Many growth factors and hormones elicit their biological response by stimulating the hydrolysis of phosphatidylinositol 4,5-P2 to produce diacylglycerol and inositol 1,4,5-P3. Adequate levels of the polyphosphoinositides used in such signaling appear to be crucial to the maintenance of proper cell function. Decreases in the levels of inositol-containing phospholipids have been associated with a variety of pathological conditions including diabetic neuropathy and Respiratory Distress Syndrome. Phosphoinositides are present not only in the plasma membrane but also occur as part of a distinct nuclear PI cycle. Nuclear PI turnover has been shown to be regulated by IGF-1 and the levels of nuclear inositol phospholipids appear to change in cells undergoing differentiation. The levels of polyphosphoinositides are determined by their relative rates of synthesis and degradation. While much research has focused on the synthesis of polyphosphoinositides, comparatively little is known of the phosphoinositide phosphatases that degrade phosphoinositides. Recent work has suggested that alterations in these enzymes may be the basis for changes in nuclear PI homeostasis associated with differentiation. Furthermore, preliminary data have indicated that activity of several enzymes involved in PI turnover and cell signaling are modulated in a cell cycle-dependent fashion suggesting the possibility that inositol phospholipid homeostasis may be modulated during the cell cycle. Based on the above considerations, the specific aims of this proposal are: 1. To clone and sequence a PI4-P 4-phosphatase. 2. To define the role of the PI 4-P phosphatase in inositol phospholipid homeostasis and cell signaling. 3. To examine the expression of PI 4-P 4-phosphatase mRNA and protein throughout the cell cycle. 4. To elucidate the role of the PI 4-P phosphatase in the nuclear PI cycle and characterize the effects of IGF's on this cycle. A PIP phosphatase from rat brain has been purified 76,000-fold in the laboratory and partially characterized. In the proposed experiments, protein sequence will be obtained from the purified PIP phosphatase and used to design probes for the cloning and sequencing of a cDNA corresponding to this enzyme. The role of the PIP phosphatase in maintaining inositol phospholipid homeostasis will then be investigated by overexpressing the PIP phosphatase in 3T3 cells and evaluating its effect on inositol phosphate and phospholipid levels. The involvement of the PIP phosphatase in the nuclear PI cycle and the effects of IGF's on this novel signaling pathway will also be investigated. Insight into the structure, function and regulation of a PIP phosphatase will aid in understanding the mechanisms involved in regulating the levels of phosphoinositides within cells.
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