PROTEIN PHOSPHATASE 2A REGULATION OF RAS SIGNALING

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.

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.