STRUCTURE AND REGULATION OF TYPE 1 PROTEIN PHOSPHATASES

1 型蛋白磷酸酶的结构和调控

• 批准号: 6147582
• 负责人: ANNA A DEPAOLI-ROACH
• 金额: $ 15.31万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-01-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6147582
• 关键词: enzyme activity; enzyme complex; enzyme inhibitors; enzyme mechanism; enzyme reconstitution; enzyme structure; epidermal growth factor; fungal genetics; glucose metabolism; glycogen; insulin; mitogen activated protein kinase; mutant; phosphoprotein phosphatase; phosphorylation; protein isoforms; protein structure function; site directed mutagenesis; transfection

DESCRIPTION: The overall goal of the principal investigator's research program is to understand the role of protein phosphatases in signal transduction pathways. Protein phosphatase type 1 (PP1) constitutes a major proportion of serine/threonine phosphatase activity in the cell. Four PP1 holoenzymes have been identified and all contain a similar catalytic subunit (CS1) but differ in the associated regulatory subunits. The function of the latter components appear to be to target the enzyme to various cellular compartments, to confer substrate specificity and to control enzyme activity. Biochemical and genetic studies have provided evidence that PP1 is involved in several cell functions, including glycogen metabolism, calcium transport, muscle contraction, gene expression, cell cycle and cell growth. Although much has been learned about the participation of these enzymes in a variety of cellular processes, knowledge of their intracellular targets and regulation of their activity is quite limited. The objectives of this project are to understand the molecular basis of the interaction between catalytic and regulatory components, to elucidate the regulatory mechanisms and to define the role in specific cell functions of two forms of type 1 holoenzyme: the ATP-Mg-dependent and the glycogen/sarcoplasmic reticulum-associated protein phosphatases. The ATP-dependent phosphatase is a cytosolic enzyme and contains inhibitor-2 as the regulatory subunit, whereas the glycogen/SR- associated form interacts with both membranes and glycogen via its regulatory component RGL. Structure/function studies will be directed at characterizing the functional domains of CS1 and the regions of interaction between CS1 and the regulatory subunits, I-2 and RGL, by utilizing the yeast two-hybrid system and in vitro reconstitution assays of bacterially expressed mutant proteins. Studies of the roles of the two phosphatases in cells will involve overexpression of CS1 together with wild type or mutated regulatory subunits (I-2 or RGL) in mammalian cells to; analyze any modulation of cellular responses to extracellular signals. In addition, the control of the phosphatases will be probed by overexpressing active or dominant negative versions of putative upstream regulatory elements such as those in the MAP kinase/ERK cascades. Success in this investigation should lead to improved understanding of the physiological role of some of the major type 1 phosphatases.

描述：主要研究者研究的总体目标 程序是了解蛋白磷酸酶在信号中的作用， 转导途径。 蛋白磷酸酶1型（PP 1）构成了一个 细胞中丝氨酸/苏氨酸磷酸酶活性的主要部分。 已鉴定出四种PP 1全酶，它们都含有类似的 催化亚基（CS 1），但在相关的调节 亚单位。 后一部分的功能似乎是针对 酶的各种细胞隔室，赋予底物 特异性和控制酶活性。 生化和遗传 研究已经提供了证据表明PP 1参与了几种细胞 功能，包括糖原代谢，钙转运，肌肉 收缩、基因表达、细胞周期和细胞生长。 虽然大部分 已经了解到这些酶参与了多种 细胞过程，了解其细胞内靶点， 对它们活动的管制相当有限。 这一目标 项目是了解分子之间的相互作用的基础 催化和调节成分，以阐明调节 机制，并确定在特定的细胞功能中的作用， 1型全酶的形式：ATP-Mg依赖型和 糖原/肌浆网相关蛋白磷酸酶。 的 ATP依赖性磷酸酶是一种胞浆酶，含有抑制剂-2 作为调节亚基，而糖原/SR相关形式 通过其调节成分与膜和糖原相互作用 RGL。 结构/功能研究将针对表征 CS 1的功能结构域和CS 1与 利用酵母双杂交技术， 系统和体外重建试验 突变蛋白 两种磷酸酶在细胞中作用的研究 将涉及CS 1与野生型或突变型CS 1一起过表达， 调节亚基（I-2或RGL），以分析任何 调节细胞对细胞外信号的反应。 此外，本发明还提供了一种方法， 磷酸酶的控制将通过过表达活性 或显性负性形式的假定上游调控元件 例如MAP激酶/ERK级联中的那些。 胜任这 研究应该导致对生理学的更好的理解。 一些主要的1型磷酸酶的作用。