title: STN8 protein kinase in Arabidopsis thaliana is specific in phosphorylation of photosystem II core proteins.
abstract: Combination of reversed genetics with analyses of in vivo protein phosphorylation in Arabidopsis thaliana revealed that STN8 protein kinase is specific in phosphorylation of N-terminal threonine residues in D1, D2, and CP43 proteins, and Thr-4 in the PsbH protein of photosystem II. Phosphorylation of D1, D2, and CP43 in the light-exposed leaves of two Arabidopsis lines with T-DNA insertions in the stn8 gene was found significantly reduced in the assays with anti-phosphothreonine antibodies. Protein phosphorylation in each of the mutants was quantified comparatively to the wild type by mass spectrometric analyses of phosphopeptides released from the photosynthetic membranes and differentially labeled with stable isotopes. The lack of STN8 caused 50-60% reduction in D1 and D2 phosphorylation, but did not change the phosphorylation level of two peptides that could correspond to light-harvesting proteins encoded by seven different genes in Arabidopsis. Phosphorylation of the PsbH protein at Thr-4 was completely abolished in the plants lacking STN8. Phosphorylation of Thr-4 in the wild type required both light and prior phosphorylation at Thr-2, indicating that STN8 is a light-activated kinase that phosphorylates Thr-4 only after another kinase phosphorylates Thr-2. Analysis of the STN8 catalytic domain suggests that selectivity of STN8 in phosphorylation of the very N-terminal residues in D1, D2, and CP43, and Thr-4 in PsbH pre-phosphorylated at Thr-2 may be explained by the long loops obstructing entrance into the kinase active site and seven additional basic residues in the vicinity of the catalytic site, as compared with the homologous STN7 kinase responsible for phosphorylation of light-harvesting proteins.
authors.affiliation: Division of Cell Biology, Linköping University, SE-581 85 Linköping, Sweden.
authors.forename: Julia P
authors.initials: JP
authors.lastname: Vainonen
authors.forename: Maria
authors.initials: M
authors.lastname: Hansson
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Vener
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D020134:Catalytic Domain; D016384:Consensus Sequence; D018744:DNA, Plant / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D017343:Genes, Plant; D008027:Light; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009154:Mutation; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D045332:Photosystem II Protein Complex / Q000737:chemistry* / Q000378:metabolism*; D018515:Plant Leaves / Q000166:cytology; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D011494:Protein Kinases / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity; D013912:Threonine / Q000737:chemistry; D020524:Thylakoids / Q000737:chemistry / Q000378:metabolism; D014357:Trypsin / Q000494:pharmacology
chemical_list: D018744:DNA, Plant; D010455:Peptides; D045332:Photosystem II Protein Complex; D010940:Plant Proteins; D013912:Threonine; D011494:Protein Kinases; C504925:STN8 protein, Arabidopsis; D014357:Trypsin