Abstract
Short-term proteasome inhibition has been shown to prevent neuronal apoptosis. However, the key pro-survival proteins that must be degraded for triggering neuronal death are mostly unknown. Here, we show that Mcl-1, an anti-apoptotic Bcl-2 family member, is degraded by the proteasome during neuronal apoptosis. Using primary cultures of cerebellar granule neurons deprived of serum and KCl, we found that ubiquitination and proteasomal degradation of Mcl-1 depended on its prior phosphorylation by GSK3, providing the first insight into post-translational regulation of Mcl-1 in neurons. In a previous study, we have reported that the E3 ubiquitin-ligase Trim17 is both necessary and sufficient for neuronal apoptosis. Here, we identified Trim17 as a novel E3 ubiquitin-ligase for Mcl-1. Indeed, Trim17 co-immunoprecipitated with Mcl-1. Trim17 ubiquitinated Mcl-1 in vitro. Overexpression of Trim17 decreased the protein level of Mcl-1 in a phosphorylation- and proteasome-dependent manner. Finally, knock down of Trim17 expression reduced both ubiquitination and degradation of Mcl-1 in neurons. Moreover, impairment of Mcl-1 phosphorylation, by kinase inhibition or point mutations, not only decreased ubiquitination and degradation of Mcl-1, but also blocked the physical interaction between Trim17 and Mcl-1. As this stabilization of Mcl-1 increased its neuroprotective effect, our data strongly suggest that Trim17-mediated ubiquitination and degradation of Mcl-1 is necessary for initiating neuronal death.
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Abbreviations
- CGN:
-
cerebellar granule neurons
- GSK3:
-
Glycogen Synthase Kinase 3
- [KCl]o:
-
extracellular concentration of KCl
- K25:
-
serum-free medium containing 25 mM KCl
- K5:
-
serum-free medium containing 5 mM KCl
- JNK:
-
c-Jun N-terminal protein kinase
- WT:
-
wild type
- GFP:
-
green-fluorescent protein
- shRNA:
-
short hairpin RNA
- DIV:
-
days in vitro
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Acknowledgements
We would like to thank the staff of the Animal facilities of the IGMM for the breeding of mice and the RIO imaging platform for technical assistance. We also thank Dr. Joseph Opferman for providing mouse mcl-1 cDNA. We are grateful to Dr. Jonathan Ham and Pr. Jean-Claude Martinou for critical reading of the manuscript. This work was supported by the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé et de la Recherche Médicale (INSERM) and Université Montpellier 2.
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Magiera, M., Mora, S., Mojsa, B. et al. Trim17-mediated ubiquitination and degradation of Mcl-1 initiate apoptosis in neurons. Cell Death Differ 20, 281–292 (2013). https://doi.org/10.1038/cdd.2012.124
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DOI: https://doi.org/10.1038/cdd.2012.124
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