title: Salt stress triggers phosphorylation of the Arabidopsis vacuolar K+ channel TPK1 by calcium-dependent protein kinases (CDPKs).
abstract: 14-3-3 proteins play an important role in the regulation of many cellular processes. The Arabidopsis vacuolar two-pore K(+) channel 1 (TPK1) interacts with the 14-3-3 protein GRF6 (GF14-λ). Upon phosphorylation of the putative binding motif in the N-terminus of TPK1, GRF6 binds to TPK1 and activates the potassium channel. In order to gain a deeper understanding of this 14-3-3-mediated signal transduction, we set out to identify the respective kinases, which regulate the phosphorylation status of the 14-3-3 binding motif in TPK1. Here, we report that the calcium-dependent protein kinases (CDPKs) can phosphorylate and thereby activate the 14-3-3 binding motif in TPK1. Focusing on the stress-activated kinase CPK3, we visualized direct and specific interaction of TPK1 with the kinase at the tonoplast in vivo. In line with its proposed role in K(+) homeostasis, TPK1 phosphorylation was found to be induced by salt stress in planta, and both cpk3 and tpk1 mutants displayed salt-sensitive phenotypes. Molecular modeling of the TPK1-CPK3 interaction domain provided mechanistic insights into TPK1 stress-regulated phosphorylation responses and pinpointed two arginine residues in the N-terminal 14-3-3 binding motif in TPK1 critical for kinase interaction. Taken together, our studies provide evidence for an essential role of the vacuolar potassium channel TPK1 in salt-stress adaptation as a target of calcium-regulated stress signaling pathways involving Ca(2+), Ca(2+)-dependent kinases, and 14-3-3 proteins.
authors.affiliation: Department of Molecular Plant Physiology and Biophysics, Julius-von-Sachs-Institute, University of Würzburg, Julius-von-Sachs-Platz 2, D-97082 Würzburg, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Latz
authors.affiliation: Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories, University of Vienna, Dr. Bohrgasse 9, A-1030 Vienna, Austria.
authors.forename: Norbert
authors.initials: N
authors.lastname: Mehlmer
authors.affiliation: Department of Molecular Plant Physiology and Biophysics, Julius-von-Sachs-Institute, University of Würzburg, Julius-von-Sachs-Platz 2, D-97082 Würzburg, Germany.
authors.forename: Simone
authors.initials: S
authors.lastname: Zapf
authors.affiliation: Department of Molecular Plant Physiology and Biophysics, Julius-von-Sachs-Institute, University of Würzburg, Julius-von-Sachs-Platz 2, D-97082 Würzburg, Germany.
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Mueller
authors.affiliation: Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories, University of Vienna, Dr. Bohrgasse 9, A-1030 Vienna, Austria.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Wurzinger
authors.affiliation: Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories, University of Vienna, Dr. Bohrgasse 9, A-1030 Vienna, Austria.
authors.forename: Barbara
authors.initials: B
authors.lastname: Pfister
authors.affiliation: Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories, University of Vienna, Dr. Bohrgasse 9, A-1030 Vienna, Austria.
authors.forename: Edina
authors.initials: E
authors.lastname: Csaszar
authors.affiliation: Department of Molecular Plant Physiology and Biophysics, Julius-von-Sachs-Institute, University of Würzburg, Julius-von-Sachs-Platz 2, D-97082 Würzburg, Germany.|College of Science, King Saud University, Riyadh 11451, Saudi Arabia.
authors.forename: Rainer
authors.initials: R
authors.lastname: Hedrich
authors.affiliation: Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories, University of Vienna, Dr. Bohrgasse 9, A-1030 Vienna, Austria.
authors.forename: Markus
authors.initials: M
authors.lastname: Teige
authors.affiliation: Department of Molecular Plant Physiology and Biophysics, Julius-von-Sachs-Institute, University of Würzburg, Julius-von-Sachs-Platz 2, D-97082 Würzburg, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Becker
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism; D020816:Amino Acid Motifs; D017360:Arabidopsis / Q000166:cytology* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D055786:Gene Knockout Techniques; D018525:Germination; D006706:Homeostasis; D008958:Models, Molecular; D009154:Mutation; D010766:Phosphorylation; D011188:Potassium / Q000378:metabolism; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D055550:Protein Stability; D021381:Protein Transport; D012492:Salts / Q000494:pharmacology*; D015398:Signal Transduction; D013312:Stress, Physiological / Q000187:drug effects*; D014617:Vacuoles / Q000378:metabolism*
chemical_list: D048948:14-3-3 Proteins; D029681:Arabidopsis Proteins; D015221:Potassium Channels; D012492:Salts; C586033:TPK1 protein, Arabidopsis; C516786:CPK3 protein, Arabidopsis; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D011188:Potassium