Abstract
Prostate apoptosis response protein 4 (Par-4) also known as PRKC apoptosis WT1 regulator is a tumor suppressor that selectively induces apoptosis in cancer cells. However, its post-translational regulation by ubiquitin-mediated proteolysis and the cellular machinery that is responsible for its proteasomal degradation are unknown. Using immunopurification and an unbiased mass spectrometry-based approach, we show that Par-4 interacts with the SPRY-domain containing E3 ubiquitin ligase Fbxo45 through a short consensus sequence motif. Fbxo45 interacts with Par-4 in the cytoplasm and mediates its ubiquitylation and proteasomal degradation. Fbxo45 silencing results in stabilization of Par-4 with increased apoptosis. Importantly, a Par-4 mutant that is unable to bind Fbxo45 is stabilized and further enhances staurosporine-induced apoptosis. Co-expression of Fbxo45 with Par-4 protects cancer cells against Par-4-induced apoptosis. Our studies reveal that Fbxo45 is the substrate-receptor subunit of a functional E3 ligase for Par-4 that has a critical role in cancer cell survival.
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Abbreviations
- Par-4:
-
Prostate apoptosis response protein 4
- PAWR:
-
PRKC apoptosis WT1 regulator
- PAM:
-
protein-associated with myc
- SCF:
-
Skp1, CUL1, F-box protein
- MYCBP2:
-
myc-binding protein 2
- NLS:
-
nuclear localization sequence
- LC-MS/MS:
-
liquid chromatography-tandem mass spectrometry
- mES:
-
mouse embryonic stem
- SOCS:
-
suppressor of cytokine signaling
- WCE:
-
whole-cell extract
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Acknowledgements
We thank Dr. Joon-Young Ahn for his assistance with the characterization of the anti-Fbxo45 polyclonal antibody. This work was funded by NIH grants R01 DE119249 and R01 CA136905 to KSJE-J and R01 CA140806 to MSL
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Chen, X., Sahasrabuddhe, A., Szankasi, P. et al. Fbxo45-mediated degradation of the tumor-suppressor Par-4 regulates cancer cell survival. Cell Death Differ 21, 1535–1545 (2014). https://doi.org/10.1038/cdd.2014.92
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DOI: https://doi.org/10.1038/cdd.2014.92
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