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

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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.