Proteomic and biochemical analysis of 14-3-3-binding proteins during C2-ceramide-induced apoptosis

FEBS J. 2010 Aug;277(16):3321-42. doi: 10.1111/j.1742-4658.2010.07730.x. Epub 2010 Jul 8.


14-3-3 is a family of proteins comprising several isoforms that, in many cases, promote cell survival by association with proapoptotic proteins. This study was designed to obtain further understanding of the 14-3-3 role in apoptosis regulation, by analyzing apoptosis-related protein-14-3-3 interactions. Western blot analysis of an eluted fraction from the 14-3-3-affinity chromatography column identified proapoptotic proteins as receptor-interacting protein 3 and Bcl-2-antagonist/killer as new phophorylation-dependent 14-3-3-binding proteins under physiological conditions. The apoptosis inducer C2-ceramide promoted decay of the 14-3-3-binding signal of protein cell extracts. Investigation of the role of 14-3-3 in C2-ceramide-induced apoptosis showed that depletion of the 14-3-3zeta isoform sensitized to cell death, whereas overexpression of this isoform delayed cell death. A combination of tandem affinity purification and liquid chromatography-tandem MS techniques identified 15 proteins involved in cell survival processes whose 14-3-3-binding status changed during C2-ceramide-induced apoptosis. Under physiological conditions, desmin was clearly identified as a new 14-3-3-interactor protein, and vasodilator-stimulated phosphoprotein, nucleophosmin and calmodulin, whose 14-3-3 binding was suggested by others on the basis of MS analysis, were confirmed here as phosphorylation-dependent 14-3-3-associated proteins. Interestingly, proteins related to the regulation of DNA double-strand break repair in the early stages of apoptosis, such as DNA-dependent protein kinase, or the regulation of cell shrinkage during apoptosis, such as vasodilator-stimulated phosphoprotein and death promoters like receptor-interacting protein 3, were identified as 14-3-3-associated proteins whose 14-3-3-binding status changed when apoptosis was initiated. The functional diversity of these identified proteins suggests that 14-3-3 may regulate the apoptotic process through new mechanisms, in addition to others previously characterized.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 14-3-3 Proteins / chemistry
  • 14-3-3 Proteins / genetics
  • 14-3-3 Proteins / isolation & purification
  • 14-3-3 Proteins / metabolism*
  • Apoptosis / drug effects*
  • Apoptosis / physiology*
  • Blotting, Western
  • Caspase 8 / metabolism
  • Cell Line, Tumor
  • Enzyme Inhibitors / pharmacology*
  • Gene Expression Regulation* / drug effects
  • Gene Knockdown Techniques
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • HeLa Cells
  • Humans
  • Mitochondria / metabolism
  • Protein Binding
  • Proteomics*
  • RNA, Small Interfering
  • Reproducibility of Results
  • Sphingosine / analogs & derivatives*
  • Sphingosine / pharmacology


  • 14-3-3 Proteins
  • Enzyme Inhibitors
  • N-acetylsphingosine
  • RNA, Small Interfering
  • Green Fluorescent Proteins
  • Caspase 8
  • Sphingosine