The goal of the current proposal is to define the mechanisms that control phosphatidylinositol (PtdIns) biosynthesis. PtdIns is a structural component of the membrane and a precursor to signalling molecules. Its biosynthetic pathway utilizes phosphatidic ac id (PtdOH) and cytidylyldiphosphodiacylglycerol (CDP-DAG). There are two mammalian genes encoding CDP-DAG synthetases (CDS1 and CDS2). In the current proposal we will test whether one CDS isoform is involved in de novo PtdIns formation whereas the other participates in the turnover of PtdIns after agonist-induced stimulation of phospholipase C. There are strong computational evidence for CDS1 and CDS2 phosphorylation, and we propose to develop isoform specific antibodies to characterize the post-translation al modifications as well as the sub-cellular distribution of the two isoforms. The second goal is to identify the enzyme(s) that regulate the distribution of PtdOH between the CDP-DAG and diacylglycerol.
Our alternative hypothesis suggests that PtdIns bio-synthesis is regulated by the supply of PtdOH, the substrate of CDS. Magnesium-dependent phosphatidate phosphohydrolase (PAP1) is the enzyme involved in the de novo pathway of PtdOH utilization. The gene(s) coding for PAP1 enzyme(s) have not been characterized. We used a bio-informatics approach to identify two novel genes as candidates for PAP1 and we propose to characterize their biochemical activities and cellular functions as well as their involvement in PtdIns biosynthesis. Propranolol and bromoenol lactone have been previously identified as PAP1 inhibitors and additional micro-array experiments are proposed to characterize the transcriptional response to them, which may lead to the identification of their target PAP1 gene(s). The results of these studies will identify novel players in PtdIns biosynthesis, advance our understanding of the mechanisms that govern mammalian PtdIns metabolism and may lead to the development of novel therapeutics.
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