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

Rufus Hards, Charles L. Howarth, Kwame Wiredu, Ian LaCroix, Juan Mercado del Valle, Mark Adamo, Arminja N. Kettenbach, Andrew J. Holland, View ORCID ProfileScott A. Gerber
doi: https://doi.org/10.1101/2021.12.08.471812
Rufus Hards
1Department of Biochemistry & Cell Biology, Geisel School of Medicine, Lebanon NH, USA
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Charles L. Howarth
1Department of Biochemistry & Cell Biology, Geisel School of Medicine, Lebanon NH, USA
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Kwame Wiredu
2Department of Molecular & Systems Biology, Geisel School of Medicine, Lebanon NH, USA
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Ian LaCroix
3Norris Cotton Cancer Center, Lebanon, NH, USA
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Juan Mercado del Valle
2Department of Molecular & Systems Biology, Geisel School of Medicine, Lebanon NH, USA
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Mark Adamo
3Norris Cotton Cancer Center, Lebanon, NH, USA
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Arminja N. Kettenbach
1Department of Biochemistry & Cell Biology, Geisel School of Medicine, Lebanon NH, USA
3Norris Cotton Cancer Center, Lebanon, NH, USA
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Andrew J. Holland
4Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA
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Scott A. Gerber
1Department of Biochemistry & Cell Biology, Geisel School of Medicine, Lebanon NH, USA
2Department of Molecular & Systems Biology, Geisel School of Medicine, Lebanon NH, USA
3Norris Cotton Cancer Center, Lebanon, NH, USA
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  • ORCID record for Scott A. Gerber
  • For correspondence: scott.a.gerber@dartmouth.edu
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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.

Competing Interest Statement

The authors have declared no competing interest.

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Posted December 10, 2021.
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Development and validation of inducible protein degradation and quantitative phosphoproteomics to identify kinase-substrate relationships
Rufus Hards, Charles L. Howarth, Kwame Wiredu, Ian LaCroix, Juan Mercado del Valle, Mark Adamo, Arminja N. Kettenbach, Andrew J. Holland, Scott A. Gerber
bioRxiv 2021.12.08.471812; doi: https://doi.org/10.1101/2021.12.08.471812
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Development and validation of inducible protein degradation and quantitative phosphoproteomics to identify kinase-substrate relationships
Rufus Hards, Charles L. Howarth, Kwame Wiredu, Ian LaCroix, Juan Mercado del Valle, Mark Adamo, Arminja N. Kettenbach, Andrew J. Holland, Scott A. Gerber
bioRxiv 2021.12.08.471812; doi: https://doi.org/10.1101/2021.12.08.471812

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