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. 2021:2329:19-27.
doi: 10.1007/978-1-0716-1538-6_2.

Substrate Phosphorylation Rates as an In Vivo Measurement of Kinase Activity

Matthew P Swaffer  1
Affiliations

Substrate Phosphorylation Rates as an In Vivo Measurement of Kinase Activity

Matthew P Swaffer. Methods Mol Biol. 2021.

Abstract

Measuring kinase activity in different in vivo contexts is crucial for understanding the mechanism and functions of protein kinases, such as the cyclin-dependent kinases (Cdks) and other cell cycle kinases. Here, I present the rationale and the experimental framework for an alternative approach to measure kinase activity that is based on estimating substrate phosphorylation rates in vivo. The approach presented was first developed for experiments performed to measure Cdk1 activity at different stages of the fission yeast S. pombe's cell cycle [Swaffer et al., Cell 167:1750-1761, 2016]. However, it also affords a more generalizable framework that can be adaptable to other systems and kinases, as long as specific, rapid, and reversible kinase inhibition is possible. Briefly this involves transient and reversible kinase inhibition to dephosphorylate kinase substrates in vivo, followed by quantitative measurements of phosphorylation after inhibition is removed.

Keywords: Cdk1; Kinase activity; Kinases; Phosphorylation.

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References

    1. Swaffer MP, Jones AW, Flynn HR, Snijders AP, Nurse P (2018) Quantitative phosphoproteomics reveals the signaling dynamics of cell-cycle kinases in the fission yeast Schizosaccharomyces pombe. Cell Rep 24(2):503–514. https://doi.org/10.1016/j.celrep.2018.06.036 - DOI - PubMed - PMC
    1. Olsen JV, Vermeulen M, Santamaria A, Kumar C, Miller ML, Jensen LJ, Gnad F, Cox J, Jensen TS, Nigg EA, Brunak S, Mann M (2010) Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis. Sci Signal 3(104):ra3. https://doi.org/10.1126/scisignal.2000475 - DOI - PubMed
    1. Sharma K, D'Souza RC, Tyanova S, Schaab C, Wisniewski JR, Cox J, Mann M (2014) Ultradeep human phosphoproteome reveals a distinct regulatory nature of Tyr and Ser/Thr-based signaling. Cell Rep 8(5):1583–1594. https://doi.org/10.1016/j.celrep.2014.07.036 - DOI - PubMed
    1. Carpy A, Krug K, Graf S, Koch A, Popic S, Hauf S, Macek B (2014) Absolute proteome and phosphoproteome dynamics during the cell cycle of Schizosaccharomyces pombe (Fission Yeast). Mol Cell Proteomics 13(8):1925–1936. https://doi.org/10.1074/mcp.M113.035824 - DOI - PubMed - PMC
    1. Coudreuse D, Nurse P (2010) Driving the cell cycle with a minimal CDK control network. Nature 468(7327):1074–1079. https://doi.org/10.1038/nature09543 - DOI - PubMed

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