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

一个独特的Cdk （Cdc28）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，尚未证实Cdk5参与细胞增殖。Cdk5通过结合p35激活，在神经发生的控制中起重要作用。Cdk5和Pho85激酶在结构和活性调控上有相似之处。它们不会被cdk活化激酶进一步激活，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突变表型，包括不能在不可发酵的碳源上生长、形态缺陷和由Pho4或Clb2过量产生引起的生长缺陷。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这两种激酶的功能：然而，与p35-Cdk5相关的未知因子调节神经元发育，以及与细胞周期蛋白- pho85复合物相互作用调节细胞周期进程和细胞形态的酵母蛋白将被确定。为了寻找更多的Pho85激酶靶点，我们进行了基因芯片分析，以鉴定受Pho85零突变影响的基因表达。我们发现一些参与碳水化合物代谢的基因受到了积极或消极的影响。其中，我们重点研究了生长所必需的UGP1基因，它催化UTP和葡萄糖1-磷酸合成udp -葡萄糖。通过northern分析和检测一个由UGP1启动子和lacZ组成的报告子的活性，我们证实在缺乏PHO85的细胞中，UGP1的表达及其启动子活性增加。从启动子中删除Pho4和Bas2结合位点或引入Pho4零突变会减少pho85零突变中报告基因活性的增加，这表明pho85激酶通过Pho4调节UGP1的表达。少

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