Abstract
The serine threonine kinase checkpoint kinase 2 (CHK2) is a DNA damage checkpoint protein important for the ATM-p53 signaling pathway. In addition to its phosphorylation, CHK2 is also ubiquitylated, and both post-translational modifications are important for its function. However, although the mechanisms that regulate CHK2 phosphorylation are well established, those that control its ubiquitylation are not fully understood. In this study, we demonstrate that the ubiquitin E3 ligase PIRH2 (p53-induced protein with a RING (Really Interesting New Gene)-H2 domain) interacts with CHK2 and mediates its polyubiquitylation and proteasomal degradation. We show that the deubiquitylating enzyme USP28 forms a complex with PIRH2 and CHK2 and antagonizes PIRH2-mediated polyubiquitylation and proteasomal degradation of CHK2. We also provide evidence that CHK2 ubiquitylation by PIRH2 is dependent on its phosphorylation status. Cells deficient in Pirh2 displayed accumulation of Chk2 and enhanced hyperactivation of G1/S and G2/M cell-cycle checkpoints. This hyperactivation was, however, no longer observed in Pirh2β/βChk2β/β cells, providing evidence for the importance of Chk2 regulation by Pirh2. These findings indicate that PIRH2 has central roles in the ubiquitylation of Chk2 and its turnover and in the regulation of its function.
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Abbreviations
- ATM:
-
ataxia telangiectasia mutated
- CHK2:
-
checkpoint kinase 2
- HA:
-
hemagglutinin
- ΞR:
-
mutant lacking the ring finger domain
- MEF:
-
mouse embryonic fibroblast
- PIRH2:
-
p53-induced protein with a RING (Really Interesting New Gene)-H2
- Ub:
-
ubiquitin
- USP28:
-
ubiquitin-specific-processing protease 28
- WT:
-
wild type
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Acknowledgements
We thank V Stambolic, S Benchimol, C Arrowsmith, L Salmena, B Raught and the Hakem laboratory members for helpful discussions. We also thank Dr. T. Mak, Dr. S. Benchimol, Dr. S. Elledge and Dr. M. Eilers for providing reagents. This work was supported by the Canadian Institute of Health Research. RH was supported by a salary award from the Canadian Institute of Health Research.
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Bohgaki, M., Hakem, A., Halaby, M. et al. The E3 ligase PIRH2 polyubiquitylates CHK2 and regulates its turnover. Cell Death Differ 20, 812β822 (2013). https://doi.org/10.1038/cdd.2013.7
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DOI: https://doi.org/10.1038/cdd.2013.7
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