STRUCTURE, FUNCTION, AND REGULATION OF CASEIN KINASE-1

描述(改编自申请人的摘要)：的总体目标 本课题旨在推导蛋白激酶配体的结构基础。 选择性和规律性。这项工作的重点是酪蛋白激酶-1(CK1) 酶家族，存在于所有真核细胞中，扮演着一种 在细胞骨架调节中的重要作用。除了他们的 正常生理意义，该酶家族中选定的成员 是介导病理性过度磷酸化的主要候选者 在神经退行性疾病中发现的细胞骨架蛋白 阿尔茨海默氏症。虽然CK1家族成员众多， 包括蛋白激酶超家族中最大的分支之一， 初步数据表明，它们在蛋白质底物的作用机制上具有共性 识别、通过磷酸化进行调节以及对 小分子抑制剂。这项提案的目标是测试这一点 生物物理学和分子生物学相结合的方法进行假说。 具体目标是：(1)建立CK1底物的基础 认可和监管。在现有CK1晶体结构的指导下， 特定模型将在体外和体内进行测试，使用 寡核苷酸定向诱变；(2)分离和鉴定 新的CK1调节激酶。一种使某一部位磷酸化的酶 与细胞周期蛋白依赖蛋白激酶的调控位点同源的将是 纯化、生化鉴定和克隆以建立其 与细胞周期蛋白依赖性激酶激活酶(CAK)的关系；以及(3) 探索新型小分子抑制剂的作用机理。这个 这些分子占据的结合位置将被识别并在 其他已知三维结构的蛋白激酶。尽管这些 实验聚焦于CK1，其结果将对 蛋白激酶家族作为一个整体。此外，全面了解 蛋白激酶结构和抑制剂的选择性将加快合理的 具有潜在应用前景的蛋白激酶选择性抑制剂的研究进展 治疗特性。

DESCRIPTION (Adapted from applicant's abstract): The broad objective of this project is to deduce the structural basis of protein kinase ligand selectivity and regulation. The work focuses on the casein kinase-1 (CK1) family of enzymes, which are present in all eukaryotic cells and play an essential role in regulation of the cytoskeleton. In addition to their normal physiological significance, selected members of this enzyme family are leading candidates for mediating the pathological hyperphosphorylation of cytoskeletal proteins found in neurodegenerative diseases such as Alzheimer's disease. Although members of the CK1 family are numerous and comprise one of the largest branches of the protein kinase superfamily, preliminary data suggest commonality in their mechanism of protein substrate recognition, regulation by phosphorylation, and sensitivity to small-molecule inhibitors. The goal of this proposal is to test this hypothesis by a combination of biophysical and molecular biological methods. The specific aims are: (1) to establish the basis of CK1 substrate recognition and regulation. Guided by an existing CK1 crystal structure, specific models will be tested in vitro and in vivo using oligonucleotide-directed mutagenesis; (2) to isolate and characterize a novel CK1 regulatory kinase. An enzyme that phosphorylates a site homologous to a regulatory site in cyclin-dependent protein kinases will be purified, characterized biochemically, and cloned to establish its relationship to the cyclin-dependent kinase activating kinase (CAK); and (3) to discover the mechanism of action of novel small molecule inhibitors. The binding site occupied by these molecules will be identified and sought in other protein kinases of known 3-dimensional structure. Although these experiments focus on CK1, the results will have broad implications for the protein kinase family as whole. In addition, a complete understanding of protein kinase structure and inhibitor selectivity will speed the rational development of protein kinase-selective inhibitors with potentially useful therapeutic properties.