Abstract
Deciphering how signaling enzymes operate within discrete microenvironments is fundamental to understanding biological processes. A-kinase anchoring proteins (AKAPs) restrict the range of action of protein kinases within intracellular compartments. We exploited the AKAP targeting concept to create genetically encoded platforms that restrain kinase inhibitor drugs at distinct subcellular locations. Local Kinase Inhibition (LoKI) allows us to ascribe organelle-specific functions to broad specificity kinases. Using chemical genetics, super resolution microscopy, and live-cell imaging we discover that centrosomal delivery of Polo-like kinase 1 (Plk1) and Aurora A (AurA) inhibitors attenuates kinase activity, produces spindle defects, and prolongs mitosis. Targeted inhibition of Plk1 in zebrafish embryos illustrates how centrosomal Plk1 underlies mitotic spindle assembly. Inhibition of kinetochore-associated pools of AurA blocks phosphorylation of microtubule-kinetochore components. This versatile precision pharmacology tool enhances investigation of local kinase biology.
Highlights
Protein kinase inhibitor drugs are an emerging class of therapeutics for a variety of clinical indications (Ferguson and Gray, 2018)
It is well established that Polo-like kinase 1 (Plk1) and Aurora A (AurA) coordinate various aspects of cell division, current drug-targeting strategies limit our ability to decode the spatiotemporal regulation of these events (Barr et al, 2004; Combes et al, 2017; Lens et al, 2010)
Designing pharmacological tools that restrict the spatial and temporal action of kinase inhibitor drugs is paramount to deciphering local kinase action
Summary
Protein kinase inhibitor drugs are an emerging class of therapeutics for a variety of clinical indications (Ferguson and Gray, 2018). Anchored protein kinase A action is constrained to within 200–400 angstroms of the AKAP (Smith et al, 2013; Smith et al, 2017) This has led to the formulation of a signaling island model where catalytic activity of anchored kinases is restricted to the immediate vicinity of select substrates (Scott and Pawson, 2009; Langeberg and Scott, 2015; Esseltine and Scott, 2013). We demonstrate that local inhibition of Plk and AurA kinases at centrosomes and kinetochores disrupts substrate phosphorylation, spindle organization, and mitotic duration Together, these studies decipher how activities of individual kinases at precisely defined microenvironments contribute to the global signaling events that underlie mitosis
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