論文 - 中道　範人

Characterization of the Prr1 response regulator with special reference to sexual development in Schizosaccharomyces pombe 査読有り

Norihito Nakamichi, Hisami Yanada, Hirofumi Aiba, Keisuke Aoyama, Ryosuke Ohmiya, Takeshi Mizuno

Biosci Biotechnol Biochem   67 巻   頁： 547-555   2003年

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The histidine (His)-to-Aspartate (Asp) phosphorelay is a paradigm of intracellular signaling systems through protein phosphorylation in both prokaryotes and eukaryotes. The fission yeast Schizosaccharomyces pombe has three histidine kinases (Phk1/Mak2, Phk2/Mak3, and Phk3/Mak1), together with two response regulators (Mcs4 and Prr1). The results of recent extensive studies suggested that these His-to-Asp phosphorelay components are involved in oxidative stress responses through the transcriptional regulation of several scavenger genes for toxic free radicals. It was also suggested that they were somehow implicated in control of both the mitotic and meiotic cell proliferations. Among these S. pombe His-to-Asp phosphorelay components, however, the function of Prr1 is less clear. We here characterized a mutant, named prr1-D418N, specifying an altered Prr1 protein that presumably acts as a gain-of-function (or constitutive-active) mutant, with special reference to sexual development. The mutant cells showed a striking phenotype in that they underwent mating even in a nitrogen-sufficient medium, under which conditions the wild-type cells hardly did so. Furthermore, the mutant cells underwent mating very rapidly in a nitrogen-deficient medium. Under anaerobic (or micro-aerobic) growth conditions, the wild-type cells were not capable of undergoing sexual development even in a nitrogen-deficient medium. The prr1-D418N cells underwent mating efficiently under such anaerobic growth conditions. Taken these together, it was suggested that the function of Prr1 is closely linked to the well-characterized signaling pathways for induction of the sexual development, in a way that this response regulator regulates a critical step of the initiation of meiosis through activating the transcription of ste11+, mam2+, and mei2+, in S. pombe.

Cell autonomous circadian waves of the APRR1/TOC1 quintet in an established cell line of Arabidopsis thaliana 査読有り

Norihito Nakamichi, Akinori Matsushika, Takafumi Yamashino, Takeshi Mizuno

Plant Cell Physiol   44 巻   頁： 360-365   2003年

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

A small family of genes, named Arabidopsis Pseudo Response Regulator (APRR), are intriguing with special reference to circadian rhythms in plants, based on the fact that one of the members (APRR1) is identical to TOC1 (Timing of CAB Expression 1) that is believed to encode a clock component. In Arabidopsis plants, each transcript of the APRR1/TOC1 quintet genes starts accumulating after dawn rhythmically and one after another at intervals in the order of APRR9 --> APRR7 --> APRR5 --> APRR3 --> APRR1/TOC1. To characterize such intriguing circadian-associated events, we employed an established Arabidopsis cell line (named T87). When T87 cells were grown in an appropriate light and dark cycle, cell autonomous diurnal oscillations of the APRR1/TOC1 quintet genes were observed at the level of transcription, as seen in intact plants. After transfer to the conditions without any environmental time cues, particularly in constant dark, we further showed that free-running circadian rhythms persisted in the cultured cells, not only for the APRR1/TOC1 quintet genes, but also other typical circadian-controlled genes including CCA1 (Circadian Clock Associated 1), LHY (Late Elongated Hypocotyl) and CCR2 (Cold Circadian Rhythm RNA Binding 2). To our knowledge, this is the first indication of cell autonomous circadian rhythms in cultured cells in Arabidopsis thaliana, which will provide us with an alternative and advantageous means to characterize the plant biological clock.


The APRR3 component of the clock-associated APRR1/TOC1 quintet is phosphorylated by a novel protein kinase belonging to the WNK family, the gene for which is also transcribed rhythmically in Arabidopsis thaliana 査読有り

Masaya Murakami-Kojima, Norihito Nakamichi, Takafumi Yamashino, Takeshi Mizuno

Plant Cell Physiol   43 巻   頁： 675-683   2002年

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

In higher plants, clock-controlled circadian rhythms are a longstanding issue in physiology, and a newly emerging paradigm of molecular biology. In the model higher plant Arabidopsis thaliana, several genes have been proposed to encode potential clock-associated components, including a member (APRR1/TOC1) of the pseudo-response regulator family. We previously showed that transcripts of the APRR1/TOC1 family start accumulating after dawn rhythmically and sequentially at approximately 2 h intervals in the order of APRR9-->APRR7-->APRR5-->APRR3-->APRR1/ TOC1. This and other results led us to propose that this APRR1/TOC1 quintet might play coordinate roles in the mechanism underlying circadian rhythms in higher plants. To gain further insight as to such an idea, we here attempt to identify proteins that interact with one of the quintet members, APRR3. The identified component is a novel protein kinase, named WNK1, which is considerably similar to, but clearly distinct from, mitogen-activated protein kinases (MAPKs). It was found that APRR3 is a substrate of this novel protein kinase, the gene for which also shows a rhythmic transcription profile that is well coincident with the APRR3 rhythm. These findings give new insight into the mechanisms underlying the circadian rhythm in A. thaliana, providing a molecular link between the putative clock component, APRR3, and WNK1, a novel protein kinase that might be implicated as a signal transducer.

His-to-Asp Phosphorelay Circuitry for Regulation of Sexual Development in Schizosaccharomyces pombe 査読有り

Norihito Nakamichi, Hisami Yamada, Ryusuke Ohmiya, Keisuke Aoyama, Hirofumi Aiba, Takeshi Mizuno

Biosci.Biotechnol.Biochem   66 巻   頁： 2663-2672   2002年

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The fission yeast Schizosaccharomyces pombe has three histidine kinases (Phk1/Mak2, Phk2/Mak3, and Phk3/Mak1), and two response regulators (Mcs4 and Prr1). The results of recent extensive studies on the S. pombe His-to-Asp phosphorelay circuitry suggested that it is involved in oxidative stress responses through the transcriptional regulation of several scavenger genes for toxic free radicals. The functions of these histidine kinases have not yet been fully characterized. Here we characterize a homothallic (h90) mutant lacking the genes for all the histidine kinases, with special reference to sexual development. Homothallic phk1/2/3delta cells underwent mating precociously in a nitrogen-deficient medium. Surprisingly, the mutant cells underwent mating even in a nitrogen-sufficient medium, under which conditions wild-type cells did so rarely if at all. Under anaerobic (or microaerobic) growth conditions, wild-type cells did not undergo sexual development even in a nitrogen-deficient medium, but the homothallic phk1/2/3delta cells mated efficiently. Oxidative reagents such as H2O2 induced sexual development in wild-type cells grown anaerobically. On the basis of these results, we propose the novel view that the S. pombe His-to-Asp phosphorelay, initiated by the Phk histidine kinases, is crucial for regulation of sexual development. This Phk-mediated signaling pathway is linked to the well-documented canonical pathway for induction of the sexual development, in that both converge at the initiation of meiosis through activation of ste11+, mam2+, and mei2+ transcription.

Compilation and characterization of a novel WNK family of protein kinases in Arabiodpsis thaliana with reference to circadian rhythms 査読有り

Norihito Nakamichi, Masaya Murakami-Kojima, Eriko Sato, Yasuko Kishi, Takafumi Yamashino, Takeshi Mizuno

Biosci Biotechnol Biochem   66 巻   頁： 2429-2436   2002年

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The complete genome sequence of Arabidopsis thaliana revealed that this higher plant has a tremendous number of protein kinases. We recently isolated a novel type of protein kinase, named AtWNK1, which shows an in vitro ability to phosphorylate the APRR3 member of the APRR1/TOC1 quintet that has been implicated in a mechanism underlying circadian rhythms in Arabidopsis. We here address two issues, one general and one specific, as to this novel protein kinase. We first asked the general question of how many WNK family members are present in this higher plant, then whether or not other members are also relevant to circadian rhythms. The results of our analyses showed that Arabidopsis has at least 9 members of the WNK1 family of protein kinases (designated here as WNK1 to WNK9), the structural design of which is clearly distinct from those of other known protein kinases, such as receptor-like kinases and mitogen-activated protein kinases. They were examined with special reference to the circadian-related APRR1/TOC1 quintet. It was found that not only the transcription of the WNK1 gene, but also those of three other members (WNK2, WNK4, and WNK6) are under the control of circadian rhythms. These results suggested that certain members of the WNK family of protein kinases might play roles in a mechanism that generates circadian rhythms in Arabidopsis.

Aberrant Expression of the Arabidopsis Circadian-Regulated APRR5 Gene Belonging to the APRR1/TOC1 Quintet Result in Early Flowering and Hypersensitive to Light in Early Photomorphogenesis 査読有り

Eriko Sato, Norihito Nakamichi, Takafumi Yamashino, Takeshi Mizuno

Plant Cell Physiol   43 巻   頁： 1374-1385   2002年

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

In Arabidopsis thaliana, the transcripts of the APRR1/TOC1 family genes each start accumulating after dawn rhythmically and one after another at intervals in the order of APRR9-->APRR7-->APRR5-->APRR3-->APRR1/TOC1 under continuous light. Except for the well-characterized APRR1/TOC1, however, no evidence has been provided that other APRR1/TOC1 family genes are indeed implicated in the mechanisms underlying circadian rhythms. We here attempted to provide such evidence by characterizing transgenic plants that constitutively express the APRR5 gene. The resulting APRR5-overexpressing (APRR5-ox) plants showed intriguing properties with regard to not only circadian rhythms, but also control of flowering time and light response. First, the aberrant expression of APRR5 in such transgenic plants resulted in a characteristic phenotype with regard to transcriptional events, in which free-running rhythms were considerably altered for certain circadian-regulated genes, including CCA1, LHY, APRR1/TOC1, other APRR1/TOC1 members, GI and CAB2, although each rhythm was clearly sustained even after plants were transferred to continuous light. With regard to biological events, APRR5-ox plants flowered much earlier than wild-type plants, more or less, in a manner independent of photoperiodicity (or under short-day conditions). Furthermore, APRR5-ox plants showed an SRL (short-hypocotyls under red light) phenotype that is indicative of hypersensitiveness to red light in early photomorphogenesis. Both APRR1-ox and APRR9-ox plants also showed the same phenotype. Therefore, APRR5 (together with APRR1/TOC1 and APRR9) must be taken into consideration for a better understanding of the molecular links between circadian rhythms, control of flowering time through the photoperiodic long-day pathway, and also light signaling-controlled plant development.

Identification and characterization of a novel gene, hos2+, the function of which is necessary for growth under high osmotic stress in fission yeast 査読有り

Norihito Nakamichi, Eiji Yamamoto, Hisami Yamada, Hirofumi Aiba, Takeshi Mizuno

Biosci Biotechnol Biochem   64 巻   頁： 2493-2496   2000年

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

hos2 mutants of the fission yeast Schizosaccharomyces pombe showed the phenotype of high osmolarity sensitivity for growth. An S. pombe strain carrying the hos2-M10 allele cannot form colonies on agar plates containing 2 M glucose, but the parental strain can do so very well, as demonstrated previously. In this study, the hos2+ gene was identified as one that encodes a small protein of 94 amino acids, which shows no sequence similarity to any other proteins in the current databases. The hos2-M10 mutation resulted in Gln-62 to TAG-termination codon. A Hos2-defective (hos2delta) strain, which we then constructed, showed the phenotype of high osmolarity sensitivity, as in the case of the original hos2-M10 mutant. For this hos2delta mutant, three multicopy suppressor genes were isolated and one of which was identified as the pgk1+ gene, encoding a phosphoglycerate kinase.