STRUCTURE, FUNCTION, AND REGULATION OF CASEIN KINASE-1

描述（改编自申请人摘要）： 本课题旨在推导蛋白激酶配体的结构基础 选择性和监管。 这项工作的重点是酪蛋白激酶-1（CK 1） 酶家族，存在于所有真核细胞中， 在调节细胞骨架中的重要作用。 除了它们 正常的生理意义，该酶家族的选定成员 是介导病理性过度磷酸化 在神经退行性疾病中发现的细胞骨架蛋白， 老年痴呆症 尽管CK 1家族的成员众多， 包括蛋白激酶超家族的最大分支之一， 初步数据表明，它们的蛋白质底物机制具有共性 识别，磷酸化调节，以及对 小分子抑制剂。 本提案的目标是测试这一点 生物物理学和分子生物学方法相结合的假说。 具体目的是：（1）建立CK 1底物的基础 承认和监管。 在现有CK 1晶体结构的指导下， 将在体外和体内测试特定模型， （2）分离和表征一种新的突变体， 新型CK 1调节激酶。 磷酸化酶使一个位点磷酸化的酶 与细胞周期蛋白依赖性蛋白激酶中调节位点同源的蛋白质将 纯化，生物化学表征，并克隆以建立其 与细胞周期蛋白依赖性激酶激活激酶（CAK）的关系;以及（3） 发现新的小分子抑制剂的作用机制。 的 将在中识别和寻找这些分子占据的结合位点 已知三维结构的其他蛋白激酶。 虽然这些 实验集中在CK 1，结果将对 蛋白激酶家族。 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 具有潜在用途的蛋白激酶选择性抑制剂的开发 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.