REGULATORY DOMAINS OF PROTEIN PHOSPHATASE 1

蛋白磷酸酶 1 的调控域

• 批准号: 2023648
• 负责人: PETER J. KENNELLY
• 金额: $ 11.12万
• 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 1999-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2023648
• 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(摘自申请者摘要)：蛋白质 磷酸化-去磷酸化是自然界中最重要的 基本重要和广泛使用的监管机制 蛋白质的功能，因此，细胞事件。 蛋白-丝氨酸/苏氨酸磷酸酶-1(PP1)代表了两种最重要的 哺乳动物中数量上重要的蛋白质-丝氨酸/苏氨酸磷酸酶 细胞。PP1是通过两种内源蛋白的可逆结合来调节的 抑制剂I-1和I-2，以及靶向蛋白，如 糖原靶向亚基G，用于将其招募到特定的 单元格中的位置。这项研究的目的是(1)确定 PP1上这些调控分子中的每一个都结合的位点，以及(2)适用 这一知识剖析了这些调节器在 调节PP1对糖原代谢关键酶的作用-- 培养细胞中的糖原合成酶和糖原磷酸化酶。这个 第一个特定目标将通过构建嵌合PP1来实现 潜在的调节相互作用结构域被结合的分子 通过基因工程获得PP1的独特同源物，PP1-ARCH来自 古生菌Sulfolobus solfararicus，即缺乏绑定能力的 真核生物衍生的调节分子，但仍有29%与 兔源PP1。这些研究将在执行和执行方面得到帮助 新近发表的PP1三维结构的解释 从兔子那里。对PP1的结构特征的知识负责 然后，与调节配体的结合将被用于构建，通过基因 工程学，改变形式的PP1-兔，每只都有缺陷的能力 绑住它的一个内生监管者。这些受监管损害的表格 将其导入肝癌和成肌细胞系，并将该细胞 挑战Forsklin，一种糖原分解诱导剂，或胰岛素， 糖原合成诱导剂。负责以下方面的监管模式 控制PP1对糖原合成酶和糖原磷酸化酶的作用 在每种情况下都将根据不能 用改变后的PP1与该调节子相互作用的细胞 触发在方向或大小上与细胞相似的反应 野生型PP1基因转染。该项目的成功完成可能会 增加对PP1如何在细胞中调节的理解，并在 分子细节。