Mps1 Mph1 Kinase Phosphorylates Mad3 to Inhibit Cdc20 Slp1-APC/C and Maintain Spindle Checkpoint Arrests

摘要

The spindle checkpoint is a mitotic surveillance system which ensures equal segregation of sister chromatids. It delays anaphase onset by inhibiting the action of the E3 ubiquitin ligase known as the anaphase promoting complex or cyclosome (APC/C). Mad3/BubR1 is a key component of the mitotic checkpoint complex (MCC) which binds and inhibits the APC/C early in mitosis. Mps1~(Mph1)kinase is critical for checkpoint signalling and MCC-APC/C inhibition, yet few substrates have been identified. Here we identify Mad3 as a substrate of fission yeast Mps1~(Mph1)kinase. We map and mutate phosphorylation sites in Mad3, producing mutants that are targeted to kinetochores and assembled into MCC, yet display reduced APC/C binding and are unable to maintain checkpoint arrests. We show biochemically that Mad3 phospho-mimics are potent APC/C inhibitors in vitro , demonstrating that Mad3p modification can directly influence Cdc20~(Slp1)-APC/C activity. This genetic dissection of APC/C inhibition demonstrates that Mps1~(Mph1)kinase-dependent modifications of Mad3 and Mad2 act in a concerted manner to maintain spindle checkpoint arrests. Author Summary When cells divide they need to ensure that a complete copy of their genetic material is transmitted to both daughter cells. Cells have evolved many controls to ensure that every division is carried out with very high fidelity. The spindle checkpoint is one such control, which acts as a surveillance system during mitosis. Defects in this checkpoint control lead to unequal segregation of DNA/chromosomes, termed aneuploidy, which is responsible for human birth defects and is very common in tumour cells. The molecular components of the spindle checkpoint, identified initially through yeast genetics, include several protein kinases. Surprisingly few of their substrates have been identified. Here we identify the checkpoint protein Mad3 as an important substrate of the Mps1~(Mph1)kinase. We show that Mps1~(Mph1)-dependent modification of Mad3 and Mad2 acts to delay cell division in situations where the genetic material would not be equally inherited by daughter cells. This delay enables the cell to correct any problems within the division machinery and thus avoid aneuploidy.