Subcellular Drug Targeting Illuminates Local Action of Polo‐like kinase 1 and Aurora A During Mitosis

Abstract

Fundamental cellular processes such as cell division, migration, metabolism, and growth rely on the precise and timely regulation of signaling proteins. Yet how individual signal transduction events at specific microenvironments drive complex cellular processes remains elusive. During mitosis, two protein kinases, polo‐like kinase 1 (Plk1) and Aurora A (AurA) share several overlapping functions. Current approaches in which small‐molecule kinase inhibitor drugs are used to ascribe function to Plk1 and AurA provide important insight into the global roles of these proteins inside the cell. Unfortunately, the indiscriminate distribution of such traditional inhibitor drugs limits our ability to resolve how these mitotic enzymes function at distinct organelles. Here we exploit the SNAP‐tag system to create genetically encoded platforms that restrain kinase inhibitor drugs at defined subcellular locations. Local Kinase Inhibition (LoKI) allows us to ascribe organelle‐specific functions to broad‐specificity kinases. Using a combination of biochemical approaches, quantitative imaging, and live‐cell microscopy we uncover that local targeting of Plk1 and AurA kinase inhibitors to centrosomes disrupts substrate phosphorylation, spindle formation, and mitotic duration more profoundly than global drug distribution in human cells. Furthermore, we show that zebrafish embryos treated with centrosome‐targeted Plk1 inhibitors have more microtubule abnormalities than those treated with a non‐localized inhibitor. Finally, we expand our approach by targeting drugs to the kinetochore and find that inhibition of kinetochore‐associated pools of AurA blocks phosphorylation of microtubule‐kinetochore components. Future work will employ LoKI to investigate how downstream substrates of Plk1 and AurA function at centrosomes and kinetochore to coordinate mitotic signaling events.