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。

• 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

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

一个独特的Cdk（Cdc 28）在酵母细胞周期的进程中起作用，但存在一个Cdk家族，其成员包括Pho 85激酶，在各种细胞事件中起作用。在哺乳动物Cdk家族成员中，尚未证明Cdk 5参与细胞增殖。cdk 5通过与p35结合而被激活，在神经发生的调控中起重要作用。Cdk 5和Pho 85激酶在结构以及活性调节方面具有相似性。它们不被Cdk激活激酶进一步激活，而Cdk 5的Y15和Pho 85的Y18的磷酸化似乎分别增强p35和Pho 80的结合。我们发现在pho 85 Δ细胞中产生的小鼠Cdk 5可以抑制pho 85 Δ突变体的一些表型，包括不能在非发酵性碳源上生长、形态缺陷以及由Pho 4或Clb 2过量产生引起的生长缺陷。我们还证明Cdk 5与Pho 85-cyclins共免疫沉淀，免疫复合物可以磷酸化P ...更多信息 ho 4，Pho 85激酶的天然底物。因此，小鼠Cdk 5是酵母Pho 85激酶的功能同源物。这一发现将进一步揭示这两种激酶的功能：然而，与p35-Cdk 5相关的调节神经元发育的未知因子，以及与cyclin-Pho 85复合物相互作用的调节细胞周期和细胞形态的酵母蛋白将被鉴定。我们进行了基因芯片分析以鉴定其表达受pho 85无效突变影响的基因。我们发现一些参与碳水化合物代谢的基因受到了影响，无论是积极的还是消极的。其中，我们重点研究了生长所必需的UGP 1基因，该基因催化UTP和葡萄糖1-磷酸合成UDP-葡萄糖。通过北方分析和检测由UGP 1启动子和lacZ组成的报告基因的活性，我们证实了在缺乏PHO 85的细胞中UGP 1表达及其启动子活性增加。从启动子中删除推定的Pho 4和Bas 2结合位点或引入Pho 4无效突变减少了Pho 85无效突变体中报告活性的增加，表明Pho 85激酶通过Pho 4调节UGP 1表达。少

项目成果

Nishizawa, M., Y.Kanaya, A.Toh-e.: "Mouse cyclin-dependent kinase (Cdk) 5 is a functional homologue of a yeast Cdk, Pho85 kinase."J.Biol.Chem.. 274. 33859-33862 (1999)

Nishizawa, M., Y.Kanaya, A.Toh-e.：“小鼠细胞周期蛋白依赖性激酶 (Cdk) 5 是酵母 Cdk Pho85 激酶的功能同源物。”J.Biol.Chem.. 274. 33859-

M.Nishizawa: "The Pho85 kinase, a member of the yeast cyclin-dependent kinase (Cdk) family, has a regulation mechanism different from Cdks functioning throughout the cell cycle"Genes to Cells. 4. 627-642 (1999)

M.Nishizawa：“Pho85 激酶是酵母细胞周期蛋白依赖性激酶 (Cdk) 家族的成员，其调节机制不同于在整个细胞周期中发挥作用的 Cdks”《基因到细胞》。