Abstract
Identification of protein kinase targets and specific inhibition of individual kinase isoforms on the protein level in planta are important techniques to elucidate signal transduction pathways. The use of ATP-binding pocket mutants, the so-called gatekeeper mutants, that accommodate N6-enlarged nucleotides and kinase inhibitors has allowed a dramatic increase in kinase isoform selectivity. In this chapter, we describe protocols for the identification and mutation of the gatekeeper residue, radiolabeling of N6-modified nucleotides, analysis of protein targets by using [32P]-labeled N6-modified nucleotides, and in vivo inhibition of kinase activity combined with subsequent molecular readouts. The chapter includes alternative approaches for the described techniques, considerations for other kinases and recommendations toward a setup of a substrate labeling experiment using total cell lysate.
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Acknowledgments
This project was supported by the Deutsche Forschungsgemeinschaft (DFG; SFB 635), the ZIP program from the German ministry of education and science, the Alexander von Humboldt-Foundation, the Max Planck Society, and a DFG long term Research Fellowship to Maik Böhmer. We thank David G. Mendoza-Cózatl for critical reading of the manuscript.
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Böhmer, M., Bölker, M., Romeis, T. (2011). Chemical Genetic Analysis of Protein Kinase Function in Plants. In: Dissmeyer, N., Schnittger, A. (eds) Plant Kinases. Methods in Molecular Biology, vol 779. Humana, Totowa, NJ. https://doi.org/10.1007/978-1-61779-264-9_15
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