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.
