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: 6188969
• 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/6188969
• 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（PP 2A）是一种 信号转导途径的重要调节因子，病毒的靶点 癌蛋白，和治疗药物设计的潜在有用的目标。 然而，PP 2A在体外的广泛底物特异性阻碍了尝试， 了解其在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）的一个PR 55/B调节亚基 PP 2A-B）作为C.优雅，我们的 遗传数据表明，SUR-6与KSR在一个共同的过程中起作用，并指导 PP 2A与特定Ras途径底物的结合。SUR-6 PP 2A-B中的两个突变 优先影响Ras介导的信号传导，对其他信号传导的影响最小。 SUR-6或PP 2A监管工艺。我们将使用C。秀丽隐翅虫外阴发育 作为一个遗传模型系统，以阐明PP 2A-B亚基如何调节PP 2A 催化活性和/或底物特异性，以及PP 2A如何调节 Ras/Raf/MEK/ERK信号级联。检验SUR-6的绝对要求 PP 2A-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 外阴诱导为了确定SUR-6 PP 2A-B如何影响PP 2A催化 活性，我们将使用RNA介导的干扰，PP 2A-C突变，PP 2A-C 转基因和SV 40小t抗原表达，以测试如何改变PP 2A 催化活性影响外阴诱导。我们还将使用RNA介导的 干扰，以测试对其他PP 2A-B调节亚基的要求 这可能会导致PP 2A的替代Ras途径底物，并测试 SUR-6的结构域对于结合PP 2A-A是重要的。测试候选人是否 蛋白质可能是PP 2A的底物，我们将测试是否特异性 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PP 2A的其他监管机构和目标 活性在外阴诱导，我们将进行酵母双杂交筛选 用于与SUR-6 PP 2A-B结合的蛋白质，并进行遗传修饰剂筛选 对于具有与sur-6突变相似的性质的突变。我们的研究 将为PP 2A的正常体内调节提供重要的见解， 它的目标，以及PP 2A活性如何被操纵用于治疗 方面的影响.