2. Academic Validation
  3. Modulation of plant growth in vivo and identification of kinase substrates using an analog-sensitive variant of CYCLIN-DEPENDENT KINASE A;1

Modulation of plant growth in vivo and identification of kinase substrates using an analog-sensitive variant of CYCLIN-DEPENDENT KINASE A;1

  • BMC Plant Biol. 2016 Sep 26;16(1):209. doi: 10.1186/s12870-016-0900-7.
Hirofumi Harashima 1 2 3,  Nico Dissmeyer 1 2 4,  Philippe Hammann 5,  Yuko Nomura 6,  Katharina Kramer 7,  Hirofumi Nakagami 6 7,  Arp Schnittger 8 9 10
Affiliations

Affiliations

  • 1 Department of Molecular Mechanisms of Phenotypic Plasticity, Institut de Biologie Moléculaire des Plantes du CNRS, IBMP-CNRS - UPR2357, Université de Strasbourg, F-67084, Strasbourg, France.
  • 2 Trinationales Institut für Pflanzenforschung, F-67084, Strasbourg Cedex, France.
  • 3 Present address: RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro, Tsurumi, Yokohama, Kanagawa, 230-0045, Japan.
  • 4 Present address: Leibniz Institute of Plant Biochemistry (IPB), Independent Junior Research Group on Protein Recognition and Degradation, Weinberg 3, D-06120, Halle, (Saale), Germany.
  • 5 Plateforme protéomique Strasbourg Esplanade, Institut de Biologie Moléculaire et Cellulaire FRC1589-CNRS, F-67084, Strasbourg, France.
  • 6 Plant Proteomics Research Unit, RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045, Japan.
  • 7 Max Planck Institute for Plant Breeding Research, Basic Immune System of Plants / Protein Mass Spectrometry, Carl-von-Linne-Weg 10, 50829, Cologne, Germany.
  • 8 Department of Molecular Mechanisms of Phenotypic Plasticity, Institut de Biologie Moléculaire des Plantes du CNRS, IBMP-CNRS - UPR2357, Université de Strasbourg, F-67084, Strasbourg, France. arp.schnittger@uni-hamburg.de.
  • 9 Trinationales Institut für Pflanzenforschung, F-67084, Strasbourg Cedex, France. arp.schnittger@uni-hamburg.de.
  • 10 Department of Developmental Biology, University of Hamburg, Biozentrum Klein Flottbek, Ohnhorststr. 18, D-22609, Hamburg, Germany. arp.schnittger@uni-hamburg.de.
PMID: 27669979 DOI: 10.1186/s12870-016-0900-7
Abstract

Background: Modulation of protein activity by phosphorylation through kinases and subsequent de-phosphorylation by phosphatases is one of the most prominent cellular control mechanisms. Thus, identification of kinase substrates is pivotal for the understanding of many - if not all - molecular biological processes. Equally, the possibility to deliberately tune kinase activity is of great value to analyze the biological process controlled by a particular kinase.

Results: Here we have applied a chemical genetic approach and generated an analog-sensitive version of CDKA;1, the central cell-cycle regulator in Arabidopsis and homolog of the yeast Cdc2/CDC28 kinases. This variant could largely rescue a cdka;1 mutant and is biochemically active, albeit less than the wild type. Applying bulky kinase inhibitors allowed the reduction of kinase activity in an organismic context in vivo and the modulation of plant growth. To isolate CDK substrates, we have adopted a two-dimensional differential gel electrophoresis strategy, and searched for proteins that showed mobility changes in fluorescently labeled extracts from Plants expressing the analog-sensitive version of CDKA;1 with and without adding a bulky ATP variant. A pilot set of five proteins involved in a range of different processes could be confirmed in independent kinase assays to be phosphorylated by CDKA;1 approving the applicability of the here-developed method to identify substrates.

Conclusion: The here presented generation of an analog-sensitive CDKA;1 version is functional and represent a novel tool to modulate kinase activity in vivo and identify kinase substrates. Our here performed pilot screen led to the identification of CDK targets that link cell proliferation control to sugar metabolism, proline proteolysis, and glucosinolate production providing a hint how cell proliferation and growth are integrated with plant development and physiology.

Keywords

Arabidopsis; Cell cycle; Kinase; Mitosis; Phosphorylation; Substrate.

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