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Regulation of gene expression by protein kinase in yeast

酵母中蛋白激酶对基因表达的调节

基本信息

批准号：
04404001
负责人：
TOH-E Akio
金额：
$ 18.56万
依托单位：
University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (A)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1994
项目状态：
已结题

项目摘要

PHO85 was first identified as a negative regulator of the PHO system in budding yeast. It encodes a protein kinase that is highly homologous to cyclindependent kinases (CDKs). Since a pho85DELTA mutation causes not only a constitutive expression of the yeast acid phosphatase but defects in normal growth rate and in the utilization of carbon sources, we assume that the Pho85 kinase mediates a signal from the nutrient availability to START progression that is regulated by the Cdc28 kinase. To test this idea, we carried out genetic and biochemical analyzes of the Pho85 kinase.PHO80 is another negative regulator of the PHO system, functioning together with PHO85 to repress PHO5. By an in vitro kinase assay using kappa-casein as substrate, we found that hyperproduced Pho80 stimulates the Pho85 kinase activity and that an immunoprecipitate containing a tagged Pho80 possessed a kinase activity that was dependent of functional PHO85. These results indicate that the Pho80 protein interacts with the Pho85 kinase to regulate its activity.To reveal the functional difference between Pho85 and Cdc28, we analyzed the functional domain (s) of the Pho85 kinase. We constructed various point mutants containing mutations in the conserved regions of CDKs. Both a Y18,22F mutation which occurred in the region corresponding to the negative phosphorylation site of cdc2, and a E53A mutation that resides in the PSTAIRE sequence are nonfunctional in vivo as well as in vitro and are dominant negative against the wild type Pho85. On the other hand, YTH (Cdc28 type), FTS,or FAA mutations in F165S166S167, the region corresponding to an active phosphorylation site of cdc2, are functional in vivo. These results indicate that Y18,22 and the PSTAIRE sequence are important for PHO85 function whereas S166 is not, and imply that the function of each domain could be different among CDKs in spite of its conservation.

PHO85首先被鉴定为芽殖酵母PHO系统的负调控因子。它编码一种与环无关激酶（CDKs）高度同源的蛋白激酶。由于pho85DELTA突变不仅导致酵母酸性磷酸酶的组成表达，而且导致正常生长速率和碳源利用的缺陷，我们假设Pho85激酶介导了一个信号，从营养可用性到由Cdc28激酶调节的START进展。为了验证这一想法，我们对Pho85激酶进行了遗传和生化分析。PHO80是phoo系统的另一个负调节因子，与PHO85一起抑制PHO5。通过使用kappa-酪蛋白作为底物的体外激酶测定，我们发现高产量的Pho80刺激Pho85激酶活性，并且含有标记Pho80的免疫沉淀物具有依赖于功能性Pho85的激酶活性。这些结果表明Pho80蛋白与Pho85激酶相互作用以调节其活性。为了揭示Pho85和Cdc28之间的功能差异，我们分析了Pho85激酶的功能域(s)。我们构建了包含CDKs保守区域突变的各种点突变体。发生在cdc2负磷酸化位点对应区域的Y18,22F突变和位于PSTAIRE序列的E53A突变在体内和体外均无功能，并且对野生型Pho85呈显性阴性。另一方面，与cdc2活性磷酸化位点对应的F165S166S167区域的YTH （Cdc28型）、FTS或FAA突变在体内具有功能。这些结果表明，Y18、22和PSTAIRE序列对PHO85的功能至关重要，而S166则不是，这意味着尽管CDKs具有保守性，但每个结构域的功能在CDKs中可能是不同的。