ROLE OF PHOSPHATIDIC ACID IN GROWTH FACTOR SIGNALING

磷脂酸是响应大量细胞外信号而产生的脂质第二信使。它在细胞内信号传递中的作用在很大程度上仍不清楚。最近的工作表明，丝氨酸/苏氨酸激酶c-Raf-1可能是磷脂酸(PA)的直接细胞内靶标。PI实验室的工作表明，胰岛素促进c-Raf-1募集到质膜的机制需要激活ARF蛋白，而ARF蛋白反过来刺激磷脂酶D(PLD)和PA的生成。此外，单独加入PA可瞬时增加c-Raf-1与质膜的结合，但不影响Raf激酶的净激活或其下游效应，即MAP激酶级联的激活。因此，提出了PA与其他因素联合作用促进c-Raf-1在膜上的募集和随后的激活的一般模型。这一模型将在PI及其合作者开发的几个工具的帮助下进行测试。将使用最先进的成像方法研究RAF-1到膜的转位，该方法基于检测到包含A.Victoria绿色荧光蛋白(GFP)全序列的Raf-1融合产物。这些构建物已经在我们的实验室生产出来，我们已经证明了它们适合于定量确定Raf-1到细胞膜的移位。我们建议将这一成像方法与标准的生化技术结合起来，以确定PLD激活和细胞内PA的产生在胰岛素和PDGF激活MAPK级联反应中的作用。特别是，我们将研究：1)胰岛素和PDGF诱导的Raf-1到质膜的移位，使用实时活细胞成像和细胞分离方法描述完整的时间过程。2)阻断ARF和PLD激活对活细胞内Raf-1转位动力学的影响。3)阻断Raf-1易位与MAPK级联调控的相关性。4)使用(A)细胞分级和(B)双标记荧光显微镜和电子显微镜技术将Raf-1转位到细胞内的膜室。5)PA转化为甘油二酯和Lyso-PA对胰岛素和PDGF调节的Raf-1转位和MAPK调节的作用。6)利用定点突变、活细胞和体外检测相结合的方法，研究Raf-1的特定区域在易位、PA结合和激活中的作用。

Phosphatidic acid is a lipid second messenger generated in response to numerous extracellular signals. Its role in intracellular signalling remains largely unknown. Recent work has suggested that the serine/threonine kinase c-Raf-1 may be a direct intracellular target for phosphatidic acid (PA). Work in the PI's laboratory has shown that the mechanisms by which insulin promote the recruitment of c-Raf-1 to the plasma membrane require the activation of ARF proteins, which in turn stimulate phospholipase D (PLD) and the generation of PA. Furthermore, the addition of PA alone increases transiently the binding of c-Raf-1 to the plasma membrane but has no effects on the net activation of Raf kinase or on its downstream effects, namely the activation of the MAP kinase cascade. Therefore, a general model according to which PA acts in conjunction with other factors to promote the recruitment of c-Raf-1 to the membrane and its subsequent activation is proposed. This model will be tested with the aid of several tools developed by the PI and his collaborators. Raf-1 translocation to the membrane will be studied using state-of-the-art imaging methods based on the detection of Raf-1 fusion products containing the full sequence of A. victoria Green Fluorescent Protein (GFP). These constructs have already been produced in our laboratory and we have shown their suitability to determine quantitatively the translocation of Raf-1 to cellular membranes. It is proposed to use this imaging approach in conjunction with standard biochemical techniques to determine the role of PLD activation and the generation of intracellular PA on the activation of the MAPK cascade by insulin and PDGF. In particular, we will study: 1) The translocation of Raf-1 to the plasma membrane induced by insulin and PDGF, describing complete time courses using both real time live cell imaging and cell fractionation approaches. 2) The effects of the blockade of ARF and PLD activation on the dynamics of Raf-1 translocation in live cells. 3) The correlation between the blockade of Raf-1 translocation and the regulation of the MAPK cascade. 4) The translocation of Raf-1 to intracellular membrane compartments using (a) cell fractionation and (b) double-labelling fluorescence microscopy and electron microscopy techniques. 5) The role of the transformation of PA into diacylglycerol and lyso-PA on insulin- and PDGF-regulated Raf-1 translocation and MAPK regulation. 6) The role of specific regions of Raf-1 in translocation, PA binding and activation using site-directed mutagenesis and a combination of live cell and in vitro assays.