Abstract
We previously reported that gliotoxin (GT), the major virulence factor of the mold Aspergillus fumigatus causing invasive aspergillosis (IA) in immunocompromised patients, induces apoptosis in a Bak-dependent manner. The signaling pathway leading to Bak activation and subsequent mitochondrial outer membrane permeabilization (MOMP) is elusive. Here, we show that GT and the supernatant of A. fumigatus (but not its GT-defective mutant) activate the JNK pathway and require a co-operative JNK-mediated BimEL phosphorylation at three sites (S100, T112 and S114) to induce apoptosis in mouse fibroblasts, human bronchial and mouse alveolar epithelial cells. Cells (i) treated with the JNK inhibitor SP600125, (ii) deleted or knocked down for JNK1/2 or Bim or (iii) carrying the BimEL triple phosphomutant S100A/T112A/S114A instead of wild-type BimEL are similarly resistant to GT-induced apoptosis. Triple-phosphorylated BimEL is more stable, redistributes from a cytoskeletal to a membrane fraction, better interacts with Bcl-2 and Bcl-xL and more effectively activates Bak than the unphosphorylated mutant. These data indicate that JNK-mediated BimEL phosphorylation at S100, T112 and S114 constitutes a novel regulatory mechanism to activate Bim in response to apoptotic stimuli.
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Abbreviations
- GT:
-
gliotoxin
- MEF:
-
mouse embryo fibroblasts
- WT:
-
wild-type
- KO:
-
knockout
- DKO:
-
double knockout
- DTT:
-
dithiothreitol
- SDS-PAGE:
-
sodium dodecyl sulfate polyacrylamide gel electrophoresis
- DMEM:
-
Dulbecco’s minimal essential medium
- FCS:
-
fetal calf serum
- poly-HEMA:
-
poly-2-hydroxyethyal methacrylate
- PH:
-
poly-HEMA
- UV:
-
ultraviolet
- GFP:
-
green fluorescent protein
- PBS:
-
phosphate buffered saline
- IRES:
-
internal ribosome entry site
- EtOH:
-
ethanol
- RT-qPCR:
-
reverse transcription quantitative polymerase chain reaction
- IP:
-
immunoprecipitation
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Acknowledgements
We thank Georg Häcker, Freiburg, for the pMIG-BimEL plasmid and the FLAG-BimEL cDNA, Andreas Strasser, WEHI, Melbourne, for Bim KO, other BH3-only KO and Bax/Bak DKO MEFs, Jean-Claude Martinou, Geneva, for recombinant tBid and Bax and Janyce A. Sugui, NIH Bethesda, for the WT and mutant Aspergillus fumigatus strains. We thank Tilman Brummer, Freiburg, for his advice and encouragements. This work was supported by the Centre of Chronic Immunodeficiency (CCI) funded by the BMBF, Germany (to AG and CB), the Spemann Graduate School of Biology and Medicine (SGBM, GSC-4) funded by the Excellence Initiative of the German Federal and State Governments, Germany (to AG, FH and CB), by the Centre for Biological Signalling Studies (BIOSS, EXC-294) funded by the Excellence Initiative, Germany (to CB and UM) and by the Deutsche Forschungsgemeinschaft (DFG ID7/4-2, to MI). RJD is an investigator of the Howard Hughes Medical Institute.
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CB, UM and MMS conceived the project. AG, FH, DOF, KW carried out the experiments. RJD contributed the knockout/in mouse lines and the pT112 Bim antibody. MI contributed the primary alveolar epithelial type II cells. CB, AG and FH prepared the figures and wrote the manuscript.
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Geissler, A., Haun, F., Frank, D. et al. Apoptosis induced by the fungal pathogen gliotoxin requires a triple phosphorylation of Bim by JNK. Cell Death Differ 20, 1317–1329 (2013). https://doi.org/10.1038/cdd.2013.78
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DOI: https://doi.org/10.1038/cdd.2013.78
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