STRUCTURE AND REGULATION OF PHOSPHOPROTEIN PHOSPHATASES

蛋白质的共价磷酸化在控制肌萎缩侧索硬化中起着核心作用。 许多细胞过程是由胰岛素和肾上腺素等激素引起的。零件 这一调节涉及负责去磷酸化的酶 蛋白质，即磷酸蛋白磷酸酶。该项目涉及以下内容之一 在细胞中发现的主要类型的蛋白磷酸酶，将被称为 “类型1”磷酸酶(这类酶的一个特征是它们的 催化亚基被耐热磷酸酶抑制剂抑制 蛋白质、抑制物-1、抑制物-2)。了解该结构和 对这些酶的调节是不完整的。主要目标将是 建立这一类的原生形式的数目和结构 酵素。应用的方法将包括(1)常规蛋白质 纯化，(2)免疫学方法，和(3)重组DNA 技巧。纯化的高分子形式的1型磷酸酶 将被表征并与结构更简单的已知酶进行比较， 即分离的催化亚基Cl和依赖于ATP的镁 磷酸酶。非催化亚基的功能作用将是 调查，特别是与酶活性控制有关的问题。 将特别注意磷酸酶的磷酸化，因为 有充分的理由相信抑制物-2的磷酸化 ATP-镁依赖的磷酸酶的组成与其 激活。由小分子介导的可能的控制将是 已评估。磷酸酶的短期细胞内调控模型 活动将使用暴露于胰岛素和/或 肾上腺素。将应用免疫学方法分析 抑制蛋白2和其他磷酸酶组分的磷酸化，以及 磷酸酶活性。这项调查的成功可以广泛地 重要性，因为这类磷酸酶很可能参与 控制许多细胞过程。任何与行为的机械性联系 荷尔蒙，特别是胰岛素，将特别对 了解胰岛素的正常作用。磷酸酶受损， 然而，这可能是某些类型糖尿病的决定因素。

Covalent phosphorylation of proteins plays a central role in the control of many cellular processes by hormones such as insulin and epinephrine. Part of this regulation involves the enzymes responsible for dephosphorylating proteins, the phosphoprotein phosphatases. This project addresses one of the main classes of protein phosphatase found in cells, what will be termed "type 1" phosphatase (one characteristic of such enzymes is that their catalytic subunit is inhibited by the heat stable phosphatase inhibitor proteins, inhibitor-1 inhibitor-2). Knowledge of the structure and regulation of these enzymes is incomplete. A primary objective will be to establish the number and structures of the native forms of this class of enzyme. Methods to be applied will include (1) conventional protein purification, (2) immunological methods, and (3) recombinant DNA techniques. Purified high molecular weight forms of type 1 phosphatase will be characterized and compared with known enzymes of simpler structure, namely the isolated catalytic subunit, Cl, and the ATP-Mg-dependent phosphatase. The functional roles of non-catalytic subunits will be investigated, particularly in relation to control of enzyme activity. Special attention will be paid to phosphorylation of phosphatases since there is good reason to believe that phosphorylation of the inhibitor-2 component of the ATP-Mg-dependent phosphatase is related to its activation. Possible controls mediated by small molecules will be evaluated. Models for the short-term intracellular control of phosphatase activity will employ mouse diaphragms exposed to insulin and/or epinephrine. Immunological methods will be applied to analyze the phosphorylation of inhibiton 2 and other phosphatase components, as well as phosphatase activity. Success in this investigation can have widespread importance since this class of phosphatase is likely to be involved in the control of many cellular processes. Any mechanistic link to the action of hormones, especially insulin, will be of particular interest in understanding how insulin functions normally. Impaired phosphatase, however, could be a determining factor in some types of diabetes.