Proteome-wide analysis of kinase phosphorylation specificity in yeast

描述（由申请人提供）：可逆蛋白磷酸化是真核生物中调节细胞行为的最常见机制之一。基因组测序工作已经发现了数百种新的可预测的蛋白激酶。对大多数来说，我们对它们的生物学功能知之甚少：只有少数蛋白质底物被确定，控制它们激活的途径通常是模糊的。靶向蛋白质组范围的生化筛选在快速提供关于相当大的蛋白质家族（如蛋白激酶）的功能信息方面具有很大的效用。我们建议系统地研究来自酿酒酵母菌的124种蛋白激酶的底物特异性。我们将通过调整我们最近描述的肽库方法来实现高通量分析。每个激酶将在酵母中过表达，纯化，并进行肽库筛选以确定其一致的磷酸化基序。我们将通过蛋白质序列数据库搜索和与其他激酶导向酵母蛋白质组学的整合来确定每种激酶的候选底物。我们将利用基序导向的磷酸化蛋白检测和亲和试剂，通过在体内证实其磷酸化位点来验证选定激酶的靶底物。这项工作将有助于我们对蛋白质激酶如何实现特异性的基本理解，并确定信号网络中的关键连接。此外，我们将为研究信号转导和磷酸化依赖性蛋白调控的研究人员提供一般资源。

DESCRIPTION (provided by applicant): Reversible protein phosphorylation is one of the most common mechanisms for regulating cell behavior in eukaryotes. Genome sequencing efforts have uncovered hundreds of new predicted protein kinases. For most, we have little insight into their biological function: protein substrates have been identified for only a minority, and pathways that control their activation are generally obscure. Targeted proteome-wide biochemical screens have great utility in rapidly providing functional information regarding sizable protein families such as protein kinases. We propose to systematically investigate the substrate specificity of each of the 124 protein kinases from Saccharomyces cerevisiae. We will accomplish this by adapting our recently described peptide library method to enable high throughput analysis. Each kinase will be overexpressed in yeast, purified, and subjected to peptide library screening to determine its consensus phosphorylation motif. We will identify candidate substrates for every kinase through protein sequence database searching and integration with other kinase-directed yeast proteomics efforts. We will validate target substrates of selected kinases by substantiating their sites of phosphorylation in vivo, making use of motif-directed phosphoprotein detection and affinity reagents. This work will contribute to our fundamental understanding of how specificity is achieved by protein kinases and identify critical connections in signaling networks. In addition, we will provide a general resource for researchers studying signal transduction and phosphorylation-dependent regulation of proteins.