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Use of Phospho-Site Substitutions to Analyze the Biological Relevance of Phosphorylation Events in Regulatory Networks

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Plant Kinases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 779))

Abstract

Biological information is often transmitted by phosphorylation cascades. However, the biological relevance of specific phosphorylation events is often difficult to determine. An invaluable tool to study the effect of kinases and/or phosphatases is the use of phospho- and dephospho-mimetic substitutions in the respective target proteins. Here, we present a generally applicable procedure of how to design, set-up, and carry out phosphorylation modulation experiments and subsequent monitoring of protein activities, taking ­cyclin-dependent kinases (CDKs) as a case study. CDKs are key regulators of cell cycle progression in all eukaryotic cells. Consequently, CDKs are controlled at many levels and phosphorylation of CDKs ­themselves is used to regulate their kinase activity. We describe in detail complementation experiments of a mutant in CDKA;1, the major cell cycle kinase in Arabidopsis, with phosphorylation-site variants of CDKA;1. CDKA;1 versions were generated either by mimicking a phosphorylated amino acid by replacing the respective residue with a negatively charged amino acid, e.g., aspartate or glutamate, or by mutating it to a non-phoshorylatable amino acid, such as alanine, valine, or phenylalanine. The genetic complementation studies were accompanied by the isolation of these kinase variants from plant extract and subsequent kinase assays to determine changes in their activity levels. This work allowed us to judge the importance of ­posttranslational regulation of CDKA;1 in plants and has shown that the molecular mechanistics of CDK function are apparently conserved across the kingdoms. However, the regulatory wiring of CDKs is ­strikingly different between plants, animals, and yeast.

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Acknowledgments

The authors thank Annika Weimer for critical reading and helpful comments on the manuscript. Frank Sprenger and Vimal Ramachandran helped with running the first kinase assays of Drosophila Cdc2 in vitro translations and shared materials at the old Institute for Genetics of the University of Cologne. We thank Aurine Verkest, Max Bush, Lazlo Bögre, and Mihály Horváth for helpful communication and assistance on the experimental ­procedures. N.D. was a fellow of the International Max Planck Research School and funded by the Max Planck Society, work in the laboratory of A.S. is supported through an ATIP grant from the Centre National de la Recherche Scientifique (CNRS) and an ERC starting grant from the European Union.

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  1. Leibniz Institute of Plant Biochemistry (IPB), Independent Junior Research Group on Protein Recognition and Degradation, Halle (Saale), Germany

    Nico Dissmeyer

  2. Institut de Biologie Moléculaire des Plantes, Centre National de la Recherche Scientifique (IBMP-CNRS), Unité Propre de Recherche, Strasbourg, France

    Arp Schnittger

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  1. Nico Dissmeyer
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  2. Arp Schnittger
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Correspondence to Nico Dissmeyer .

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  1. Leibniz Institute of Plant Biochemistry, Weinberg 3, Halle (Saale), 06120, Sachsen-Anhalt, Germany

    Nico Dissmeyer

  2. rue du Général Zimmer 12, Strasbourg, F-67000, France

    Arp Schnittger

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Dissmeyer, N., Schnittger, A. (2011). Use of Phospho-Site Substitutions to Analyze the Biological Relevance of Phosphorylation Events in Regulatory Networks. 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_6

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  • DOI: https://doi.org/10.1007/978-1-61779-264-9_6

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