Cervone, Nando (2017) Evidence that Maintenance and Resolution of the Spindle Assembly Checkpoint does not require Activity of Protein Phosphatase 2A. [Tesi di dottorato]

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Mitosis is the critical stage of the cell cycle deputed to form two daughter cells by the faithful separation of replicated chromosomes of a mother cell into two identical sets, daughter cells genomes. Inappropriate resolution of sister chromatids, prior to proper bipolar attachments, during mitosis, leads to random chromosomes segregation and aneuploidy, a deleterious condition often associated with various pathological conditions (Weaver & Cleveland, 2006). To prevent errors in chromosome segregation, a safeguard pathway, the spindle assembly checkpoint (SAC), is activated by improper kinetochore-spindle microtubule attachments and prevents activation of the mitosis exit-promoting ubiquitin-ligase Anaphase Promoting Complex/Cyclosome (APC/C) to delay mitosis exit until all the chromosomes become correctly bi-orientated at the equator of the metaphase plate (Lampson et al., 2004). Several protein kinases and phosphatases play essential roles in these processes presumably by controlling the phosphorylation status of a number of proteins involved in SAC control. There is a balance between kinase and phosphatase activities and this balance is constantly changing during the time required for spindle assembly completion (Visconti et al., 2013). Protein phosphatases as protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), are well known to be required for mitosis completion, however, whether and how activity of these phosphatases is involved in SAC control is still incompletely understood. PP2A, in particular, is a holoenzyme composed by a scaffolding (A), a catalytic (C) and a regulatory (B) subunit. The B subunit consists of four distinct subfamilies (B, B’, B” and B”’) acting as target modulators to provide substrate specificity; holoenzyme formation, in all its three subunits, is fundamental for PP2A action (Wurzenberger & Gerlich, 2011; Rogers et al., 2016). PP2A has been suggested to contribute to SAC resolution and to the metaphaseto- anaphase transition, namely through experiments based on small interfering 6 RNAs-mediated genetic knockdown of the various PP2A subunits (Schmitz et al., 2010; Wurzenberger & Gerlich, 2011; Kruse et al., 2013; Nijlenhuis et al., 2014). However, the contribution of the catalytic activity of PP2A in SAC control has not been directly investigated. In this work we investigated the role of PP2A activity in SAC regulation by using inhibitors of PP2A catalytic activity, like Okadaic acid (OA) and LB 100, without interfering with holoenzyme formation. We found compelling evidence that the catalytic activity of PP2A does not substantially affect SAC maintenance or resolution in human cells.

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