title: Reconstitution of an Initial Step of Phototropin Signaling in Stomatal Guard Cells.
abstract: Phototropins are light-activated receptor kinases that mediate a wide range of blue light responses responsible for the optimization of photosynthesis. Despite the physiological importance of phototropins, it is still unclear how they transduce light signals into physiological responses. Here, we succeeded in reproducing a primary step of phototropin signaling in vitro using a physiological substrate of phototropin, the BLUS1 (BLUE LIGHT SIGNALING1) kinase of guard cells. When PHOT1 and BLUS1 were expressed in Escherichia coli and the resulting recombinant proteins were incubated with ATP, white and blue light induced phosphorylation of BLUS1 but red light and darkness did not. Site-directed mutagenesis of PHOT1 and BLUS1 revealed that the phosphorylation was catalyzed by phot1 kinase. Similar to stomatal blue light responses, the BLUS1 phosphorylation depended on the fluence rate of blue light and was inhibited by protein kinase inhibitors, K-252a and staurosporine. In contrast to the result in vivo, BLUS1 was not dephosphorylated in vitro, suggesting the involvement of a protein phosphatase in the response in vivo. phot1 with a C-terminal kinase domain but devoid of the N-terminal domain, constitutively phosphorylated BLUS1 without blue light, indicating that the N-terminal domain has an autoinhibitory action and prevents substrate phosphorylation. The results provide the first reconstitution of a primary step of phototropin signaling and a clue for understanding the molecular nature of this process.
authors.affiliation: Department of Biology, Faculty of Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395 Japan kenrcb@kyushu-u.org.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Takemiya
authors.affiliation: Department of Biology, Faculty of Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395 Japan kenrcb@kyushu-u.org.
authors.forename: Ayaka
authors.initials: A
authors.lastname: Doi
authors.affiliation: Department of Biology, Faculty of Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395 Japan kenrcb@kyushu-u.org.
authors.forename: Sayumi
authors.initials: S
authors.lastname: Yoshida
authors.affiliation: Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka, 599-851 Japan Present address: Department of Physics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Kanagawa, 223-8522 Japan. kenrcb@kyushu-u.org.
authors.forename: Koji
authors.initials: K
authors.lastname: Okajima
authors.affiliation: Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka, 599-851 Japan kenrcb@kyushu-u.org.
authors.forename: Satoru
authors.initials: S
authors.lastname: Tokutomi
authors.affiliation: Department of Biology, Faculty of Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395 Japan kenrcb@kyushu-u.org.
authors.forename: Ken-Ichiro
authors.initials: K
authors.lastname: Shimazaki
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000502:physiology* / Q000528:radiation effects; D029681:Arabidopsis Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002227:Carbazoles / Q000494:pharmacology; D003624:Darkness; D026121:Indole Alkaloids / Q000494:pharmacology; D008027:Light; D055537:Light Signal Transduction; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism*; D010788:Photosynthesis; D056949:Phototropins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018524:Phototropism; D054046:Plant Stomata / Q000235:genetics / Q000502:physiology* / Q000528:radiation effects; D017346:Protein Serine-Threonine Kinases; D011994:Recombinant Proteins
chemical_list: D029681:Arabidopsis Proteins; C000612260:BLUS1 protein, Arabidopsis; D002227:Carbazoles; D026121:Indole Alkaloids; D010750:Phosphoproteins; D056949:Phototropins; D011994:Recombinant Proteins; C049985:staurosporine aglycone; D010770:Phosphotransferases; C103646:NPH1 protein, Arabidopsis; D017346:Protein Serine-Threonine Kinases
keywords: Arabidopsis; Blue light; Phosphorylation; Phototropin; Stomata