Substrate selectivity of diacylglycerol kinase in PDGF-stimulated 3T3 cells

The authors investigated the properties of Diacylglycerol (DAG) Kinase in 3T3 cells. PDGF treatment caused an increase in DAG mass, an increase in incorporation of 32P into phosphatidic acid (PA) and phosphatidylinositol (PI), and an increase in the rate of phosphorylation of membrane DAG in vitro. The mechanism of enhanced phosphorylation of DAG was studied with dicaprylin (diC10) as a probe. Cells were prelabeled with 32P and treated with PDGF or carrier. DiC10 was added to the cell medium before harvesting. With PDGF treatment, the radioactivity in endogenous PA increased fourfold, whereas the radioactivity in PA10 and PI10 was consistently decreased. To verify that the PDGF effect on PA10 formation in intact cells was due to reduced phosphorylation of diC10 by DAG kinase, cells were treated with PDGF and/or diC10, freeze-thawed, and then incubated with Mg[32P]ATP. The rate of phosphorylation of cell-associated diC10 was decreased 50% by PDGF treatment. This effect could not be explained by decreased intracellular levels of diC10, or by saturation of DAG kinase with endogenous DAGs. Therefore, it seemed that endogenous DAGs, derived from PI, might be better substrates for DAG kinase than is diC10. In studies of the properties of DAG kinase with pure DAGs in mixed detergent micelles, they found that the enzyme phosphorylated arachidonoyl-DAG more readily than diC10. The selectivity of DAG kinase may play a key role in the formation of arachidonoyl species of PI