POLO kinase inhibits Protein Phosphatase 1 to promote the Spindle Assembly Checkpoint and prevent aneuploidy

Protein phosphatase 1 (PP1) is essential for spindle assembly checkpoint (SAC) silencing and mitotic exit, but its regulation during mitosis remains ill-defined. Here, we demonstrate in vitro and in Drosophila cells that the mitotic kinase POLO phosphorylates PP187B at a conserved residue (T286) within a pocket implicated in the recognition of RVxF-containing target proteins. Phosphorylation of T286 inhibits PP187B binding to the RVxF motif of the SAC kinase MPS1, dampening the dephosphorylation of MPS1 T-loop. Phosphorylation of T286 is dynamically regulated during mitosis. It occurs at unattached/tensionless kinetochores and decreases as chromosomes congress. Expression of phosphomimetic PP187BT286D prevents MPS1 inactivation in metaphase and causes a SAC-dependent delay of anaphase onset. Conversely, an unphosphorylatable PP187BT286A mutant impairs MPS1 activation at unattached kinetochores and weakens the SAC. In vivo, larval neuroblasts expressing PP187BT286 phosphomutants exhibit frequent chromosome mis-segregation and aneuploidy. Thus, our findings identify POLO-mediated phosphorylation of PP187B as a critical regulatory strategy that fine-tunes phosphatase activity to ensure a robust and timely SAC and prevent genome instability.