title: Mutations in N-terminal flanking region of blue light-sensing light-oxygen and voltage 2 (LOV2) domain disrupt its repressive activity on kinase domain in the Chlamydomonas phototropin.
abstract: Phototropin is a light-regulated kinase that mediates a variety of photoresponses such as phototropism, chloroplast positioning, and stomata opening in plants to increase the photosynthetic efficiency. Blue light stimulus first induces local conformational changes in the chromophore-bearing light-oxygen and voltage 2 (LOV2) domain of phototropin, which in turn activates the serine/threonine (Ser/Thr) kinase domain in the C terminus. To examine the kinase activity of full-length phototropin conventionally, we employed the budding yeast Saccharomyces cerevisiae. In this organism, Ser/Thr kinases (Fpk1p and Fpk2p) that show high sequence similarity to the kinase domain of phototropins exist. First, we demonstrated that the phototropin from Chlamydomonas reinhardtii (CrPHOT) could complement loss of Fpk1p and Fpk2p to allow cell growth in yeast. Furthermore, this reaction was blue light-dependent, indicating that CrPHOT was indeed light-activated in yeast cells. We applied this system to a large scale screening for amino acid substitutions in CrPHOT that elevated the kinase activity in darkness. Consequently, we identified a cluster of mutations located in the N-terminal flanking region of LOV2 (R199C, L202L, D203N/G/V, L204P, T207I, and R210H). An in vitro phosphorylation assay confirmed that these mutations substantially reduced the repressive activity of LOV2 on the kinase domain in darkness. Furthermore, biochemical analyses of the representative T207I mutant demonstrated that the mutation affected neither spectral nor multimerization properties of CrPHOT. Hence, the N-terminal flanking region of LOV2, as is the case with the C-terminal flanking Jα region, appears to play a crucial role in the regulation of kinase activity in phototropin.
authors.affiliation: Department of Botany, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Aihara
authors.affiliation: Division of Molecular Interaction, Institute for Genetic Medicine, Hokkaido University Graduate School of Life Science, Sapporo 060-0815, Japan, and.
authors.forename: Takaharu
authors.initials: T
authors.lastname: Yamamoto
authors.affiliation: Department of Biological Sciences, Graduate School of Science, Osaka Prefecture University, Osaka 599-8531, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Okajima
authors.affiliation: Department of Botany, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Yamamoto
authors.affiliation: Department of Botany, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.
authors.forename: Tomomi
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: Department of Biological Sciences, Graduate School of Science, Osaka Prefecture University, Osaka 599-8531, Japan.
authors.forename: Satoru
authors.initials: S
authors.lastname: Tokutomi
authors.affiliation: Division of Molecular Interaction, Institute for Genetic Medicine, Hokkaido University Graduate School of Life Science, Sapporo 060-0815, Japan, and.
authors.forename: Kazuma
authors.initials: K
authors.lastname: Tanaka
authors.affiliation: Department of Botany, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan,. Electronic address: nagatani@physiol.bot.kyoto-u.ac.jp.
authors.forename: Akira
authors.initials: A
authors.lastname: Nagatani
mesh_terms: D016825:Chlamydomonas reinhardtii / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010766:Phosphorylation / Q000235:genetics; D056949:Phototropins / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
chemical_list: D056949:Phototropins