Proteome-wide analysis of kinase phosphorylation specificity in yeast

描述（由申请人提供）：可逆的蛋白质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预测蛋白激酶。对于大多数人来说，我们对它们的生物学功能知之甚少：只确定了少数蛋白质底物，并且控制它们激活的途径通常不清楚。靶向蛋白质组范围的生物化学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实用性。我们建议系统地研究从酿酒酵母的124个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这一点，以实现高通量分析。每种激酶将在酵母中过表达，纯化，并进行肽文库筛选以确定其共有磷酸化基序。我们将通过蛋白质序列数据库搜索和与其他激酶指导的酵母蛋白质组学工作的整合来确定每个激酶的候选底物。我们将通过证实其在体内的磷酸化位点，利用基序导向的磷蛋白检测和亲和试剂来验证所选激酶的靶底物。这项工作将有助于我们对蛋白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的蛋白质调节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.