The long term goal of this research is to determine what role polyphosphoinositides phosphorylated at the D-3 position of the inositol ring play in growth factor stimulation of cells. This laboratory recently discovered three new polyphosphoinositides not in the pathway for generation of inositol-1,4,5-trisphosphate. The enzyme which produces these lipids was discovered because of a tight association with several protein-tyrosine kinases, including the platelet derived growth factor (PDGF) receptor. This association occurs within 15 seconds of PDGF addition to quiescent fibroblasts. In vitro this activity phosphorylates phosphatidylinositol (PI) to produce phosphatidylinositol-3-phosphate (PI- 3-P). When phosphatidylinositol-4-phosphate (PI-4-P) is used as a substrate, a lipid whose structure appears to be phosphatidylinositol-3,4- bisphosphate (PI-3-4-P2) is produced and when phosphatidylinositol-4,5- bisphosphate (PI-4,5-P2) is added, phosphatidylinositol-trisphosphate (PIP3) is produced. We have evidence that a single enzyme phosphorylates all three substrates at the D-3 position. These results along with observations that the same PI kinase activities associate with the middle t transforming gene product of polyoma virus but fail to associate with transformation defective mutants suggest that this novel pathway mediates an important signal in growth and transformation. PI-3-P has now been found in a variety of proliferating cells from yeast to mammalian fibroblasts, and the PI-3,4-P2 and PIP3 have been shown to be produced in cells stimulated with PDGF or transformed with polyoma virus. The focus of this research will be to elucidate the metabolic pathway and determine whether this is a novel signal transduction system parallel to the inositol-1,4,5-trisphosphate system.
The specific aims of this proposal are to 1) complete the structural analysis of these lipids, 2) investigate changes in vivo levels of these lipids in response to specific growth factors and oncogenes, 3) determine the pathways for synthesis and degradation of these lipids, and 4) determine the effects of these lipids on cell morphology and physiology. Our preliminary studies with mutated oncogenes and growth factor receptors strongly argue that synthesis of the D-3 phosphorylated polyphosphoinositides is a critical event in stimulation of cell growth. Understanding this pathway should shed light on the biochemical basis of cell growth and oncogenesis.
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