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发挥作用，但存在一个CDK家族，其成员包括Pho85激酶，在各种细胞事件中发挥作用。缺乏Pho85激酶的酵母细胞表现出多效性，包括pho基因的结构性表达，糖原积累，细胞形态异常，生长速度缓慢，以及在没有CLN1和CLN2的情况下不能生长。已知Pho85可与10种细胞周期蛋白相互作用，但对哪一种激酶-细胞周期蛋白组合负责哪种细胞功能知之甚少。为了明确Pho85-Cyclin复合体的细胞靶点，我们构建了10个Pho85-Cyclin融合基因，并用各自的融合基因替换染色体上的PhO85位点，使酵母细胞产生独特的Pho85-Cyclin融合蛋白，使我们能够利用DNA微阵列来研究相应的激酶-Cyclin复合体的靶基因。首先，我们从…的能力来研究融合蛋白的功能更多补体Pho85突变表型。我们发现产生Pho85-Ph80的细胞可以抑制PHO5，产生Pho85-Pc11的细胞在没有CLN1和CLN2的情况下可以生长，产生Pho85-Pc18的细胞不积累糖原，这表明这些融合蛋白可以作为相应的复合体发挥作用。为了揭示Pho85和每个Pho85-Cyclin复合体的直接靶基因，我们构建了四环素诱导的PHO85和PHO85-Cyclin融合基因的表达系统，并将表达载体导入pko85 Pho80突变细胞。诱导后，在0~4h内定期提取RNA，转化成cRNA，用DNA芯片(GeneChip)进行表达分析。当PHO85被诱导时，观察到SWI5、CHS2、EGT2、PCL9、TSL1、PCL7和GSY1的诱导以及MAK11、DRS1和PRS3的抑制。PHO85-PHO80的诱导也得到了类似的结果，提示PHO85的大多数靶基因可能受PHO85-PHO80复合体的调控。然而，PCL7是由Pho85诱导的，而不是由Pho85-Pho80诱导的，这表明PCL7受激酶-周期蛋白复合体(ES)的调控，而不是Pho85-Pho80。综上所述，这种方法被证明是有用的，以区分10个不同的Pho85-Cyclin络合物的目标。较少

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. Yeast cells lacking Pho85 kinase display pleiotropic phaenotypes, including constitutive expression of PHO genes, accumulation of glycogen, abnormal cell morphology, slow growth rate, and inability to grow in the absence of CLN1 and CLN2. Pho85 is known to interact with 10 cyclin-lke proteins, but only a little is known regarding which kinase-cyclin combination is responsible for which cellular function. To clarify cellular targets of respective Pho85-cyclin complex, we constructed ten Pho85-cyclin fusion genes and replace the chromosomal PHO85 locus with respective fusion gene, so that the yeast cell produces unique Pho85-cyclin fusion protein, enabling us to study target genes of the corresponding kinase-cyclin complex using a DNA microarray.First, we studied function of the fusion proteins by their ability to … More complement pho85 mutant phenotypes. We found that cells producing Pho85-Pho80 could repress PHO5, those producing Pho85-Pc11 could grow in the absence of CLN1 and CLN2, and Pho85-Pc18 producing cells did not accumulate glycogen, which indicate that these fusion proteins can function as corresponding complex.To reveal immediate target genes of Pho85 and each Pho85-cyclin complex, we constructed an expression system of PHO85 and PHO85-cyclin fusion genes induced by tetracycline, and introduced the expression plasmid into pko85 pho80 mutant cells. After iduction of the kinase or kinase-cyclin fusion gene, RNA was extracted periodically from 0 h to 4 h, and was converted to cDNA and then to cRNA, which was subjected to expression analysis using a DNA chip (GeneChip). When PHO85 was induced, induction of SWI5, CHS2, EGT2, PCL9, TSL1, PCL7, and GSY1, and repression of MAK11, DRS1, and PRS3 were observed. Induction of PHO85-PHO80 gave similar results, suggesting a possibility that most target genes of PHO85 are regulated by PHO85-PHO80 complex. However, PCL7 was induced by Pho85 but not by Pho85-Pho80, suggesting that PCL7 is regulated by a kinase-cyclin complex (es) other than Pho85-Pho80. Taken together, this approach was proven to be useful to distinguish the targets of ten different Pho85-cyclin complexes. Less