PC METABOLISM IN NUCLEAR ENVELOPE SIGNAL TRANSDUCTION

描述(改编自申请人的摘要)：拉本博士和 他的同事们最近提出了一种假设，即核脂 新陈代谢是核信号级联的一个组成部分，他 定义为核膜信号转导或巢。这个 在这项提议中要检验的核心的、新颖的假设是 在质膜上启动的信号被发送到细胞核并 触发特定的核脂代谢作为不可或缺的组成部分 质膜到细胞核的信号转导的级联。他们的 研究表明，核磷脂酰胆碱(PC)代谢，以及 激活PKC，是Nest的重要组成部分。这些研究 在这项建议中，检查核内PC代谢和PKC的激活 有丝分裂原α-凝血酶在静止的成纤维细胞中的诱导。 这些研究代表了第一系列旨在 明确定义了这部小说中涉及的分子成分 信号通路。这种级联反应的缺陷可能会导致 包括肿瘤在内的疾病。 这些研究是在三个具体目标下组织的。的目标是 特异目的I鉴定谷氨酸蛋白结合蛋白(S)(GBP) 负责将α-凝血酶受体偶联到核PC 一是新陈代谢，二是确定MAPK(丝裂原激活的 蛋白激酶)是这种激活的中间成分。这个 α-凝血酶受体与GBP偶联，α-凝血酶被激活 MAPK，一种与核信号转导有关的激酶。在这个目标中， 研究人员将检验一种假说，即一种 α-凝血酶受体的特异性GBP(S)导致激活 MAPK的激活，导致核PC水解酶的激活。英镑(S) 与α-凝血酶受体偶联并参与诱导核 将通过以下方法确定PC水解度： 反义介导的光反应性GTP消融特定GBP 模拟的。MAPK在这些事件中的作用将由以下因素决定 检测α-凝血酶激活核PC的能力 稳定转染可诱导显性基因的细胞中的水解酶 MAPK的阴性变种。在特定目标II中，他们将确定， 核型磷脂酶原激活酶S的鉴定与分离 Nest参与的水解物。他们将第一次隔离 时间，真核PC水解酶的新方法拉本博士 已经为表达克隆和使用一种新的PC耦合而开发 亲和层析用琼脂糖树脂。在具体目标三中， 研究人员将探索核DGs的生物学作用 通过识别核PKC底物增加和PKC的激活 使用PKC抑制剂、体外磷酸化和序列分析。

DESCRIPTION (adapted from the applicant's abstract): Dr. Raben and his colleagues have recently advanced the hypothesis that nuclear lipid metabolism is a component of nuclear signaling cascades which he defined as nuclear envelope signal transduction or NEST. The central, novel hypothesis to be tested in this proposal is that signals initiated at the plasma membrane are sent to the nucleus and trigger specific nuclear lipid metabolism as an integral component of plasma membrane-to-nucleus signal transduction cascades. Their studies indicate that nuclear phosphatidylcholine (PC) metabolism, and activation of PKC, are important components of NEST. The studies in this proposal examine nuclear PC metabolism and PKC activation induced in quiescent fibroblasts by alpha-thrombin, a potent mitogen. These studies represent the first series of experiments aimed at explicitly defining the molecular components involved in this novel signaling pathway. Defects in this cascade may lead to pathological condition including neoplasias. The studies are organized under three specific aims. The goal of Specific Aim I is to identify the GTP-binding protein(s) (GBP) responsible for coupling the alpha-thrombin receptor to nuclear PC metabolism and secondly, to determine whether MAPK (mitogen-activated protein kinase) is an intermediate component in this activation. The alpha-thrombin receptor couples to GBPs, and alpha- thrombin activates MAPK, a kinase implicated in nuclear signal transduction. In this aim, the investigators will test the hypothesis that the activation of a specific GBP(s) by the alpha-thrombin receptor results in the activation of a MAPK, leading to the activation of nuclear PC hydrolysis. GBP(s) coupled to the alpha-thrombin receptor and involved in induced nuclear PC hydrolysis will be identified by methods which will include antisense-mediated ablation of specific GBPs with a photoreactive GTP analog. The role of MAPK in these events will be determined by examining the ability of alpha-thrombin to activate nuclear PC hydrolysis in cells stably transfected with an inducible dominant- negative variant of MAPK. In Specific Aim II, they will identify, characterize and isolate the enzyme(s) responsible for nuclear PC hydrolysis involved in NEST. They will isolate, for the first time, eukaryotic PC hydrolyzing enzymes using new methods Dr. Raben has developed for expression cloning and using a new PC-coupled sepharose resin for affinity chromatography. In specific aim III, the investigators will explore the biological roles of nuclear DGs increases and PKC activation by identifying the nuclear PKC substrates using PKC inhibitors, in vitro phosphorylation, and sequence analyses.