The objectives of this proposal are to elucidate (in cell culture models and cell-free systems) the biological role of phosphatidylcholine-derived diacylglycerol (DAG) in cell signal transduction and the mechanism by which phorbol esters stimulate this pathway. Preliminary results indicate that phorbol esters stimulate a phosphatidylcholine (PC) specific phospholipase C (PL-C) activity in nontransformed rat embryo fibroblasts (REF- NT) causing a 2- to 3-fold increase in DAG levels. However, this response was not observed in the corresponding transformed cell lines. The levels of PC hydrolase (PL-C) activity in lysates of both cell types were similar approximately 2.6 nmol/mg protein/hr). These results indicate that the lack of responsiveness in the transformed cell line is not due to loss of the enzyme, but may be due to either a lesion in the phorbol ester receptor/protein kinase C (PKC) pathway or in biochemical regulation of the transformed cell PC PL-C activity. In order to determine if alterations in regulation of PKC may contribute to the nonresponsiveness, we propose to compare the two cell lines with respect to the biochemical properties of PKC and phorbol ester binding activities. Type-specific antibodies prepared to three types of rabbit brain PKC will be used to characterize potential differences in PKC types in the two cell lines. We will also compare the biochemical properties of PC PL-C from the two cell lines. Furthermore, we have identified brain cytosol as a tissue source from which the enzyme can be purified. Both the cell lysates and the purified enzyme will be used in studies to test the effect of PKC-mediated phosphorylation on PC hydrolase activity. Each of three types of purified brain PKC will be used in these studies. The role of PC hydrolysis in regulation of growth and differentiation is not yet known, although a potential role in cell growth is suggested because a natural mitogenic agonist, vasopressin, also stimulates PC hydrolysis in these cells. Furthermore, the generality of the observed difference in responsiveness of REF-NT and REF-T cells will be tested by using several nontransformed cell lines and their virally or chemically transformed counterparts. A thorough characterization of the lipid metabolic pathways and the enzymes involved, assessment of physiological effectors of the pathway, and elucidation of the mechanism of phorbol ester activation will help establish the role of this putative second messenger system in normal and disease processes.
