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 PP1α87B at a conserved residue (T286) within a pocket implicated in the recognition of RVxF-containing target proteins. Phosphorylation of T286 inhibits PP1α87B 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 PP1α87B ^T286D prevents MPS1 inactivation in metaphase and causes a SAC-dependent delay of anaphase onset. Conversely, an unphosphorylatable PP1α87B ^T286A mutant impairs MPS1 activation at unattached kinetochores and weakens the SAC. In vivo , larval neuroblasts expressing PP1α87B ^T286 phosphomutants exhibit frequent chromosome mis-segregation and aneuploidy. Thus, our findings identify POLO-mediated phosphorylation of PP1α87B as a critical regulatory strategy that fine-tunes phosphatase activity to ensure a robust and timely SAC and prevent genome instability.