REGULATORY DOMAINS OF PROTEIN PHOSPHATASE 1

蛋白磷酸酶 1 的调控域

• 批准号: 2857255
• 负责人: PETER J. KENNELLY
• 金额: $ 11.55万
• 依托单位: VIRGINIA POLYTECHNIC INST AND ST UNIV
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2857255
• 关键词: active sites; chemical models; chimeric proteins; enzyme activity; enzyme inhibitors; enzyme structure; glycogen synthase; phosphorylases; phosphorylation; protein kinase; protein structure function; protein tyrosine phosphatase; tissue /cell culture; transfection

DESCRIPTION (Adapted from applicant's abstract): Protein phosphorylation-dephosphorylation represents one of nature's most fundamentally important and widely used mechanisms for the regulation of protein function and, consequently, cellular events. Protein-serine/threonine phosphatase-1 (PP1) represents one of the two most quantitatively important protein-serine/threonine phosphatases in mammalian cells. PP1 is regulated via the reversible binding of two endogenous inhibitors, I-1 and I-2, and to targeting proteins such as the glycogen-targeting subunit, G, which serve to recruit it to specific locations in the cell. The objectives of this study are to (1) identify the sites on PP1 to which each of these regulatory molecules bind, and (2) apply this knowledge to dissect the functional roles that these regulators play in modulating PP1 action toward the key enzymes of glycogen metabolism -- glycogen synthase and glycogen phosphorylase -- in cultured cells. The first specific aim will be accomplished by constructing chimeric PP1 molecules in which potential regulatory interaction domains are incorporated by genetic engineering onto a unique homolog of PP1, PP1-arch from the archaeon Sulfolobus solfataricus, that is lacks the ability to bind eukaryotically-derived regulatory molecules but is still 29% identical to PP1 from rabbit. These studies will be aided in execution and interpretation by the recently published three-dimensional structure of PP1 from rabbit. Knowledge of the structural features of PP1 responsible for binding to regulatory ligands will then be used to construct, by genetic engineering, altered forms of PP1-rabbit each defective in their ability to bind one of its endogenous regulators. These regulationally-impaired forms will be transfected into hepatoma and myoblast cell lines, and the cells challenged with forskolin, an inducer of glycogenolysis, or insulin, an inducer of glycogen synthesis. The regulatory modalities responsible for controlling PP1 action toward glycogen synthase and glycogen phosphorylase under each circumstance will be identified by virtue of the inability of cells transfected with an altered PP1 to interact with that regulator to trigger a response similar in direction or magnitude to that seen in cells transfected with wild-type PP1. Successful completion of this project may increase the understanding of how PP1 is regulated in cells, and do so in molecular detail.

描述（改编自申请人的摘要）：蛋白质 磷酸化-去磷酸化代表了自然界最重要的作用之一 根本上重要且广泛使用的监管机制 蛋白质功能以及细胞事件。 蛋白质-丝氨酸/苏氨酸磷酸酶-1 (PP1) 是最重要的两种酶之一 哺乳动物中数量重要的蛋白质丝氨酸/苏氨酸磷酸酶 细胞。 PP1 通过两个内源性蛋白的可逆结合进行调节 抑制剂 I-1 和 I-2，以及靶向蛋白质，例如 糖原靶向亚基 G，用于将其募集到特定的位置 细胞中的位置。 本研究的目标是 (1) 确定 PP1 上每个调节分子结合的位点，以及 (2) 适用 这些知识可以剖析这些监管机构在其中发挥的功能作用 调节 PP1 对糖原代谢关键酶的作用—— 糖原合成酶和糖原磷酸化酶——在培养细胞中。 这 第一个具体目标将通过构建嵌合PP1来实现 包含潜在调控相互作用域的分子 通过基因工程转化为 PP1 的独特同源物，即 PP1-arch 古细菌 Sulfolobus solfataricus，缺乏结合能力 真核衍生的调节分子，但仍有 29% 相同 兔子的PP1。 这些研究将有助于执行和 最近发表的PP1三维结构解读 来自兔子。 了解 PP1 的结构特征 然后与调节配体的结合将用于通过遗传构建 工程，PP1-兔的改变形式，每个形式都有缺陷 结合其内源性调节因子之一。 这些受到监管损害的形式 将被转染到肝癌和成肌细胞系中，并且这些细胞 接受毛喉素（一种糖原分解诱导剂）或胰岛素（一种 糖原合成诱导剂。 监管模式负责 控制 PP1 对糖原合成酶和糖原磷酸化酶的作用 在每种情况下都将由于无法识别 转染改变的 PP1 的细胞可与该调节因子相互作用 触发与细胞中观察到的方向或幅度相似的反应 转染野生型PP1。 该项目的成功完成可能 增加对 PP1 在细胞中如何调节的了解，并在 分子细节。