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发挥作用，但存在一个 Cdk 家族，其成员（包括 Pho85 激酶）在多种细胞事件中发挥作用。在哺乳动物 Cdk 家族成员中，Cdk5 尚未被证明参与细胞事件。通过与 p35 结合激活的 Cdk5 在神经发生的控制中发挥重要作用，Cdk5 和 Pho85 激酶在结构及其活性调节方面具有相似性。它们不会被 Cdk 激活激酶进一步激活，而 Cdk5 的 Y15 和 Pho85 的 Y18 的磷酸化似乎分别增强 p35 和 Pho80 的结合，我们发现 pho85Δ 细胞中产生的小鼠 Cdk5 可以抑制一些 pho85Δ 突变表型，包括失败。我们还证明了在不可发酵碳源上生长、形态缺陷以及 Pho4 或 Clb2 过量产生的生长缺陷。 Cdk5 与 Pho85-cyclins 共免疫沉淀，并且免疫复合物可以磷酸化 Pho85 激酶的天然底物 P … More ho4，因此小鼠 Cdk5 是酵母 Pho85 激酶的功能同源物，这一发现将进一步揭示两者的功能。激酶：与 p35-Cdk5 相关以调节神经元发育的未知因素，以及与细胞周期蛋白-Pho85 复合物相互作用的酵母蛋白为了寻找更多Pho85激酶的靶标，我们进行了基因芯片分析，以鉴定其表达受pho85无效突变影响的基因，我们发现一些与碳水化合物代谢有关的基因。其中，我们重点关注对生长至关重要的 UGP1 基因，该基因催化 UTP 和葡萄糖 1-磷酸合成 UDP-葡萄糖，并通过 Northern 分析和测定了报告基因的活性。通过对 UGP1 启动子和 lacZ 的分析，我们证实在缺乏 PHO85 的细胞中 UGP1 表达及其启动子活性增加，从启动子中删除推定的 Pho4 和 Bas2 结合位点或引入 pho4 无效突变会减少报告基因的增加。 Pho85 无效突变体中的活性，表明 Pho85 激酶通过 Pho4 调节 UGP1 表达。