caa a22 4500 467894698 CHVBK 20180406152759.0 cr unu---uuuuu 170328e20060501xx s 000 0 eng 10.1007/s11103-006-0014-y doi (NATIONALLICENCE)springer-10.1007/s11103-006-0014-y Cell Cycle Regulated D3-type Cyclins form Active Complexes with Plant-specific B-type Cyclin-dependent Kinase in vitro [Elektronische Daten] [Kazue Kawamura, James Murray, Atsuhiko Shinmyo, Masami Sekine] Tobacco (Nicotiana tabacum L.) cv Bright Yellow-2 (BY-2) cells are the most highly synchronizable plant cell culture, and previously we used them to analyze cell cycle regulation of cyclin-dependent kinases (CDKs) containing the cyclin binding motifs PSTAIRE (CDKA) and PPTA/TLRE (CDKB). Here we describe the analysis of tobacco CycD3 cyclins whose transcripts predominantly accumulate during G2 to M phase, which represents a unique feature of this type of cyclin D in plants. Although protein levels of CycD3s fluctuate with different patterns during the cell cycle, kinase assays revealed that the CycD3-associated kinases phosphorylate histone H1 and the tobacco retinoblastoma related protein (NtRBR1) with two peaks at the G1/S and G2/M boundaries. In vitro pull-down assays revealed that cell cycle-regulated CycD3s bind to CDKA, but more weakly than does CycD3;3, and that they also bind to CDKB and the CDK inhibitor NtKIS1a. Mutations in the cyclin box of the CycD3s showed that two amino acids are required for binding with CDKA and NtKIS1a, but no diminished interaction was observed with CDKB. A reconstituted kinase assay was adapted for use with bacterially produced GST-CycD3s, and kinase activity could be activated by incubation of extracts from exponentially growing BY-2 cells. Such activated complexes contained CDKA and CDKB, and the reconstituted GST-CycD3 mutants, retaining binding ability to CDKB, showed kinase activity, suggesting that these cell cycle-regulated CycD3s form active complexes with both A- and B-type CDKs in vitro. Springer, 2006 cyclin nationallicence cell cycle nationallicence kinase assay nationallicence BY-2 : Bright Yellow-2 nationallicence CDK : cyclin-dependent serine/threonine protein kinase nationallicence GST : glutathione S-transferase nationallicence ORF : open reading frame nationallicence Rb : retinoblastoma nationallicence RBR : Rb-related protein nationallicence Kawamura Kazue Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), Takayama 8916-5, 630-0101, Ikoma, Nara, Japan aut Murray James Institute of Biotechnology, University of Cambridge, Tennis Court Road, CB2 1QT, Cambridge, UK aut Shinmyo Atsuhiko Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), Takayama 8916-5, 630-0101, Ikoma, Nara, Japan aut Sekine Masami Department of Bioproduction Science, Faculty of Bioresources and Environmental Sciences, Ishikawa Prefectural University, Suematsu 1-308, 921-8836, Nonoichimachi, Ishikawa, Japan aut Plant Molecular Biology Kluwer Academic Publishers 61/1-2(2006-05-01), 311-327 0167-4412 61:1-2<311 2006 61 11103 https://doi.org/10.1007/s11103-006-0014-y text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s11103-006-0014-y text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Kawamura Kazue Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), Takayama 8916-5, 630-0101, Ikoma, Nara, Japan aut NATIONALLICENCE 700 1- Murray James Institute of Biotechnology, University of Cambridge, Tennis Court Road, CB2 1QT, Cambridge, UK aut NATIONALLICENCE 700 1- Shinmyo Atsuhiko Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), Takayama 8916-5, 630-0101, Ikoma, Nara, Japan aut NATIONALLICENCE 700 1- Sekine Masami Department of Bioproduction Science, Faculty of Bioresources and Environmental Sciences, Ishikawa Prefectural University, Suematsu 1-308, 921-8836, Nonoichimachi, Ishikawa, Japan aut NATIONALLICENCE 773 0- Plant Molecular Biology Kluwer Academic Publishers 61/1-2(2006-05-01), 311-327 0167-4412 61:1-2<311 2006 61 11103 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer