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ANALYSIS OF A NEW FACTOR RELATED WITH THE SIGNAL TRANSDUCTION IN FISSION YEAST

裂殖酵母信号转导新因子的分析

基本信息

批准号：
10660087
负责人：
KAWAMUKAI Makoto
金额：
$ 2.3万
依托单位：
SHIMANE UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10660087/
关键词：
RAS cAMP Signal transduction fission yeast 14-3-3 Byr2 slal 14・3・3 cAMP

项目摘要

Schizosaccharomyces pombe expresses two homologs of mammalian 14-3-3 genes : rad24, which is involved in checkpoint control, and rad25, the function of which is considered to be supplementary to rad24 activity. In this study, we present evidence that both Rad24 and Rad25 act as negative regulators of Byr2 (MAPKKK). Overexpression of either rad24 or rad25 reduced sporulation efficiency in homothallic wild-type cells. In contrast, the sporulation efficiency of rad24 and rad25 null cell was higher than that of wild-type cells. Deletion of rad24 or rad25 increased sporulation efficiency in ras1 null cells, but not in byr2 or ste4 null cells. Rad24 and Rad25 had no effect on the activity of constitutively active Byr1S214DT218D. Rad24 and Rad25 bound to both the N-terminal and the C-terminal domains of Byr2 in the yeast two-hybrid system. The formation of complexes in vivo was also confirmed by immuno-coprecipitation. Furthermore, we showed that an antibody against phospho-p44/42 MAP kinase recognized elevated phosphorylation levels of Spk1 protein (MAPK) in rad24 and rad25 null cells, and that the mRNA level of mam2, which is regulated by Ras1, was clearly elevated in ras1 rad24 and ras1 rad25 null cells relative to ras1 null cells. The cellular localization of Rad24, Rad25 and Byr2 was examined using fusion to green fluorescent protein (GFP). While Byr2-GFP translocated from the cytoplasm to the plasma membrane in response to nutrient starvation, Rad24-GFP and Rad25-GFP remained in the cytoplasm. Our of all results consistently indicate that Rad24 and Rad25 bind to Byr2 in the cytoplasm and negatively regulate Byr2 signaling in the sexual development in S.pombe.

粟酒裂殖酵母表达哺乳动物14-3-3基因的两个同源物：rad 24，其参与检查点控制，和rad 25，其功能被认为是rad 24活性的补充。在这项研究中，我们提出的证据表明，Rad 24和Rad 25作为负调控Byr 2（MAPKKK）。rad 24或rad 25的过表达降低了同宗配合野生型细胞中的孢子形成效率。相反，rad 24和rad 25缺失细胞的产孢效率高于野生型细胞。缺失rad 24或rad 25增加了ras 1缺失细胞的产孢效率，但在byr 2或ste 4缺失细胞中不增加产孢效率。Rad 24和Rad 25对组成型活性Byr 1 S214 DT 218 D的活性没有影响。在酵母双杂交系统中，Rad 24和Rad 25与Byr 2的N-末端和C-末端结构域结合。免疫共沉淀法也证实了体内复合物的形成。此外，我们发现，磷酸化p44/42 MAP激酶的抗体识别升高的磷酸化水平的Spk 1蛋白（MAPK）在rad 24和rad 25无效的细胞，和mam 2的mRNA水平，这是由Ras 1调节，明显升高ras 1 rad 24和ras 1 rad 25无效的细胞相对于ras 1无效的细胞。使用与绿色荧光蛋白（GFP）的融合来检查Rad 24、Rad 25和Byr 2的细胞定位。虽然Byr 2-GFP响应于营养饥饿而从细胞质易位到质膜，但Rad 24-GFP和Rad 25-GFP保留在细胞质中。我们的所有结果一致表明，Rad 24和Rad 25绑定到Byr 2的细胞质和负调控Byr 2信号在S. pombe的性发育。