Abstract
Forkhead box O3 (FOXO3) is a multispecific transcription factor that is responsible for multiple and conflicting transcriptional programs such as cell survival and apoptosis. The protein is heavily post-translationally modified and there is considerable evidence that post-transcriptional modifications (PTMs) regulate protein stability and nuclear–cytosolic translocation. Much less is known about how FOXO3 PTMs determine the specificity of its transcriptional program. In this study we demonstrate that exposure of hepatocytes to ethanol or exposure of macrophages to lipopolysaccharide (LPS) induces the c-Jun N-terminal kinase (JNK)-dependent phosphorylation of FOXO3 at serine-574. Chromatin immunoprecipitation (ChIP), mRNA and protein measurements demonstrate that p-574-FOXO3 selectively binds to promoters of pro-apoptotic genes but not to other well-described FOXO3 targets. Both unphosphorylated and p-574-FOXO3 bound to the B-cell lymphoma 2 (Bcl-2) promoter, but the unphosphorylated form was a transcriptional activator, whereas p-574-FOXO3 was a transcriptional repressor. The combination of increased TRAIL (TNF-related apoptosis-inducing ligand) and decreased Bcl-2 was both necessary and sufficient to induce apoptosis. LPS treatment of a human monocyte cell line (THP-1) induced FOXO3 S-574 phosphorylation and apoptosis. LPS-induced apoptosis was prevented by knockdown of FOXO3. It was restored by overexpressing wild-type FOXO3 but not by overexpressing a nonphosphorylatable S-574A FOXO3. Expression of an S-574D phosphomimetic form of FOXO3 induced apoptosis even in the absence of LPS. A similar result was obtained with mouse peritoneal macrophages where LPS treatment increased TRAIL, decreased Bcl-2 and induced apoptosis in wild-type but not FOXO3−/− cells. This work thus demonstrates that S-574 phosphorylation generates a specifically apoptotic form of FOXO3 with decreased transcriptional activity for other well-described FOXO3 functions.
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Abbreviations
- FOXO3:
-
forkhead box O3
- PTM:
-
post-transcriptional modification
- LPS:
-
lipopolysaccharide
- ChIP:
-
chromatin immunoprecipitation
- TRAIL:
-
TNF-related apoptosis-inducing ligand
- Bcl-2:
-
B-cell lymphoma 2
- PrxIII:
-
peroxiredoxin III
- SOD2:
-
superoxide dismutase 2
- Bim:
-
B-cell lymphoma 2 interacting mediator of cell death
- TUNEL:
-
terminal deoxynucleotidyl transferase dUTP nick end labeling
- LDH:
-
lactate dehydrogenase
- JNK:
-
c-Jun N-terminal kinase
- PUMA:
-
p53 upregulated modulator of apoptosis
- Noxa:
-
phorbol-12-myristate-13-acetate-induced protein 1
- ADH:
-
alcohol dehydrogenase
- HCV:
-
hepatitis C virus
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Acknowledgements
This study was supported by grant AA012863 from the National Institute on Alcoholism and Alcohol Abuse, grants P20 GM103418 and P20 GM103549 from the National Institute of General Medical Sciences from the National Institutes of Health and a fellowship grant from the Biomedical Research Training Program of the University of Kansas Medical Center to ZL. The human hepatocytes used in this study were derived from samples provided by the University of Kansas Liver Center Tissue Bank. We thank Drs. Jameson Forster, Tim Schmitt and Bashar Abdulkarim for their assistance in obtaining these specimens and Dr. Michele Pritchard for helpful discussions. Supplementary Information accompanies this paper on Cell Death and Differentiation website (http://www.nature.com/cdd).
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Li, Z., Zhao, J., Tikhanovich, I. et al. Serine 574 phosphorylation alters transcriptional programming of FOXO3 by selectively enhancing apoptotic gene expression. Cell Death Differ 23, 583–595 (2016). https://doi.org/10.1038/cdd.2015.125
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DOI: https://doi.org/10.1038/cdd.2015.125
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