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Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death
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Phosphorylation of Bad is not essential for PKB-mediated survival signaling in hemopoietic cells

S W Wang1, T A Denny, U P Steinbrecher

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

Apoptosis : an International Journal on Programmed Cell Death
|April 22, 2005
PubMed

Insights

Cytokine-dependent cell survival does not require Bad phosphorylation. Specifically, the PI 3-kinase/PKB pathway activation can occur independently of Bad phosphorylation at Ser136.

Area of Science:

  • Cellular and Molecular Biology
  • Immunology
  • Biochemistry

Background:

  • The protein Bad plays a crucial role in preventing apoptosis, a process critical for cell survival.
  • Phosphorylated Bad is known to inhibit apoptosis, suggesting its phosphorylation is vital for cell survival signaling.
  • The PI 3-kinase (phosphatidylinositol 3-kinase) pathway is implicated in cell survival, and its role via PKB (Protein Kinase B) in Bad phosphorylation is under investigation.

Purpose of the Study:

  • To investigate the phosphorylation of Bad at key regulatory serine residues in hemopoietic cells dependent on cytokines.
  • To determine if the survival signaling effect of the PI 3-kinase pathway is mediated by PKB phosphorylation of Bad.
  • To examine the relationship between cytokine signaling, specific Bad phosphorylation sites, and cell survival.

Main Methods:

  • Overexpression of Bad and active PKB to assess Bad phosphorylation at Ser136.
  • Analysis of endogenous Bad phosphorylation at Ser136 in MC/9 mast cells and bone marrow-derived macrophages.
  • Stimulation of cells with cytokines (IL-3, GM-CSF, IL-4) to investigate Bad phosphorylation at Ser112 and Ser155 in relation to MEK/erk and PI 3-kinase pathways.
  • Assessment of ceramide's effect on Bad phosphorylation.

Main Results:

  • Overexpression of Bad or active PKB led to increased Bad phosphorylation at Ser136.
  • Endogenous Bad phosphorylation at Ser136 was not detected in MC/9 mast cells or murine bone marrow-derived macrophages.
  • Cytokines IL-3 and GM-CSF induced Bad phosphorylation at Ser112 and Ser155, associated with MEK/erk pathway activation.
  • IL-4, activating PI 3-kinase but not MEK/erk, did not induce phosphorylation at these sites, despite promoting cell survival.
  • Ceramide did not affect Bad phosphorylation status.

Conclusions:

  • Bad phosphorylation at major regulatory sites is not essential for cytokine-dependent cell survival.
  • Activation of the PI 3-kinase/PKB pathway can be dissociated from Bad phosphorylation at Ser136.
  • Specific cytokine-induced signaling pathways differentially regulate Bad phosphorylation sites, independent of overall cell survival outcomes.

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