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Regulation of the protein phosphatase scaffold RepoMan during the cell cycle
Book - Dissertation
Protein kinases and phosphatases are antagonistically acting enzymes that cooperate to control the speed, amplitude and specificity of phosphorylation signals in numerous cellular processes, including mitosis. Early mitotic events are characterized by bulk phosphorylation of specific proteins by kinases such as CDK1/Cyclin B and Aurora B. The mitotic exit depends on protein phosphatases, mainly PP1 and PP2A, which remove phosphate groups from mitotic phosphoproteins and re-establish the interphase state. We are interested in the antagonistically acting enzymes Aurora B and PP1:RepoMan. During prometaphase, PP1-RepoMan dephosphorylates Histone 3 at Threonine 3 along the chromosome arms to promote the centromeric enrichment of Aurora B. This is dynamically opposed by CDK1-mediated phosphorylation of the PP1-binding domain of RepoMan and by Aurora B itself, which inhibits RepoMan binding to histones. The reciprocal regulation between RepoMan and Aurora B results in the enrichment of Aurora B at the centromeres, where the kinase is maximally active and prevents erroneous kinetochore-microtubules attachments. During anaphase, Aurora B localizes to the spindle midzone and PP1-RepoMan is massively recruited to the chromosomes, where it catalyzes the bulk dephosphorylation of mitotic-exit substrates. Aurora-B overexpression in cancer is often associated with chromosome mis-segregation. The main molecular pathways that regulate Aurora-B abundance in cells are well understood. Moreover, small molecule inhibitors of Aurora B are being used in clinical trials to treat cancer patients. RepoMan is also frequently overexpressed in human cancers. However, it is not known if RepoMan and Aurora B are co-overexpressed and, importantly, which mechanisms control RepoMan levels in tumor cells. By using publicly available datasets, we found that RepoMan and Aurora-B co-overexpression correlated with tumor aggressiveness in some types of tumors. Then, by using biochemical approaches we showed that RepoMan levels oscillate similarly to Aurora B, increasing in G2/M phase and declining at the mitotic exit and in interphase. Interestingly, both RepoMan and Aurora B mRNA levels are controlled by the transcription factor FOXM1 and their protein downregulation is mainly controlled by ubiquitin-mediated proteasomal degradation. During mitotic exit and early G1, RepoMan and Aurora B are both targeted by the ubiquitin ligase APC/C-CDH1, while during G1/S phase their levels are kept low by E3 complex SCF-FBXW7. The existence of a cell-cycle co-regulation of RepoMan:Aurora B pair indicate that cells should benefit from having balanced levels of the two enzymes. Mathematical modeling revealed that co-oscillations in RepoMan:Aurora B level generate a dynamic bistable system that is more robust to stochastic changes. A theoretical model also predicted that cells with high RepoMan concentration are more vulnerable to treatment with Aurora-B inhibitors, which was validated experimentally. We speculate that tumor cells use multiple ways to keep the balance between RepoMan and Aurora B in order to proliferate with mild/advantageous levels of aneuploidy. Disturbing this balance, chemically or genetically, might be a novel and more specific therapeutic approach for the treatment of cancer patients.