Prediction and Validation of Phospho-Regulatory Sites in Crop Plant Proteomes

维管植物的基因组编码超过1000种蛋白激酶。长期目标是确定作物蛋白质组中所有的磷酸化调控位点。这些地点代表了植物生长各个方面的潜在控制点，包括对生物和非生物胁迫的反应。重点是使用生物信息学来预测水稻蛋白质组中磷酸调节的潜在重要位点的子集，然后使用基于肽的实验策略来开始验证这些预测。四个具体目标是：1。改进生物信息学工具，以识别植物中参与磷酸化信号转导的基序.对植物蛋白质组中所有含丝氨酸和苏氨酸的亚序列进行聚类分析.设计肽阵列平台，用于激酶活性的体外分析.识别能够破坏体内信号通路的肽（将测试超过50种）。所提出的研究的独特性和强度取决于基本原理，即两种植物蛋白质组的计算比较可以用作“噪音过滤器”，以识别重要的序列基序，这些序列基序是单子叶植物和双子叶植物之间保守的信号通路的基础。这种过滤提供了实施“肽干扰”作为一个强大的新方法来理解和操纵植物细胞磷酰组的动态的关键基础。所有被发现编码“肽破坏物”或作为特定激酶活性的报告基因的基因的DNA构建体将被存放在俄亥俄州州立大学的ABRC，并且将免费获得。

The genomes of vascular plants encode more than 1000 protein kinases. The long-term goal is todefine all the regulatory phosphorylation sites in crop plant proteomes. Such sites represent potential control points for all aspects of plant growth, including responses to biotic and abiotic stress. The focus is to use bioinformatics to predict a subset of potentially important sites of phospho-regulation in the rice proteome, and then use a peptide-based experimental strategy to start validating those predictions. The four Specific Aims are:1. Improve bioinformatics tools to identify motifs involved in phospho-signaling in plants.2. Conduct a clustering analysis of all Ser and Thr containing sub-sequences in plant proteomes.3. Design peptide array platform to profile kinase activities in vitro.4. Identify peptides that can disrupt in vivo signaling pathways (more than 50 will be tested).The uniqueness and strength of the proposed research rests upon the underlying rationale that acomputational comparison of two plant proteomes can be used as a 'noise filter' to identifyimportant sequence motifs that are fundamental to signaling pathways conserved between monocots and dicots. This filtering provides the critical foundation for implementing 'peptide-disruptors' as a powerful new approach to understanding and manipulating the dynamics of the plant cell phosphorylome. DNA constructs for all genes found to encode a 'peptide disruptor' or function as a reporter for a particular kinase activity will be deposited at the ABRC at Ohio State University, and will be freely available.