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

蛋白激酶是所有细胞中必不可少的蛋白质，它通过磷酸化作用向靶蛋白添加带电的磷酸基团，从而发出细胞功能变化的信号。靶蛋白的磷酸化改变了它们的结构，从而改变了它们的活性，改变了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。尽管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个问题，将直接的遗传分析和面包酵母中重组DNA的使用与使用化学方法--质谱学来识别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家族中的一种酵母蛋白激酶Yck 2的靶标，它可以在哺乳动物细胞中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和那些降低了Yck 2蛋白激酶活性的突变细胞中的那些蛋白，将确定Yck 2的靶标。那些在正常细胞中被磷酸化，但在突变细胞中没有被磷酸化或磷酸化程度较低的蛋白质将被认为是潜在的靶标。其他方法将被用来测试潜在目标是否为真实目标。将使用的质谱学方法提供了磷酸化蛋白质的身份以及磷酸化的位置。因此，Yck-2蛋白激酶的靶标以及其他蛋白激酶的靶标都将被识别。这些信息将被收集到一个数据库中，供其他研究人员使用。这个项目的后续目标是研究目标蛋白如何受到磷酸化的影响，并确定每个目标蛋白在细胞寿命的哪个时间点被磷酸化。通过这种方式，我们可以了解Yck-2蛋白激酶的功能。这个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，以及将获得的关于其他蛋白激酶靶标的信息，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广泛研究蛋白质方法的科学教育和培训。

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.