Use of quantitative proteomics to evaluate substrates and functional modules of the aurora and polo-like kinases.

Abstract

e13530 Background: Protein kinases are rapidly emerging as an important class of targets for cancer chemotherapy, including the mitotic kinases Aurora A, Aurora B, and Polo-like kinase 1 (Plk1), which are frequently overexpressed in many solid tumors. In this work, we mapped in vivo phosphorylation sites to these three kinases through the use of innovative methods in quantitative proteomics. Using our approach, we connected 815 phosphorylation sites on 609 proteins to the specific activities of these three kinases in preclinical model systems. Methods: HeLa cells in culture were arrested in mitosis and treated separately with the selective kinase inhibitors MLN8054, AZD1152, and BI2536 to inhibit the activity of the Aurora A, Aurora B, and Plk1 enzymes, respectively. Subsequently, the phosphoproteomes of these treated cells were compared to control cells using stable-isotope labeling methods in quantitative proteomics. Inhibition of enzyme activity resulted in a decrease in the relative intensities of substrate phosphorylation sites relative to the control when analyzed by mass spectrometry. Results: Using bioinformatics methods, we classify 115, 157, and 543 phosphorylation loci as substrates of Aurora A, Aurora B, and Plk1, respectively. We observe distinct linear motifs for each of these datasets which recapitulate previous reports of consensus motifs for each kinase, but also extend the motif elements and in some cases generate new substrate motifs. We observe cross-regulation of Plk1 and Aurora A with each other, but not with Aurora B. Furthermore, we find novel substrate classes that suggest potential roles for these kinases in oncogenesis. Conclusions: This work has uncovered new functional roles, established novel substrate recognition motifs, and generated a subset of candidate biomarkers and additional entry points for therapeutic intervention for these clinically relevant cancer targets. In addition, our approach provides an analytical template for further use in dissecting other kinase signaling events in translational research. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration AstraZeneca, Boehringer Ingelheim Millennium