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

doi:10.1074/jbc.273.32.20517

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

From the ^‡Department of Molecular Physiology and Biophysics, the ^‖Howard Hughes Medical Institute, and the Departments of ^§Biochemistry and ^¶Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0295

Abstract

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.

Footnotes

↵* Supported in part by Grants T32KD07061–24 (to J. A. H.) and R01CA75195 (to G. C.) from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵** Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed. Tel.: 615-322-6494; Fax: 615-322-4381; E-mail: john.exton{at}mcmail.vanderbilt.edu.
Abbreviations:

PDGF

platelet-derived growth factor

PDGFR

PDGF receptor

PLD

phospholipase D

MEF

mouse embryo fibroblast

PLC-γ1

phospholipase C

PIP₂

phosphatidylinositol 4,5-bisphosphate

DAG

diacylglycerol

IP₃

inositol-1,4,5-trisphosphate

PKC

protein kinase C

PA

phosphatidic acid

DMEM

Dulbecco’s modified Eagle’s medium

OAG

1-oleoyl-2-acetyl-sn-glycerol

PBS

phosphate-buffered saline

Ins

inositol

InsP_x

inositol phosphates

P-Tyr

phosphotyrosine

EGF

epidermal growth factor

MAP kinase

mitogen-activated protein kinase

PtdBut

phosphatidylbutanol.
- Received April 7, 1998.
- Revision received May 7, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.