Structural Biology of Tyrosine Kinase Regulation

描述（由申请人提供）：非受体激酶Src家族包括在人体中鉴定的8种不同蛋白质。这些蛋白质在多种细胞类型的信号转导中发挥广泛的作用，并涉及许多疾病，包括癌症，HIV感染，类风湿性关节炎和I型糖尿病（DM1）。在动物模型中，抑制Hck可预防糖尿病的发作，表明该蛋白质可作为抗DM 1药物开发的靶点。过多的人类酪氨酸激酶使得高特异性抑制剂的设计变得困难。本项目的长期目标是对Hck调控的结构基础有一个全面、详细的了解，这将有助于设计高度特异性的Hck抑制剂。这类药物在类风湿性关节炎和DM 1的治疗中应具有广泛的适用性。 该试验项目的具体目标是（1）建立细菌表达系统，以产生适合于通过溶液状态NMR光谱分析的Hck，以及（2）使用这些分子启动NMR研究。具体目标1需要稳定同位素标记的重组HCK和体外磷酸化。具体目标2需要Hck的1H、13C和15N共振的分配以及主链15N弛豫数据的收集和分析。这些研究将建立酪氨酸激酶的结构和动力学的综合分析，利用核磁共振的可行性。

DESCRIPTION (provided by applicant): The Src-family of nonreceptor kinases includes eight distinct proteins identified in humans. These proteins play wide-ranging roles in signal transduction in a variety of cell types, and are implicated in numerous diseases including cancer, HIV infection, rheumatoid arthritis, and Type I diabetes (DM1). Inhibition of Hck prevents onset of diabetes in animal models, suggesting this protein as a target for anti- DM1 drug development. The plethora of human tyrosine kinases makes design of high-specificity inhibitors difficult. The long term objective of this project is to develop a comprehensive, detailed understanding of the structural basis of Hck regulation which will be of use in the design of highly specific Hck inhibitors. Such drugs should have broad applicability in the treatment of rheumatoid arthritis and DM1. The specific aims of this pilot project are (1) to establish a bacterial expression system to produce Hck suitable for analysis by solution-state NMR spectroscopy and (2) to initiate NMR studies using these molecules. Specific aim 1 entails stable-isotope labeling of recombinant Hck and in vitro phosphorylation. Specific aim 2 entails assignment of 1H, 13C, and 15N resonance of Hck and collection and analysis of backbone 15N relaxation data. These studies will establish the feasibility of comprehensive analysis of tyrosine kinase structure and dynamics using NMR.