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The regulation of phosphoinositide phosphorylation was studied in Swiss 3T3 cells that were stimulated by platelet-derived growth factor (PDGF). Studies with intact cells showed that the mitogen increased the incorporation of 32P into phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns-P), and phosphatidylinositol 4,5-bisphosphate (PtdIns-P2) during the cell cycle, with distinct peaks of incorporation for all three phosphoinositides after 1 h, and for PtdIns and PtdIns-P2 after 20 h. Direct measurements of the activities of PtdIns kinase and PtdIns-P kinase in freeze-thawed cells revealed that the activity of PtdIns kinase was rate-limiting for the synthesis of PtdIns-P2. Maximal activities of PtdIns kinase and PtdIns-P kinase, with exogenous substrates, were unchanged during the 1st h of PDGF treatment, but doubled during the next 24 h. The increase in PtdIns kinase activity began within 2-4 h, exceeded the increase in cell protein, and was abolished by cycloheximide, which suggests that the enzyme was induced specifically in response to PDGF. The increase in activity of PtdIns-P kinase paralleled the increase in cell protein. Dose-response curves for PDGF showed that the activities of PtdIns kinase and PtdIns-P kinase at 24 h increased in proportion to the extent of mitogenic stimulation of the cells. Our results support the conclusion that the activities of PtdIns kinase and PtdIns-P kinase increase in response to PDGF, but only after several hours of cell cycle traverse.

We studied the influence of platelet-derived growth factor (PDGF) on diacylglycerol phosphorylation in Swiss 3T3 cells. Rates of incorporation of 32P into phosphatidic acid (PA) and phosphatidylinositol (PtdIns) were determined in prelabeled cells into which sn-1,2-didecanoylglycerol (diC10) had been introduced. PDGF stimulated the formation of [32P]PA and -PtdIns from endogenous substrates but decreased the formation of [32P]PA10 and -PtdIns10. Direct measurements of diacylglycerol phosphorylation in lysates of quiescent and stimulated cells showed that PDGF stimulated the phosphorylation of endogenous diacylglycerol 2-fold in parallel with diacylglycerol accumulation but decreased by 50% the phosphorylation of diC10. Total diacylglycerol kinase activity, measured in a mixed micellar assay, was not changed by PDGF treatment. The maximum activity of diacylglycerol kinase exceeded that needed to phosphorylate all of the endogenous diacylglycerol, suggesting that the PDGF-dependent increase in diacylglycerol mass would account for the increase in PA formation. The increased mass of diacylglycerol also could explain the inhibition of diC10 phosphorylation, via substrate competition. The predominant species of endogenous diacylglycerol was 1-stearoyl-2-arachidonoyl-glycerol (18:0/20:4 diacylglycerol). In mixed micelles, the rate of phosphorylation of 18:0/20:4 diacylglycerol was 8-fold higher than that of diC10, and the 18:0/20:4 species competed with diC10 for phosphorylation. Studies showed that a membrane-bound enzyme accounted for the PDGF effect on PA formation; there was no evidence for translocation of cytosolic enzyme to the membrane. The results support these conclusions: 1) PDGF stimulates the phosphorylation of cellular diacylglycerol by promoting a transient accumulation of this lipid. 2) The stimulated phosphorylation is catalyzed by a diacylglycerol kinase that preferentially phosphorylates 18:0/20:4 diacylglycerol over diC10. 3) The diacylglycerol kinase responsible for the PDGF effect is membrane-bound.