title: Identification of features regulating OST1 kinase activity and OST1 function in guard cells.
abstract: 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-signaling pathways. In Arabidopsis (Arabidopsis thaliana) guard cells, OPEN STOMATA 1 (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 toward understanding ABA signal transduction. Here, we report successful purification of OST1 produced in Escherichia coli: The protein is active and autophosphorylates. Using mass spectrometry, we identified five 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 (glutamine-303 to proline-318) and the ABA-specific box (leucine-333 to methionine-362). Site-directed mutagenesis and serial deletions reveal that serine (Ser)-175 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 that are critical for OST1 function in ABA signaling, although not required for OST1 kinase activity: Ser-7, Ser-18, and Ser-29 and the ABA-specific box. Ser-7, Ser-18, Ser-29, and Ser-43 represent putative targets for regulatory phosphorylation and the ABA-specific box may be a target for the binding of signaling partners in guard cells.
authors.affiliation: Institut des Sciences du Végétal, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette cedex, France. belin@isv.cnrs-gif.fr
authors.forename: Christophe
authors.initials: C
authors.lastname: Belin
authors.forename: Pierre-Olivier
authors.initials: PO
authors.lastname: de Franco
authors.forename: Clara
authors.initials: C
authors.lastname: Bourbousse
authors.forename: Stéphane
authors.initials: S
authors.lastname: Chaignepain
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Schmitter
authors.forename: Alain
authors.initials: A
authors.lastname: Vavasseur
authors.forename: Jérôme
authors.initials: J
authors.lastname: Giraudat
authors.forename: Hélène
authors.initials: H
authors.lastname: Barbier-Brygoo
authors.forename: Sébastien
authors.initials: S
authors.lastname: Thomine
mesh_terms: D000040:Abscisic Acid / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000166:cytology / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D012694:Serine / Q000502:physiology; D015398:Signal Transduction
chemical_list: D029681:Arabidopsis Proteins; D011993:Recombinant Fusion Proteins; D012694:Serine; D000040:Abscisic Acid; D011494:Protein Kinases; C472139:OST1 protein, Arabidopsis