Plant Physiol. Journal of Pharmacology and Experimental Therapeutics
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Published on June 9, 2006; 10.1104/pp.106.079327

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Received February 24, 2006
Returned for revision March 20, 2006
Accepted June 1, 2006

Identification of features regulating OST1 kinase activity and OST1 function in guard cells

Christophe Belin *, Pierre-Olivier de Franco , Clara Bourbousse , Stéphane Chaignepain , Jean-Marie Schmitter , Alain Vavasseur , Jérôme Giraudat , Hélène Barbier-Brygoo , and Sébastien Thomine

Institut des Sciences du Végétal, CNRS, 1 avenue de la terrasse, 91198 Gif-sur-Yvette cedex, France
Institut Européen de Chimie et Biologie, 2 rue Robert Escarpit, 33607 Pessac, France
Commissariat à l'Energie Atomique, Direction des Sciences du Vivant, Laboratoire des Echanges Membranaires et Signalisation, 13108 St. Paul lez Durance Cedex, France

* Corresponding author; email: belin{at}isv.cnrs-gif.fr.

The phytohormone abscisic acid (ABA) mediates drought responses in plants and in particular triggers stomatal closure. Snf1-related kinase 2 (SnRK2) proteins from several plant species have been implicated in ABA signalling pathways. In Arabidopsis thaliana guard cells, OST1/SRK2E/SnRK2-6 is a critical positive regulator of ABA signal transduction. A better understanding of the mechanisms responsible for SnRK2 protein kinase activation is thus a major goal towards understanding ABA signal transduction. Here, we report the first successful purification of OST1 produced in E.coli: the protein is active and autophosphorylates. Using mass spectrometry, we identify 5 target residues of autophosphorylation in recombinant OST1. Sequence analysis delineates two conserved boxes located in the carboxy-terminal moiety of OST1 after the catalytic domain: the "SnRK2-specific" box (Gln303 to Pro318) and the "ABA-specific" box (Leu333 to Met362). Site directed mutagenesis and serial deletions reveal that Ser175 in the activation loop and the "SnRK2-specific" box are critical for the activity of recombinant OST1 kinase. Targeted expression of variants of OST1 kinase in guard cells uncovered additional features which are critical for OST1 function in ABA signalling although not required for OST1 kinase activity: serines 7, 18 and 29 and the "ABA-specific" box. Serines 7, 18, 29 and 43 represent putative targets for regulatory phosphorylations and the "ABA-specific" box may be a target for the binding of signalling partners in guard cells.




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