ROLE OF PHOSPHATIDIC ACID IN GROWTH FACTOR SIGNALING

磷脂酸是响应多种细胞外信号而产生的脂质第二信使。它在细胞内信号传导中的作用在很大程度上仍然未知。最近的研究表明，丝氨酸/苏氨酸激酶c-Raf-1可能是磷脂酸（PA）的直接细胞内靶点。PI实验室的工作表明，胰岛素促进c-Raf-1向质膜募集的机制需要激活ARF蛋白，而ARF蛋白反过来刺激磷脂酶D （PLD）和PA的生成。此外，单独添加PA会短暂地增加c-Raf-1与质膜的结合，但不会影响Raf激酶的净激活或其下游效应，即MAP激酶级联的激活。因此，我们提出了一个通用模型，根据该模型，PA与其他因素共同作用，促进c-Raf-1向膜募集并随后激活。这个模型将在PI和他的合作者开发的几个工具的帮助下进行测试。通过检测含有维多利亚绿荧光蛋白（A. victoria Green Fluorescent Protein， GFP）全序列的Raf-1融合产物，我们将使用最先进的成像方法研究Raf-1在膜上的易位。这些结构已经在我们的实验室中产生，我们已经证明了它们的适用性，可以定量地确定Raf-1向细胞膜的易位。建议将这种成像方法与标准生化技术结合使用，以确定PLD激活和细胞内PA的产生对胰岛素和PDGF激活MAPK级联的作用。特别是，我们将研究：1)胰岛素和PDGF诱导的Raf-1向质膜的易位，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激活对活细胞中Raf-1易位动力学的影响。3)阻断Raf-1易位与调控MAPK级联的相关性。4)使用(a)细胞分离和(b)双重标记荧光显微镜和电子显微镜技术观察Raf-1在细胞膜内的易位。5) PA转化为二酰基甘油和lyso-PA对胰岛素和pdgf调控的Raf-1易位和MAPK调控的作用。6)利用定点诱变和活细胞和体外联合试验，研究Raf-1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.