Relevance of the phosphatase subunit PR130 for checkpoint kinase signaling
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Abstract
Cancer cells depend on continuous cell proliferation and efficient cell cycle checkpoints to prevent the induction of cell death. In the clinic, there are different strategies to eliminate cancer cells effectively. The strong dependence on cell proliferation can be exploited by inhibition of Cyclin-Dependent Kinases (CDKs), the main drivers of cell cycle progression, which force cancer cells into cell cycle arrest. In this context, the two CDK inhibitors WEE1 and p21 play a major role by regulating CDK activity. Another method is the induction of replication stress since cancer cells are very susceptible to it due to their fast cell division rate. Sensing replication stress and DNA damage involves phosphorylation and therefore activation of the checkpoint kinases Ataxia telangiectasia mutated (ATM), ATM and RAD3-related (ATR), Checkpoint Kinase 2 (CHK2) and CHK1, which slow down the cell cycle and initiate Deoxyribonucleic Acid (DNA) repair, or if the damage cannot be repaired, induce apoptosis. Phosphorylation and acetylation are posttranslational modifications, which control not only signaling pathways but also gene expression. The two histone deacetylases (HDAC), HDAC1 and HDAC2, have been found to control the expression of the Protein Phosphatase 2A (PP2A) B-type subunit PR130, which is involved in the regulation of various cellular processes such as DNA repair, apoptosis, cell proliferation and autophagy. Moreover, the PP2A-PR130 complex plays an important role in the DNA damage response since it deactivates ATM via dephosphorylation of S1981 upon replication stress. Recent studies showed that colorectal cancer cells devoid of PR130 had higher levels of pCHK1 and arrested earlier at the G1/S phase boundary than wild-type cells upon replication stress. Combinatorial treatment with the HDAC inhibitor (HDACi) entinostat, however, reduced phosphorylation of CHK1 independent of the PR130 status of the cells. This shows that the PP2A-PR130 complex is indirectly involved in the regulation of CHK1 phosphorylation. In this thesis it was investigated how CHK1 phosphorylation is controlled by PR130 and HDACi. Proteome and RNA sequencing analyses revealed higher levels of the cell cycle regulator p21 in PR130-deficient cells compared to wild-type cells. Moreover, it was shown that the ATM-p53-axis controls the expression of p21. Additionally, class I HDACi increased levels of p21, which binds and deactivates CDK2, leading to diminished CHK1 phosphorylation upon replication stress. Furthermore, two novel targets of the PP2A-PR130 complex, pp21-S130 and pWEE1-T190, were identified. Both proteins are directly connected to higher CDK2 activity: pp21-S130 cannot bind to CDK2 and pWEE1-T190 is not able to phosphorylate it. The results showed that PR130 controls CHK1 phosphorylation twofold, on a transcriptional level via p21 and by posttranslational modifications via dephosphorylation of pp21-S130 and pWEE1-T190. Furthermore, it was shown that class I HDACi dysregulate DNA repair proteins important for homologous recombination, thereby promoting non-homologous end-joining, resulting in Replication Timing Regulatory Factor 1 (RIF1) foci formation in a PR130-dependent manner. This gives new insights into how HDACi and PR130 control the DNA repair pathway choice upon DNA replication stress. All in all, the results demonstrate that the PP2A-PR130 complex is a key player in cell cycle regulation, DNA damage signaling and DNA repair pathways. This versatility explains the frequent mutation of the B-type subunit in cancer and underlines it as a potentially interesting target in clinical research.