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Underphosphorylated BAD interacts with diverse antiapoptotic Bcl-2 family proteins to regulate apoptosis

J Bae1, S Y Hsu, C P Leo

  • 1Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, CA 94305-5317, USA.

Apoptosis : an International Journal on Programmed Cell Death
|August 3, 2001
PubMed

Insights

The proapoptotic protein BAD, crucial for cell death, interacts with antiapoptotic Bcl-2 proteins. This interaction, conserved across species, is regulated by BAD phosphorylation and helps control apoptosis.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • Survival factors regulate apoptosis by activating kinases that phosphorylate the proapoptotic protein BAD.
  • Phosphorylated BAD binds to 14-3-3 proteins, inhibiting BAD-mediated apoptosis.
  • The interaction between BAD and anti- or proapoptotic Bcl-2 family proteins requires systematic analysis.

Purpose of the Study:

  • To systematically analyze the interaction between BAD and various antiapoptotic Bcl-2 proteins.
  • To investigate the role of BAD phosphorylation in its interaction with Bcl-2 family members.
  • To determine the evolutionary conservation of BAD's interaction domains.

Main Methods:

  • Yeast two-hybrid protein interaction assays were employed to study BAD and Bcl-2 protein interactions.
  • Nonphosphorylated recombinant BAD expressed in bacteria was used to assess direct interactions.
  • Apoptosis assays involving coexpression of BAD with Bcl-2 family members were performed.
  • Comparative analysis of BAD orthologs from zebrafish to human was conducted.

Main Results:

  • BAD exhibited negligible interaction with proapoptotic Bcl-2 proteins.
  • Underphosphorylated BAD interacted with all tested antiapoptotic Bcl-2 proteins (Bcl-2, Bcl-w, Bcl-xL, Bfl-1/A1, Mcl-1, Ced-9, BHRF-1).
  • Direct interactions between recombinant BAD and antiapoptotic Bcl-2 members were confirmed.
  • Coexpression with Bcl-2, Bcl-w, and Bfl-1 blocked BAD-induced apoptosis.
  • The 14-3-3 binding site and BH3 domain of BAD are conserved from zebrafish to humans.

Conclusions:

  • The highly conserved BAD protein interacts with diverse antiapoptotic Bcl-2 family members to regulate apoptosis.
  • BAD phosphorylation status critically influences its interaction with antiapoptotic Bcl-2 proteins.
  • These findings elucidate a conserved mechanism for apoptosis regulation involving BAD and Bcl-2 family proteins.

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