293T cell lines.
  • Overexpression of Bad, Bcl-2, Bcl-x(L), and chimeric proteins.
  • Analysis of Bad phosphorylation status using Western blotting.
  • Assessment of Bad subcellular localization via cell fractionation and Western blotting.
  • Induction of apoptosis via IL-3 deprivation.

Main Results:

  • IL-3 stimulation induced Bad phosphorylation at Ser-112, leading to its translocation from mitochondria to the cytoplasm in Bcl-x(L)-expressing cells.
  • Bad overexpression sensitized Bcl-x(L)-expressing cells to IL-3 deprivation-induced apoptosis, while having no effect on Bcl-2-expressing cells.
  • Bcl-2 expression prevented Bad phosphorylation at Ser-136 and Ser-155, sequestering Bad in mitochondrial membranes, unlike Bcl-x(L).
  • Chimeric proteins revealed that the N-terminal region of Bcl-x(L) is crucial for mediating IL-3-induced Bad phosphorylation and cytosolic localization.

Conclusions:

  • Growth factor-mediated phosphorylation of Bad is a critical mechanism contributing to the cytoprotective function of Bcl-x(L).
  • Bcl-2 sequesters Bad in the mitochondria, preventing its phosphorylation and subsequent pro-apoptotic signaling, independent of IL-3.
  • The differential regulation of Bad by Bcl-x(L) and Bcl-2 highlights distinct pathways controlling cell survival and apoptosis in response to growth factor signaling.

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