PLK1 inhibition delays mitotic entry revealing changes to the phosphoproteome of mammalian cells early in division

Abstract Polo-like kinase 1 (PLK1) is a conserved regulator of cell division. During mitotic prophase, PLK1 contributes to the activation of the cyclin-dependent kinase 1 (CDK1). However, the exact functions of PLK1 in prophase remain incompletely understood. Here, we show that PLK1 inhibition in synchronous G2 cell populations of multiple mammalian cell lines delays or prevents mitotic entry with high variability between individual cells. Using a mathematical model, we recapitulate this phenomenon and provide an explanation for the observed phenotypic variability. We show that PLK1-inhibited cells are delayed in a prophase-like state with low CDK1 activity that increases slowly and gradually over hours. These cells display progressively condensing chromosomes, increased microtubule dynamics, and reorganization of the actin cortex, while the nuclear envelope remains intact. We characterize this state further by phosphoproteomics, revealing phosphorylation of regulators of chromatin organization and the cytoskeleton consistent with the cellular phenotypes. Together, our results indicate that PLK1 inhibition stabilizes cells in a prophase-like state with low CDK1 activity displaying a specific set of early mitotic phosphorylation events.