Analysis of Platelet-derived Growth Factor-induced Phospholipase D Activation in Mouse Embryo Fibroblasts Lacking Phospholipase C-γ1

Platelet-derived growth factor (PDGF) activates phospholipase D (PLD) in mouse embryo fibroblasts (MEFs). In order to investigate a role for phospholipase C-γ1 (PLC-γ1), we used targeted disruption of the Plcg1 gene in the mouse to develop Plcg1+/+ andPlcg1−/− cell lines. Plcg1+/+ MEFs treated with PDGF showed a time- and dose-dependent increase in the production of total inositol phosphates that was substantially reduced inPlcg1−/− cells. Plcg1+/+ cells also showed a PDGF-induced increase in PLD activity that had a similar dose dependence to the PLC response but was down-regulated after 15 min. Phospholipase D activity, however, was markedly reduced in Plcg1−/−cells. The PDGF-induced inositol phosphate formation and the PLD activity that remained in the Plcg1−/− cells could be attributed to the presence of phospholipase C-γ2 (PLC-γ2) in the Plcg1−/− cells. The PLC-γ2 expressed in the Plcg1−/− cells was phosphorylated on tyrosine in response to PDGF treatment, and a small but significant fraction of the Plcg1−/− cells showed Ca2+ mobilization in response to PDGF, suggesting that the PLC-γ2 expressed in the Plcg1−/− cells was activated in response to PDGF. The inhibition of PDGF-induced phospholipid hydrolysis in Plcg1−/− cells was not due to differences in the level of PDGF receptor or in the ability of PDGF to cause autophosphorylation of the receptor. Upon treatment of the Plcg1−/− cells with oleoylacetylglycerol and the Ca2+ ionophore ionomycin to mimic the effect of PLC-γ1, PLD activity was restored. The targeted disruption ofPlcg1 did not result in universal changes in the cell signaling pathways of Plcg1−/− cells, because the phosphorylation of mitogen-activated protein kinase was similar inPlcg1+/+ and Plcg1−/−cells. Because increased plasma membrane ruffles occurred in bothPlcg1+/+ and Plcg1−/−cells following PDGF treatment, it is possible neither PLC nor PLD are necessary for this growth factor response. In summary, these data indicate that PLC-γ is required for growth factor-induced activation of PLD in MEFs.