REGULATION OF PROTEIN DEPHOSPHROYLATION

蛋白磷酸酶2A（PP 2A）是丝氨酸/苏氨酸的主要形式。 磷酸酶在许多细胞类型。生物化学和遗传学证据表明 PP 2A参与了许多细胞信号通路， 包括控制细胞生长和增殖的那些。天然 酶是由催化亚基（C）、支架（C）和催化亚基（C）组成的异源三聚体。 亚基（A）和调节亚基（R）。A和C亚基是 普遍表达并形成共同的AC核心复合物。有 多个调节亚基家族， 酶的形式。不同调节亚单位与核心的关联 复合物改变酶活性和底物特异性。改变 PP 2A活性通过蛋白质-蛋白质相互作用发挥重要作用， 调节信号转导途径。例如，DNA的相互作用 具有AC核心复合物的肿瘤病毒癌蛋白是必需的，但 足够的转化。我们的目标是找出 PP 2A调节亚基，确定其分子基础 与核心酶的相互作用，确定它们对酶的影响 活性和特异性，并确定它们如何控制磷酸化 细胞蛋白质。互补DNA克隆对应于不同的 将分离PP 2A调节亚基的种类和同种型， 表征了调节亚单位在大肠杆菌中的表达和分布 小鼠组织将通过北方分析、RNA酶保护、 和免疫印迹。调控亚基的全长cDNA将被克隆。 在细菌或Sf 9细胞中表达，并测定它们与 PP 2A在体外使用重建系统。的体内作用 MAP激酶途径的调节亚基，AP-1转录 因子、细胞增殖和PP 2A的细胞内定位 将通过在哺乳动物细胞中表达异源亚基来确定。 PP 2A活性和特异性的控制的结构基础， 将确定调节亚基。化学修饰， 诱变，酵母双杂交系统将用于鉴定 与A和C亚基相互作用的调节亚基上的位点。 与PP 2A相互作用的新的调节亚基和其他蛋白质将 使用双杂交系统和亲和层析进行鉴定， 谷胱甘肽S-转移酶-A亚基融合蛋白柱。

Protein phosphatase 2A (PP2A) is a major form of serine/threonine phosphase in many cell types. Biochemical and genetic evidence have shown that PP2A is involved in a number of cellular signalling pathways, including those controlling cell growth and proliferation. The native enzyme is a heterotrimer composed of a catalytic subunit (C), a scaffold subunit (A), and a regulatory subunit (R). The A and C subunits are ubiquitously expressed and form a common AC core complex. There are multiple families of regulatory subunits that generate a diversity of enzyme forms. Association of different regulatory subunits with the core complex alters enzymatic activity and substrate specificity. Alteration of PP2A activity by protein-protein interactions plays an important role in modulating signal transduction pathways. For example, interaction of DNA tumor virus oncoproteins with the AC core complex is necessary but not sufficient from transformation. Our goals are to identify the repertoire of PP2A regulatory subunits, determine the molecular basis for their interaction with the core enzyme, determine their effects on enzyme activity and specificity, and determine how they control phosphorylation of cellular proteins. Complimentary DNA clones corresponding to different classes and isoforms of PP2A regulatory subunits will be isolated and characterized. The expression and distribution of regulatory subunits in mouse tissues will be determined by Northern analysis, RNase protection, and immunoblotting. Full length cDNAs for the regulatory subunits will be expressed in bacteria or Sf9 cells and assayed for their interaction with PP2A in vitro using reconstitution systems. The in vivo effects of regulatory subunits on the MAP kinase pathway, the AP-l transcription factor, cell proliferation, and the intracellular localization of PP2A will be determined by expressing heterologous subunits in mammalian cells. The structural basis for the control of PP2A activity and specificity by regulatory subunits will be determined. Chemical modification, mutagenesis, and the yeast two-hybrid system will be used to identify sites on the regulatory subunits that interact with the A and C subunits. Novel regulatory subunits and other proteins that interact with PP2A will be identified using the two-hybrid system and affinity chromatography on glutathione S-transferase-A subunit fusion protein columns.