We recently identified phosphatidylcholine as a novel source of diglyceride (DG) mediators; the long-term goal of the proposed project is to define the role of these species of DG in the stimulus-response coupling of normal and neoplastic cells. The proposed investigations will focus on the specificity and regulation of the phospholipase C that degrades choline phosphoglycerides (PC) and on the role of PC-derived DG species as intracellular second-messengers. We will identify the molecular species of DG formed by this pathway and determine the ability of different molecular species to activate protein kinase C. In related studies we will investigate the role of the PC-specific phospholipase C in the turnover of cellular phospholipids.
The specific aims of this proposal are: 1) To determine the specificity of phospholipase C for different subclasses and molecular species of PC. The PC fraction of many cell types contains a mixture of alkyl-PC and acyl-PC. We have shown that degradation of PC results in a mixture of acyl-DG and alkyl-DG. The relative amounts of these subclasses of DG will be determined after cell stimulation. 2) To continue studies on the activity of phospholipase C by in vitro assay. These studies will determine the substrate specificity, cofactor requirements and subcellular localization of the PC degrading phospholipase C. 3) To determine the structural requirements for protein kinase C activation or inhibition by DG. We have shown that alkyl-DGs are inhibitors of PKC activation by, acyl-DG. We will continue these studies using the molecular species of DG observed after cell stimulation and separate the isozymes of PKC to test the effects of alkyl- and acyl-DG on each isoenzyme. 4) To determine the role of phospholipase C in the conversion of PC to ethanolamine phosphoglycerides (PE). In preliminary studies we found that alkyl-PC can be converted to alkenyl-PE by a previously undefined pathway; further studies will be performed to determine the pathway of-conversion and investigate the role of phospholipase C. The combination of the proposed experiments will further define the role of PC-derived DG in cellular regulation. The experiments will also provide further insight into the mechanism of action of phorbol diester tumor promoters and the cellular response to growth factors. It is anticipated that these studies will lead to a better understanding of the role of PC turnover in growth control.
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