ROLE OF PHOSPHATIDIC ACID IN GROWTH FACTOR SIGNALING

磷脂酸是响应于许多细胞外信号而产生的脂质第二信使。 其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，这反过来刺激磷脂酶D（PLD）和PA的产生。 此外，单独加入PA会瞬时增加c-Raf-1与质膜的结合，但对Raf激酶的净活化或其下游效应（即MAP激酶级联的活化）没有影响。 因此，提出了一个一般模型，根据该模型，PA与其他因素一起促进c-Raf-1向膜的募集及其随后的激活。 该模型将在PI及其合作者开发的几种工具的帮助下进行测试。Raf-1易位到膜将使用最先进的成像方法进行研究，该方法基于检测含有A.维多利亚绿色荧光蛋白（GFP）。 这些构建体已经在我们的实验室中产生，并且我们已经显示它们适合于定量地确定Raf-1向细胞膜的易位。 建议使用这种成像方法结合标准的生物化学技术，以确定PLD激活的作用和细胞内PA的产生对胰岛素和PDGF激活MAPK级联的影响。 特别地，我们将研究：1）Raf-1在胰岛素和PDGF诱导下向质膜的移位，使用真实的时间活细胞成像和细胞分级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)Raf-1易位的阻断与MAPK级联调节之间的相关性。 4)使用（a）细胞分级分离和（B）双标记荧光显微镜和电子显微镜技术将Raf-1易位至细胞内膜区室。 5)PA转化为甘油二酯和溶血PA对胰岛素和PDGF调节的Raf-1易位和MAPK调节的作用。 6)Raf-1的特定区域在易位，PA结合和激活中的作用，使用定点诱变和活细胞和体外试验的组合。

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.