STRUCTURE AND REGULATION OF TYPE 1 PROTEIN PHOSPHATASES

描述：首席调查员研究的总体目标 计划是了解蛋白磷酸酶在信号中的作用 转导通路。蛋白磷酸酶1(PP1)是一种 细胞中丝氨酸/苏氨酸磷酸酶活性的主要比例。 已鉴定出四种PP1全酶，它们都含有类似的 催化亚单位(CS1)，但相关的调控不同 亚单位。后一个组件的功能似乎是为了达到目标 酶作用于不同的细胞隔间，提供底物 特异性和控制酶活性。生化和遗传学 研究提供的证据表明，PP1参与了几个细胞 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。虽然很多 已经了解到这些酶在各种情况下的参与 对细胞过程的了解，对细胞内目标的了解 对他们活动的监管相当有限。这样做的目的是 该项目是为了了解相互作用的分子基础 催化和调节成分，以阐明调节 并明确两种细胞功能在特定细胞功能中的作用 1型全酶的形式：依赖于ATP的镁和 糖原/肌浆网相关蛋白磷酸酶。这个 ATP依赖的磷酸酶是一种胞浆酶，含有抑制物-2 作为调节亚单位，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/功能研究将致力于表征 CS1的功能结构域及与CS1相互作用的区域 利用酵母双杂交技术获得调控亚基I-2和Rg1 细菌表达的系统和体外重组试验 突变蛋白质。两种磷酸酶在细胞中作用的研究 会与野生型或突变型一起过表达CS1 哺乳动物细胞中的调节亚单位(I-2或RGL)；分析任何 调节细胞对细胞外信号的反应。此外, 磷酸酶的调控将通过过度表达活性来探索。 或假定的上游监管要素的显性负面版本 例如MAP激酶/ERK级联中的那些。在这方面取得了成功 调查应该导致对生理的更好的理解 一些主要的1型磷酸酶的作用。

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.