Structural Biology of Tyrosine Kinase Regulation

描述(由申请人提供)：非受体激酶的Src家族包括在人类中发现的八种不同的蛋白质。这些蛋白在各种细胞类型的信号转导中发挥着广泛的作用，并与许多疾病有关，包括癌症、艾滋病毒感染、类风湿性关节炎和I型糖尿病(DM1)。抑制HCK可以在动物模型中预防糖尿病的发生，这表明这种蛋白质是抗DM1药物开发的靶点。过多的人类酪氨酸激酶使得设计高特异性的抑制剂变得困难。该项目的长期目标是全面、详细地了解HCK调节的结构基础，这将用于设计高度特异的HCK抑制剂。这类药物在治疗类风湿性关节炎和DM1方面应该有广泛的适用性。 这个试点项目的具体目标是(1)建立一个细菌表达系统，以生产适合于溶液状态核磁共振光谱分析的HCK，以及(2)启动使用这些分子的核磁共振研究。具体目标1需要重组人绒毛膜促性腺激素的稳定同位素标记和体外磷酸化。具体目标2需要指定HCK的1H、13C和15N共振，并收集和分析主干15N弛豫数据。这些研究将建立利用核磁共振对酪氨酸激酶结构和动力学进行综合分析的可行性。

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.