PHOSPHATIDYLCHOLINE METABOLISM IN RAS-TRANSFECTED CELLS

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This proposal seeks to investigate the mechanisms of altered phosphatidylcholine metabolism in cells transfected by the human H-ras oncogene. Phosphatidylcholine hydrolysis has been implicated in pathways of signal transduction, and the protein product of the ras oncogene is thought to play a role in a signal transduction pathway. Our recent studies on phosphatidylcholine metabolism in C3H10T1/2 murine fibroblasts transfected with the H-ras cells have revealed four marked differences between normal and ras-transfected cells: elevated phosphocholine levels elevated choline kinase activity, elevated glycerophosphocholine levels, and decreased cytidylyltransferase activity. Our working hypothesis is that ras transformation results, directly or indirectly, in activation of a phospholipase A. As a mechanism for increasing phosphatidylcholine synthesis in response to the increased degradation of phosphatidylcholine, choline kinase activity is increased. Cytidylyltransferase activity is then decreased by way of fine-tuning the pathway. In this proposal we will test various aspects of this hypothesis. We will put the ras gene under the control of an inducible promoter to allow determination of the temporal order in which these changes take place upon expression of ras. The activities of phospholipases A, C, and D will be determined in control C3H10T1/2 cells and in ras-infected cells. The mechanisms by which the activities of choline kinase and cytidylyltransferase change in ras-transformed cells will be investigated. In addition, we will address the possible role of phospholipase A activation in the mechanism of transformation by ras. We will determine if inhibition of phospholipase A activity in a suppressor or ras transformation can also suppress phospholipase A activities in ras- transformed cells. Results from these experiments will help elucidate mechanisms of coordinate regulation of phosphatidylcholine biosynthesis, and lead toward an understanding of the role of phospholipase activity in ras transformation.