PROTEIN PHOSPHATASE 2A REGULATION OF RAS SIGNALING

蛋白质磷酸酶 2A 对 RAS 信号传导的调节

• 批准号: 6514658
• 负责人: Meera Sundaram
• 金额: $ 17.83万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6514658
• 关键词: Caenorhabditis elegans; biological signal transduction; developmental genetics; enzyme activity; enzyme induction /repression; enzyme substrate; gene mutation; genetic models; genetically modified animals; guanine nucleotide binding protein; mitogen activated protein kinase; phosphoprotein phosphatase; simian virus 40; virus antigen; yeast two hybrid system

DESCRIPTION: (Applicant's Description) Protein Phosphatase 2A (PP2A) is an important regulator of signal transduction pathways, a target of viral oncoproteins, and a potentially useful target for therapeutic drug design. However, PP2A's broad substrate specificity in vitro has hampered attempts to understand its normal regulation and functionally relevant substrates in vivo. We identified a PR55/B regulatory subunit of Protein Phosphatase 2A (SUR-6 PP2A-B) as a positive modulator of Ras signaling in C. elegans, and our genetic data suggest that SUR-6 acts in a common process with KSR and directs PP2A to a specific Ras pathway substrate. Two mutations in SUR-6 PP2A-B preferentially affect Ras-mediated signaling, with minimal effects on other SUR-6- or PP2A-regulated processes. We will use C. elegans vulva development as a genetic model system to elucidate how PP2A-B subunits regulate PP2A catalytic activity and/or substrate specificity, and how PP2A regulates the Ras/Raf/MEK/ERK signaling cascade. To test the absolute requirements for SUR-6 PP2A-B during Ras-mediated vulval induction, we will isolate sur-6 null alleles and use a mosaic analysis strategy to determine their effects on vulval induction. To determine how SUR-6 PP2A-B influences PP2A catalytic activity, we will use RNA-mediated interference, PP2A-C mutations, PP2A-C transgenes and SV4O small t antigen expression to test how altering PP2A catalytic activity influences vulval induction. We will also use RNA-mediated interference to test the requirements for other PP2A-B regulatory subunits that might direct PP2A to alternative Ras pathway substrates, and test which domains of SUR-6 are important for binding to PP2A-A. To test if candidate proteins are likely substrates of PP2A, we will test if specific phospho-acceptor site mutants can bypass the requirement for SUR-6 during vulva induction. Finally, to identify other regulators and targets of PP2A activity during vulval induction, we will conduct a yeast two-hybrid screen for proteins that bind to SUR-6 PP2A-B, and conduct genetic modifier screens for mutations with properties similar to those of sur-6 mutations. Our studies will provide important insights into the normal in vivo regulation of PP2A and its targets, and how PP2A activity might be manipulated for therapeutic effects.

描述：（申请人的描述）蛋白磷酸酶 2A (PP2A) 是一种 信号转导途径的重要调节因子，是病毒的靶点 癌蛋白，以及治疗药物设计的潜在有用靶标。 然而，PP2A 广泛的体外底物特异性阻碍了 了解其体内的正常调节和功能相关底物。 我们鉴定了蛋白磷酸酶 2A (SUR-6) 的 PR55/B 调节亚基 PP2A-B）作为线虫中 Ras 信号传导的正调节剂，我们的 遗传数据表明 SUR-6 与 KSR 共同作用并指导 PP2A 结合特定 Ras 途径底物。 SUR-6 PP2A-B 的两个突变 优先影响 Ras 介导的信号传导，对其他信号影响最小 SUR-6 或 PP2A 监管流程。我们将使用线虫外阴发育 作为遗传模型系统来阐明 PP2A-B 亚基如何调节 PP2A 催化活性和/或底物特异性，以及 PP2A 如何调节 Ras/Raf/MEK/ERK 信号级联。测试 SUR-6 的绝对要求 PP2A-B 在 Ras 介导的外阴诱导过程中，我们将分离 sur-6 null 等位基因并使用镶嵌分析策略来确定它们对 外阴诱导。确定 SUR-6 PP2A-B 如何影响 PP2A 催化 活性，我们将使用 RNA 介导的干扰、PP2A-C 突变、PP2A-C 转基因和 SV4O 小 t 抗原表达来测试如何改变 PP2A 催化活性影响外阴诱导。我们还将使用 RNA 介导的 干扰测试其他 PP2A-B 监管子单元的要求 可能将 PP2A 引导至替代 Ras 途径底物，并测试哪些 SUR-6 的结构域对于与 PP2A-A 的结合非常重要。测试是否是候选人 蛋白质可能是 PP2A 的底物，我们将测试是否具体 磷酸受体位点突变体可以绕过 SUR-6 的要求 外阴诱导。 最后，确定 PP2A 的其他监管机构和目标 外阴诱导期间的活性，我们将进行酵母双杂交筛选 针对与 SUR-6 PP2A-B 结合的蛋白质，并进行遗传修饰筛选 对于具有与 sur-6 突变类似的特性的突变。我们的研究 将为 PP2A 的正常体内调节提供重要的见解 它的目标，以及如何操纵 PP2A 活性进行治疗 影响。