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Proteomic and biochemical analysis of 14‐3‐3‐binding proteins during C2‐ceramide‐induced apoptosis
Author(s) -
PozueloRubio Mercedes
Publication year - 2010
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2010.07730.x
Subject(s) - apoptosis , ceramide , biology , microbiology and biotechnology , programmed cell death , inhibitor of apoptosis domain , gene isoform , tandem affinity purification , calmodulin , affinity chromatography , biochemistry , caspase , gene , enzyme
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 14‐3‐3ζ 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. Structured digital abstract• A list of the large number of protein–protein interactions described in this article is available via the MINT article ID MINT‐7899808