Analysis of platelet-derived growth factor-induced phospholipase D activation in mouse embryo fibroblasts lacking phospholipase C-gamma1.
Platelet-derived growth factor (PDGF) activates phospholipase D (PLD) in mouse embryo fibroblasts (MEFs). In order to investigate a role for phospholipase C-gamma1 (PLC-gamma1), we used targeted disruption of the Plcg1 gene in the mouse to develop Plcg1(+/+) and Plcg1(-/-) 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 in Plcg1(-/-) 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-gamma2 (PLC-gamma2) in the Plcg1(-/-) cells. The PLC-gamma2 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-gamma2 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-gamma1, PLD activity was restored. The targeted disruption of Plcg1 did not result in universal changes in the cell signaling pathways of Plcg1(-/-) cells, because the phosphorylation of mitogen-activated protein kinase was similar in Plcg1(+/+) and Plcg1(-/-) cells. Because increased plasma membrane ruffles occurred in both Plcg1(+/+) 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-gamma is required for growth factor-induced activation of PLD in MEFs.
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0295, USA.
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Hess
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Ji
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Carpenter
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Exton
D000818:Animals; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002462:Cell Membrane / Q000502:physiology; D004075:Diglycerides / Q000494:pharmacology; D004622:Embryo, Mammalian / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005347:Fibroblasts; D018390:Gene Targeting; D007295:Inositol Phosphates / Q000378:metabolism; D015759:Ionomycin / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D051966:Phospholipase C gamma; D010739:Phospholipase D / Q000378:metabolism*; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D015398:Signal Transduction / Q000502:physiology; D010738:Type C Phospholipases / Q000378:metabolism
D004075:Diglycerides; D007295:Inositol Phosphates; D007527:Isoenzymes; D010982:Platelet-Derived Growth Factor; D015759:Ionomycin; D004815:Epidermal Growth Factor; C038237:1-oleoyl-2-acetylglycerol; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D010739:Phospholipase D; D002118:Calcium