title: Conserved phosphorylation sites in the activation loop of the Arabidopsis phytosulfokine receptor PSKR1 differentially affect kinase and receptor activity.
abstract: PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser(696) and Ser(698) in the JM (juxtamembrane) region and probably Ser(886) and/or Ser(893) in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser(717) in the JM, and at Ser(733), Thr(752), Ser(783), Ser(864), Ser(911), Ser(958) and Thr(998) in the kinase domain. The LC-ESI-MS/MS spectra provided support that up to three sites (Thr(890), Ser(893) and Thr(894)) in the AL were likely to be phosphorylated in vitro. These sites are evolutionarily highly conserved in PSK receptors, indicative of a conserved function. Site-directed mutagenesis of the four conserved residues in the activation segment, Thr(890), Ser(893), Thr(894) and Thr(899), differentially altered kinase activity in vitro and growth-promoting activity in planta. The T899A and the quadruple-mutated TSTT-A (T890A/S893A/T894A/T899A) mutants were both kinase-inactive, but PSKR1(T899A) retained growth-promoting activity. The T890A and S893A/T894A substitutions diminished kinase activity and growth promotion. We hypothesize that phosphorylation within the AL activates kinase activity and receptor function in a gradual and distinctive manner that may be a means to modulate the PSK response.
authors.affiliation: Entwicklungsbiologie und Physiologie der Pflanzen, Christian-Albrechts-Universität Kiel, Am Botanischen Garten 5, 24118 Kiel, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Hartmann
authors.affiliation: Systematische Proteomics und Bioanalytik, Christian-Albrechts-Universität Kiel, Niemannsweg 11, 24105 Kiel, Germany.
authors.forename: Dennis
authors.initials: D
authors.lastname: Linke
authors.affiliation: Entwicklungsbiologie und Physiologie der Pflanzen, Christian-Albrechts-Universität Kiel, Am Botanischen Garten 5, 24118 Kiel, Germany.
authors.forename: Christine
authors.initials: C
authors.lastname: Bönniger
authors.affiliation: Systematische Proteomics und Bioanalytik, Christian-Albrechts-Universität Kiel, Niemannsweg 11, 24105 Kiel, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Tholey
authors.affiliation: Entwicklungsbiologie und Physiologie der Pflanzen, Christian-Albrechts-Universität Kiel, Am Botanischen Garten 5, 24118 Kiel, Germany msauter@bot.uni-kiel.de.
authors.forename: Margret
authors.initials: M
authors.lastname: Sauter
mesh_terms: D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; C515018:PSKR1 protein, Arabidopsis; D011956:Receptors, Cell Surface; D017346:Protein Serine-Threonine Kinases
keywords: Arabidopsis thaliana; PSKR1; activation loop; leucine-rich receptor receptor-like kinase; peptide signalling; phytosulfokine