Quantitative Phosphoproteomics Identifies Substrates and Functional Modules of Aurora and Polo-Like Kinase Activities in Mitotic Cells

Combining quantitative phosphoproteomics and selective kinase inhibition yields previously unknown substrates and functions of two families of mitotic kinases and refinement of their recognition motifs. Building the Corpus of Substrates of Mitotic Kinases Mitosis is a complex process involving duplication of DNA, nuclear membrane dissolution, construction of a mitotic spindle, proper segregation of chromosomes, and, finally, creation of two new cells—each with a complete set of genomic material. Protein phosphorylation plays a critical role in this process and is mediated predominantly by three sets of kinases: the cyclin-dependent kinase–cyclin complex Cdk1/cyclinB, the Aurora family (Aurora A and B), and the Polo-like kinase (Plk) family, especially Plk1. To explore the targets of these kinases, Kettenbach et al. combined specific small-molecule kinase inhibitors with large-scale quantitative phosphoproteomics of mitotic mammalian cells. Their data enable refinement of the motifs recognized by these kinases and suggest previously unknown functions for these kinases, as well as serve as a useful resource for future exploration of these essential mitotic regulators. Mitosis is a process involving a complex series of events that require careful coordination. Protein phosphorylation by a small number of kinases, in particular Aurora A, Aurora B, the cyclin-dependent kinase–cyclin complex Cdk1/cyclinB, and Polo-like kinase 1 (Plk1), orchestrates almost every step of cell division, from entry into mitosis to cytokinesis. To discover more about the functions of Aurora A, Aurora B, and kinases of the Plk family, we mapped mitotic phosphorylation sites to these kinases through the combined use of quantitative phosphoproteomics and selective targeting of kinase activities by small-molecule inhibitors. Using this integrated approach, we connected 778 phosphorylation sites on 562 proteins with these enzymes in cells arrested in mitosis. By connecting the kinases to protein complexes, we associated these kinases with functional modules. In addition to predicting previously unknown functions, this work establishes additional substrate-recognition motifs for these kinases and provides an analytical template for further use in dissecting kinase signaling events in other areas of cellular signaling and systems biology.