REGULATORY DOMAINS OF PROTEIN PHOSPHATASE 1

描述（改编自申请人摘要）：蛋白质 磷酸化-去磷酸化是自然界中 具有根本重要性和广泛使用的监管机制， 蛋白质的功能，并因此，细胞事件。 蛋白丝氨酸/苏氨酸磷酸酶-1（PP 1）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 PP 1是通过两个内源性的 抑制剂，I-1和I-2，以及靶向蛋白质，如 糖原靶向亚基G，其用于将其募集到特异性 在细胞中的位置。 本研究的目的是（1）确定 这些调节分子各自结合的PP 1上的位点，以及（2）应用 利用这些知识来剖析这些监管机构所扮演的职能角色 调节PP 1对糖原代谢关键酶的作用-- 糖原合成酶和糖原磷酸化酶。 的 第一个具体目标将通过构建嵌合PP 1来实现 潜在的调节相互作用结构域被掺入的分子 通过基因工程改造到PP 1的独特同源物上，PP 1-arch来自 古细菌硫磺硫化叶菌，即缺乏结合能力， 真核生物衍生的调控分子，但仍有29%相同， PP 1来自兔子。 这些研究将在执行中得到帮助， 最近发表的PP 1三维结构的解释 从兔子。 了解PP 1的结构特征， 然后将与调节配体的结合用于构建，通过遗传学方法， 工程改造，PP 1-兔的改变形式，每种都有缺陷， 与其内源性调节因子结合。 这些调节障碍的形式 将其转染到肝癌和成肌细胞系中， 挑战与毛喉素，诱导糖原分解，或胰岛素， 糖原合成诱导剂。 负责以下事项的监管模式 控制PP 1对糖原合成酶和糖原磷酸化酶的作用 在每一种情况下，将通过无法识别 用改变的PP 1转染的细胞与该调节因子相互作用， 触发与细胞中所见的方向或幅度相似的反应 用野生型PP 1转染。 该项目的成功完成可能 增加对PP 1如何在细胞中调节的理解，并在 分子细节

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.