Protein Arrays: Phosphorylation-dependent Interactions

描述(申请人提供)：蛋白质-蛋白质相互作用是核心 生物系统中的信号转导过程。跨膜 受体酪氨酸激酶(RTKs)在细胞的许多方面发挥着重要作用 成长和行为。激活的RTK，包括一个研究得很好的 PDGF-β受体，已被证明能结合十几个信号 含有SRE同源2(SH2)结构域的转导分子。这些 相互作用取决于特定的磷酸酪氨酸残基的识别 通过各种SH2结构域对上下文进行排序。在这项拟议的研究中， 选定的重组SH2结构域将在大肠杆菌中表达，并 亲和层析纯化。在衍生化玻璃上测试蛋白质阵列 幻灯片将使用来自PI3-K的SH2结构域进行优化，基于其 与人工合成的磷酸肽结合。其他重组SH2结构域将 被排列并允许与表达V-sis的细胞提取物相互作用 一种激活型死亡或野生型PDGF受体。我们预测，这种方法将 允许快速读出PDG上哪些特定的酪氨酸被磷酸化 受体。这些研究的目的是优化阵列和结合参数，并 从而发展了一种新的高通量检测磷酸化的策略 依赖蛋白质：蛋白质相互作用。 建议的商业应用： 将开发商业SH2结构域阵列，以帮助高通量筛选 新的相互作用涉及细胞信号转导和/或可能的新的激酶。 承接SH2蛋白交易的高通量筛选项目。

DESCRIPTION (provided by applicant): Protein-protein interactions are central to the process of signal transduction in biological systems. Transmembrane receptor tyrosine kinase (RTKs) play important roles in many aspects of cell growth and behavior. Activated RTKs, including one of the well-studied PDGF-beta receptor, have been shown to bind more than a dozen signal transduction molecules that contain Sre homology 2 (SH2) domains. These interactions depend on the recognition of phosphotyrosine residues in specific sequence contexts by the various SH2 domains. In the proposed research, selected recombinant SH2 domain constructs will be expressed in E. coli and purified by affinity chromatography. Test protein arrays on derivatized glass slides will be optimized with an SH2 domain from PI3-kinase based on its binding to a synthetic phosphopeptide. Additional recombinant SH2 domains will be arrayed and allowed to interact with extracts of cells expressing V-sis with a kinase dead or wild type PDGF receptor. We predict that this method will allow a rapid readout of which specific tyrosines are phosphorylated on the PDG receptor. These studies will aim to optimize array and binding parameters and thus to develop a new strategy for high throughput detection of phosphorylation dependent protein: protein interactions. PROPOSED COMMERCIAL APPLICATION: Commercial SH2 domain arrays will be developed to aid high throughput screening of novel interactions involved in cellular signal transduction and or possible novel kinases. Contracting in projects for high throughput screening of SH2 protein transactions.