ANALYSIS OF PLATELET-DERIVED GROWTH FACTOR-INDUCED PHOSPHOLIPASE-D ACTIVATION IN MOUSE EMBRYO FIBROBLASTS LACKING PHOSPHOLIPASE C-GAMMA-1

Platelet-derived growth factor (PDGF) activates phospholipase D (PLD) in mouse embryo fibroblasts (MEFs). In order to investigate a role for phospholipase C-yl (PLC-gamma 1), we used targeted disruption of the Plcg1 gene in the mouse to develop Plcg1(+/+) and Plcg1(-/-) cell line s. Plcg1(+/+) MEFs treated with PDGF showed a time- and dose-dependent increase in the production of total inositol phosphates that was subs tantially reduced in Plcg1(-/-) cells, PLcg1(+/+) cells also showed a PDGF-induced increase in PLD activity that had a similar dose dependen ce to the PLC response but was down-regulated after 15 min. Phospholip ase D activity, however, was markedly reduced in PLcg1(-/-) cells. The PDGF-induced inositol phosphate formation and the FLD activity that r emained in the Plcg1(-/-) cells could be attributed to the presence of phospholipase C-gamma 2 (PLC-gamma S) in the Plcg1(-/-) cells, The PL C-gamma 2 expressed ire the Plcg1(-/-) cells was phosphorylated on tyr osine in response to PDGF treatment, and a small but significant fract ion of the Plcg1(-/-) cells showed Ca2+ mobilization in response to PD GF, suggesting that the PLC-gamma 2 expressed in the Plcg1(-/-) cells was activated in response to PDGF. The inhibition of PDGF-induced phos pholipid hydrolysis in Plcg1(-/-) cells was not due to differences in the level of PDGF receptor or in the ability of PDGF to cause autophos phorylation of the receptor. Upon treatment of the Plcg1(-/-) cells wi th oleoyalacetylglycerol and the Ca2+ ionophore ionomycin to mimic the effect of PLC-gamma 1, PLD activity was restored. The targeted disrup tion of Plcg1 did not result in universal changes in the cell signalin g pathways of Plcg(1-/)- cells, because the phosphorylation of mitogen -activated protein kinase was similar in PLcg1(+/+) and Plcg1(-/-) cel ls. Because increased plasma membrane ruffles occurred in both Plcg1(/+) and Pbcg1(-/-) cells following PDGF treatment, it is possible neit her PLC nor PLD are necessary for this growth factor response. In summ ary, these data indicate that PLC-gamma is required for growth factor- induced activation of PLD in MEFs.