Identification of Substrates and Functions for the Yeast Yck1/2 Protein Kinases

酵母 Yck1/2 蛋白激酶的底物和功能鉴定

• 批准号: 0517204
• 负责人: Lucy Robinson
• 金额: --
• 依托单位: LSU Health Sciences Center -Shreveport
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0517204&HistoricalAwards=false
• 关键词: Identification; Substrates; Functions; Yeast; Yck1

Protein kinases are essential proteins in all cells that act to signal changes in cellular functions by phosphorylation, adding charged phosphate groups to target proteins. Phosphorylation of target proteins modifies their structure, and therefore alters their activities, their interactions with protein or small molecule partners, and/or their locations within cells. Although protein kinases play key regulatory roles in many cellular processes, it has been difficult to identify the precise target proteins for most protein kinases. This project addresses this problem on a broad scale, combining the straightforward genetic analysis and recombinant DNA usage in the baker's yeast Saccharomyces cerevisiae with the ability to identify phosphorylated proteins using a chemical method, mass spectrometry. The major goal of the project is to identify the targets of a yeast protein kinase in the casein kinase 1 family, Yck2, which is replaceable by similar protein kinases in mammalian cells. The targets of Yck2 will be identified by comparing phosphorylated proteins in normal cells with those in mutant cells that have reduced activity of the Yck2 protein kinase. Those proteins that are phosphorylated in normal cells but are not phosphorylated or less phosphorylated in the mutant cells will be considered potential targets. Other methods will be used to test whether the potential targets are real targets. The mass spectrometry method that will be used provides the identity of the phosphorylated protein as well as the position of the phosphorylation. Therefore, both targets of the Yck2 protein kinase as well as targets of other protein kinases will be identified. This information will be collected in a database for use by other researchers. The subsequent goals of this project are to study how the target proteins are affected by phosphorylation, and to determine at what point in the life of the cell each target is phosphorylated. In this way, the functions of the Yck2 protein kinase can be understood. This project is important to the scientific community because of the method used, which can be applied to other protein kinases, as well as the information that will be gained about targets of other protein kinases, which will be useful to researchers studying those proteins. The project will also provide scientific education and training in methods of studying proteins on a broad scale to high school and college students as well as graduate students.

蛋白激酶是所有细胞中的必需蛋白质，其通过磷酸化作用，将带电磷酸基团添加到靶蛋白质中，从而发出细胞功能变化的信号。 靶蛋白的磷酸化修饰了它们的结构，并因此改变了它们的活性、它们与蛋白质或小分子伴侣的相互作用和/或它们在细胞内的位置。 虽然蛋白激酶在许多细胞过程中起着关键的调节作用，但很难确定大多数蛋白激酶的精确靶蛋白。 该项目在广泛的范围内解决了这一问题，将面包酵母酿酒酵母中的直接遗传分析和重组DNA使用与使用化学方法质谱法鉴定磷酸化蛋白质的能力相结合。 该项目的主要目标是确定酪蛋白激酶1家族Yck 2中酵母蛋白激酶的靶点，Yck 2在哺乳动物细胞中可被类似的蛋白激酶取代。 通过比较正常细胞中的磷酸化蛋白质与Yck 2蛋白激酶活性降低的突变细胞中的磷酸化蛋白质，将鉴定Yck 2的靶点。 那些在正常细胞中磷酸化但在突变细胞中不磷酸化或磷酸化较少的蛋白质将被认为是潜在的靶标。 将使用其他方法来测试潜在目标是否是真实的目标。 将使用的质谱法提供磷酸化蛋白的鉴别以及磷酸化的位置。 因此，将鉴定Yck 2蛋白激酶的靶标以及其他蛋白激酶的靶标。 这些信息将被收集在数据库中，供其他研究人员使用。 该项目的后续目标是研究靶蛋白如何受到磷酸化的影响，并确定每个靶蛋白在细胞生命的哪个阶段被磷酸化。 通过这种方式，可以理解Yck 2蛋白激酶的功能。 该项目对科学界很重要，因为所使用的方法可以应用于其他蛋白激酶，以及将获得有关其他蛋白激酶靶点的信息，这将对研究这些蛋白质的研究人员有用。 该项目还将向高中生和大学生以及研究生提供蛋白质研究方法的科学教育和培训。