Diglyceride occupies a central position in the synthesis of phospholipids and as a signal in response to growth factors and other agonists. Diglyceride accumulates in cells that have undergone transformation in response to oncogenes such as ras. The accumulation of diglyceride activates protein kinase C (PKC), which has been implicated in the regulation of both normal growth and abnormal responses such as in transformation. Manipulations that prevent the generation of diglyceride block activation of PKC and mitogenesis in model systems (e.g. transfection with ras), as do inhibitors of PKC. There are several pathways by which diglyceride can be metabolized. This project focuses on metabolism by hydrolysis catalyzed by two different enzymes. Diglyceride lipase catalyzes the hydrolysis of the fatty acid from the sn-1 position and monoglyceride lipase acts on the resultant monoglyceride. This pathway could shut off the signal for activation of protein kinase C and thereby regulate cellular responses. Additionally, this pathway has been proposed as a mechanism by which arachidonic acid might be released. This essential fatty acid can be converted into a large family of oxygenated products known as eicosanoids that have diverse, potent physiological actions. Moreover, arachidonic acid itself has been implicated in the regulation of cellular growth due to its effect on GAP. Previous studies from many laboratories have supported the hypothesis that the diglyceride (DG) and monoglyceride lipases can metabolize diglyceride. However, the relative importance of this pathway to others in lipid metabolism has been controversial. In preliminary experiments we have shown that the concentrations of diglyceride change at different growth states of cells. Additionally, exogenous phospholipases that generate diglyceride within the cells caused a variety of responses. Finally, we have purified the DG lipase from human platelets to near homogeneity and find that it is closely related, or identical, to another lipase. We propose to complete the purification and determine whether the diglyceride lipase is a unique enzyme, and then characterize it. A cDNA for the appropriate enzyme will be cloned and expressed in mammalian cells. A variety of experiments will determine whether this enzyme plays a crucial role in the metabolism of diglyceride in response to transformation with an oncogenic form of ras, whether the activation of PKC is regulated by the DG lipase, and whether cellular release of arachidonic acid (or its metabolites) depends on this pathway. Similar studies will be carried out with the monoglyceride lipase - purification and characterization, and cloning of the cDNA. Its effects will be examined in analogous experiments in appropriate cell systems. These studies should define the role that hydrolysis of diglycerides and monoglycerides plays in regulating functions including growth and inflammation.
