Abstract
Bfl-1/A1 is generally recognized as a Bcl-2-related inhibitor of apoptosis. We show that Bfl-1 undergoes constitutive ubiquitin/proteasome-mediated turnover. Moreover, while Bfl-1 suppresses apoptosis induced by staurosporine or cytokine withdrawal, it is proapoptotic in response to tumor necrosis factor (TNF) receptor activation in FL5.12 pro-B cells. Its anti- versus proapoptotic effect is regulated by two proteolytic events: (1) its constitutive proteasome-mediated turnover and (2) its TNF/cycloheximide (CHX)-induced cleavage by μ-calpain, or a calpain-like activity, coincident with acquisition of a proapoptotic phenotype. In vitro studies suggest that calpain-mediated cleavage of Bfl-1 occurs between its Bcl-2 homology (BH)4 and BH3 domains. This would be consistent with the generation of a proapoptotic Bax-like BH1–3 molecule. Overall, our studies uncovered two new regulatory mechanisms that play a decisive role in determining Bfl-1's prosurvival versus prodeath activities. These findings might provide important clues to counteract chemoresistance in tumor cells that highly express Bfl-1.
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Abbreviations
- STS:
-
staurosporine
- TNF:
-
tumor necrosis factor
- BH:
-
Bcl-2 homology
- TM:
-
transmembrane
- CHX:
-
cycloheximide
- zVAD-fmk:
-
carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone
- GFP:
-
green fluorescent protein
- ALLN:
-
N-acetyl-L-leucyl-L-leucyl-norleucinal
- ALLM:
-
N-acetyl-L-leucyl-L-leucyl-methioninal
- IL-3:
-
interleukin-3
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Acknowledgements
We thank C Labrie, J-Y Springael, E White and R Sundararajan for the gifts of cells and reagents. We are very grateful to E White, R Sundararajan and members of the Gélinas laboratory for discussion and suggestions during the course of this work, to C Gauthier-Rouvière for allowing some experiments to be carried out in her laboratory, and to Petra Pham and Hyejeong Choi for help with FACS analysis. We also thank E White, Y Fan, J Dutta, N Gupta and G Fan for helpful comments on the manuscript. This work was supported by Public Health Service Grant CA83937 from the National Cancer Institute to CG. We acknowledge partial support from the Flow Cytometry Core Facility of the Cancer Institute of New Jersey. JFK was a postdoctoral fellow of the New Jersey Commission on Cancer Research and The Foundation of the UMDNJ. MJS was partially supported by NIH predoctoral training grant in Biochemistry and Molecular Biology GM08360.
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Kucharczak, J., Simmons, M., Duckett, C. et al. Constitutive proteasome-mediated turnover of Bfl-1/A1 and its processing in response to TNF receptor activation in FL5.12 pro-B cells convert it into a prodeath factor. Cell Death Differ 12, 1225–1239 (2005). https://doi.org/10.1038/sj.cdd.4401684
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DOI: https://doi.org/10.1038/sj.cdd.4401684
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