Development and validation of inducible protein degradation and quantitative phosphoproteomics to identify kinase-substrate relationships

Abstract

Phosphorylation signaling is an essential post-translational regulatory mechanism that governs almost all eukaryotic biological processes and is controlled by an interplay between protein kinases and phosphatases. Knowledge of direct substrates of kinases provides evidence of mechanisms that relate activity to biological function. Linking kinases to their protein substrates can be achieved by inhibiting or reducing kinase activity and quantitative comparisons of phosphoproteomes in the presence and absence of kinase activity. Unfortunately, most of the human kinases lack chemical inhibitors with selectivity required to unambiguously assign protein substrates to their respective kinases. Here, we develop and validate a chemical proteomics strategy for linking kinase activities to protein substrates via targeted protein degradation and quantitative phosphoproteomics and apply it to the well-studied, essential mitotic regulator polo-like kinase 1 (Plk1). We leveraged the Tir1/auxin system to engineer HeLa cells with endogenously homozygous auxin-inducible degron (AID)-Plk1). We used HeLa cells and determined the impact of AID-tagging on Plk1 activity, localization, protein interactors, and substrate motifs. Using quantitative proteomics, we show that of over 8,000 proteins quantified, auxin addition is highly selective for degrading AID-Plk1 in mitotic cells. Comparison of phosphoproteome changes in response to chemical Plk1 inhibition to auxin-induced degradation revealed a striking degree of correlation. Finally, we explored basal protein turnover as a potential basis for clonal differences in auxin-induced degradation rates for AID-Plk1 cells. Taken together, our work provides a roadmap for the application of AID technology as a general strategy for the kinome-wide discovery of kinase-substrate relationships.

Inducible degradation-coupled phosphoproteomics identifies PP2ARts1as a novel eisosome regulator

“…It would be suitable for use with isobaric labeling reagents, like the tandem mass tag (TMT) system, to characterize time-resolved responses to stimuli or stress after target degradation. While we were conducting our work, other groups also demonstrated the ability to target phosphatases or kinases for degradation via the AID system and measure changes in the phosphoproteome in S. cerevisiae and human cells (49, 50, 70, 71).…”

Phosphoproteomic Approaches for Identifying Phosphatase and Kinase Substrates

“…PP2A-Cdc55 was also targeted using AID to reveal potential substrates in S. cerevisiae by another group [131]. AID phosphoproteomics has also been applied successfully to the human Polo kinase Plk1, comparing the effects of chemical inhibition to AID-mediated inhibition, with a high correlation of down-regulated phosphosites observed between the two approaches [132]. Furthermore, AID-coupled phosphoproteomics has been applied to human Protein Phosphatase 6, demonstrating the ability to identify specific changes in the phosphoproteome and novel PP6 substrates without significant changes to the general proteome [133].…”