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Plant Kinases pp 185-197 | Cite as

Sensitizing Plant Protein Kinases to Specific Inhibition by ATP-Competitive Molecules

  • Dor Salomon
  • Chao Zhang
  • Kevan M. Shokat
  • Guido SessaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 779)

Abstract

The highly conserved nature of the protein kinase catalytic domain and the low permeability of plant cell membranes pose a challenge to the development of specific inhibitors that target individual protein kinases in vivo. Here, we describe a chemical-genetic approach to specifically sensitize individual plant kinases to cell-permeable small molecules that do not inhibit wild-type kinases. In this approach, a single amino-acid substitution is introduced in the ATP-binding site of the enzyme enabling specific binding of ATP-competitive molecules. Cell-permeable molecules can then be used to specifically target the sensitized allele in transgenic Arabidopsis thaliana plants that do not express the wild-type form of the kinase. This strategy provides a useful tool for the functional characterization of protein kinases in planta and for the dissection of the signaling pathways in which they are involved.

Key words

Protein kinase PP1 Gatekeeper ATP-competitive molecules Analog-sensitive Arabidopsis Kinase activity assay Transgenic plants 

Notes

Acknowledgments

We thank Tal Sherman for his advice on Arabidopsis plant transformations. This work was supported by the US–Israel Binational Agricultural Research and Development Fund (BARD; grant no. IS-4159-08C to G.S.) and by the Binational Science Foundation (BSF; grant no. 2007091 to G.S. and K.M.S.).

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Dor Salomon
    • 1
  • Chao Zhang
    • 2
    • 3
  • Kevan M. Shokat
    • 2
    • 3
  • Guido Sessa
    • 1
    Email author
  1. 1.Department of Plant SciencesTel-Aviv UniversityTel-AvivIsrael
  2. 2.Howard Hughes Medical InstituteChevy ChaseUSA
  3. 3.Department of Cellular and Molecular PharmacologyUniversity of CaliforniaSan FranciscoUSA

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