PROTEIN PHOSPHATASE 2A REGULATION OF RAS SIGNALING

描述：(申请人描述)蛋白磷酸酶2A(PP2A)是一种 重要的信号转导调节因子，病毒的靶标 癌蛋白，以及治疗药物设计的潜在有用靶点。 然而，PP2A在体外广泛的底物专一性阻碍了尝试 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PR55/B调节亚单位 PP2A-B)作为线虫RAS信号的正向调节器，以及我们的 遗传数据表明，SUR-6与KSR在共同的过程中起作用，并指导 PP2A与特定的RAS途径底物结合。SUR-6 PP2A-B基因的两个突变 优先影响RAS介导的信号，对其他信号的影响最小 SUR-6-或PP2A调控的过程。我们将利用线虫的外阴发育 作为解释PP2A-B亚基如何调控PP2A的遗传模型系统 催化活性和/或底物专一性，以及PP2A如何调节 RAS/RAF/MEK/ERK信令级联。测试SUR-6的绝对要求 PP2A-B在RAS介导的外阴诱导过程中，我们将分离到SuR-6空 并使用镶嵌分析策略来确定它们对 外阴引产。确定SUR-6 PP2A-B对PP2A催化的影响 活性，我们将使用RNA介导的干扰，PP2A-C突变，PP2A-C 检测转基因和SV4O小T抗原表达对PP2A的影响 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调节亚基的要求 这可能会将PP2A引导到替代的RAS途径底物上，并测试 SUR-6的结构域对于与PP2A-A结合很重要。测试候选人是否 蛋白质可能是PP2A的底物，我们将测试是否具有特异性 磷酸受体位点突变体可以绕过对SUR-6的要求 外阴引产。最后，确定PP2A的其他监管机构和目标 在外阴诱导过程中，我们将进行酵母双杂交筛选 用于与SUR-6 PP2A-B结合的蛋白质，并进行遗传修饰物筛选 用于具有类似于SUR-6突变特性的突变。我们的研究 将为PP2A和PP2A在体内的正常调控提供重要的见解 它的靶点，以及PP2A活性可能如何被操纵用于治疗 效果。

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.