Regulation of yeast cell cycle by Cdk family and its application for drug discovery.

Cdk家族对酵母细胞周期的调节及其在药物发现中的应用。

基本信息

批准号：
11660096
负责人：
NISHIZAWA Masafumi
金额：
$ 2.43万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11660096/
关键词：
Cell cycle Yeast Cyclin-dependent kinase Carbon metabolism Gene chip G_1期 M期糖新生系グリコーゲン代謝系

项目摘要

A unique Cdk (Cdc28) functions in the progression of yeast cell cycle, but there exists a Cdk family whose members, including Pho85 kinase, function in various cellular events. Among mammalian Cdk family members, Cdk5 is not yet demonstrated to be involved in cell proliferation. Cdk5, activated by binding of p35, plays an important role in control of neurogenesis. Cdk5 and Pho85 kinases share similarities in structure as well as in the regulation of their activity. They are not further activated by Cdk-activating kinase, whereas phosphorylation of Y15 of Cdk5 and Y18 of Pho85 appears to enhance binding of p35 and Pho80, respectively. We found that mouse Cdk5 produced in pho85Δ cells could suppress some of pho85Δ mutant phenotypes including failure to grow on nonfermentable carbon sources, morphological defects, and growth defect caused by Pho4 or Clb2 overproduction. We also demonstrated that Cdk5 coimmunoprecipitated with Pho85-cyclins, and that the immunocomplex could phosphorylate P … More ho4, a native substrate of Pho85 kinase. Thus mouse Cdk5 is a functional homologue of yeast Pho85 kinase. This discovery will lead to further revelation of the function of the two kinases : yet unknown factors that associate with p35-Cdk5 to regulate neuronal developments, and yeast proteins interacting with cyclin-Pho85 complex to regulate cell-cycle progression and cell morphology will be identified.To search more targets of Pho85 kinase, we carried out gene chip analysis to identify genes whose expression is affected by a pho85 null mutation. We found that some genes involved in the carbohydrate metabolism were affected, either positively or negatively. Among them, we focused on the UGP1 gene which is essential for growth, and catalyzes the synthesis of UDP-glucose from UTP and glucose 1-phosphate. By northern analysis and assaying the activity of a reporter composed of the UGP1 promoter and lacZ, we confirmed that UGP1 expression and its promoter activity were increased in the cells lacking PHO85. A deletion of putative Pho4 and Bas2 binding sites from the promoter or an introduction of a pho4 null mutation diminished an increase in the reporter activity in a pho85 null mutant, suggesting that Pho85 kinase acts through Pho4 to regulate UGP1 expression. Less

独特的CDK（CDC28）在酵母细胞周期的进展中起作用，但存在一个CDK家族，其成员（包括Pho85激酶）在各种细胞事件中起作用。在哺乳动物CDK家族成员中，CDK5尚未证明与细胞增殖有关。通过p35的结合激活的CDK5在神经发生的控制中起着重要作用。 CDK5和PHO85激酶在结构以及其活性调节方面具有相似性。它们没有通过CDK激活激酶进一步激活，而PHO85的CDK5和Y18的磷酸化似乎分别增强了p35和PHO80的结合。我们发现在Pho85δ细胞中产生的小鼠CDK5可以抑制一些PHO85Δ突变体表型，包括未在不可发酵的碳源上生长，形态缺陷和由PHO4或CLB2引起的生长缺陷过量产生。我们还证明了CDK5与Pho85-Cyclins共免疫沉淀，并且免疫复合物可以磷酸化P…更多HO4，这是PHO85激酶的天然底物。 This discovery will lead to further relation of the function of the two kinases: yet unknown factors that associate with p35-Cdk5 to regulate neuronal developments, and yeast proteins interacting with cyclin-Pho85 complex to regulate cell-cycle progression and cell morphology will be identified.To search more targets of Pho85 kinase, we carried out gene chip analysis to identify genes whose expression is affected by a PHO85无效突变。我们发现，涉及碳水性代谢的一些基因受到积极或负面影响。其中，我们专注于对生长至关重要的UGP1基因，并催化从UTP和1-磷酸葡萄糖中的UDP-葡萄糖合成。通过北方分析并断言由UGP1启动子和LACZ组成的报告基因的活性，我们证实了缺乏PHO85的细胞中UGP1表达及其启动子活性的增加。从启动子中删除了假定的PHO4和BAS2结合位点，或引入PHO4 NAULL突变减少了PHO85 NULL突变体中报告基因活性的增加，这表明PHO85激酶通过Pho4起作用以调节UGP1表达。较少的