Phospho-regulation of the spindle assembly checkpoint
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Mitosis is a highly regulated process by which a cell duplicates and distributes its chromosomal DNA into two identical daughter cells equally. Equal distribution of the chromosomes is crucial for accurate propagation of genetic information. This is essential for maintaining viability and preventing genomic instability that can potentially lead to cancer. In order to avoid unequal distribution of chromosomes, cells employ a surveillance mechanism called the spindle assembly checkpoint (SAC). The SAC is an inhibitory signalling network, which delays segregation of chromosomes, until they have stably attached to spindle microtubules through their multi-protein platforms, known as kinetochores. The main target of the SAC is the anaphase promoter complex/ cyclosome (APC/C), an E3 ubiquitin ligase. Specifically APC/C and its activator Cdc20 are inhibited by the main effector of the SAC, called the mitotic checkpoint complex (MCC). The MCC consists of Cdc20, Mad2, Mad3 and Bub3 (except S. pombe) proteins, which are recruited to the unattached kinetochores to promote MCC assembly. Once the chromosomes stably attach to the spindle, the SAC is turned off, MCC disassembles, and APC/CCdc20 is released from the inhibition. Activated APC/CCdc20 then targets its two main substrates, securin and cyclin B, for proteasomal degradation, and thereby triggers anaphase onset and mitotic exit. SAC signalling involves many protein components, whose activities are essentially regulated by direct protein-protein interactions and/ or post-translational modifications. One of these major modifications is phosphorylation, which is mediated by the SAC kinases such as Aurora B, Mps1 and Bub1. A number of studies have characterised SAC related substrates of Aurora B and Mps1 kinases in several model organisms. On the other hand, Bub1 kinase activity has been thought to play a key role in chromosome bi-orientation and more of an auxiliary role in SAC activation. The aim of this study is to investigate the importance of Bub1 kinase activity for SAC response in fission yeast Schizosaccharomyces pombe (S. pombe). SAC activation assays, using various degrees of spindle perturbation, have demonstrated that Bub1 kinase activity plays an important role in SAC maintenance. In order to examine the pathways downstream of Bub1, we set out to indicate Bub1 substrates which may be involved in SAC signalling. According to studies in various species, Cdc20 appears to be a prominent candidate, whose phosphorylation by Cdk1 and Bub1 kinases has been reported to regulate its mitotic activity. To investigate whether Cdc20 is phosphorylated by Bub1 in vitro, we purified recombinant S. pombe proteins from insect cells. Subsequent kinase assays identified Cdc20 as an in vitro substrate of Bub1, and the phosphorylated sites in Cdc20 were mapped by mass spectrometry. To address if this phospho-modification is involved in SAC regulation, phosphorylation mutants of Cdc20 were analysed in terms of their abilities to activate and silence SAC in vivo. Results show that phosphorylation of Cdc20 C-terminus promotes SAC maintenance in response to spindle damage. Furthermore, the mutations mimicking Bub1-mediated phosphorylation of Cdc20 Cterminus restore the SAC defects in the absence of Bub1 kinase activity. In addition, we purified S. pombe mitotic checkpoint complex (MCC) from insect cells, and analysed the interactions between its components (Cdc20, Mad2 and Mad3) by cross-linking mass spectrometry. Crystal structure of S.pombe MCC has been determined recently, which lacks Mad3 C-terminus and flexible C-terminal tail of Cdc20. Using an MCC with full length Mad3, we identified novel interactions between the C-terminal tails Mad3 and Cdc20, which are in close proximity to the identified Cdc20 phosphorylation sites. Briefly, in this study we confirm the previously known roles of Bub1 kinase activity (chromosome bi-orientation). Moreover, we propose a new pathway (in addition to the well-established H2A pathway) mediated by Cdc20, that may be important to maintain the SAC response.