Howard Hughes Medical Institute , Vanderbilt University School of Medicine , Nashville , Tennessee 37232-0295 .
J Biol Chem 269 : 27823-6 ( 1994)
Abstract
Platelet-derived growth factor ( PDGF ) stimulates phosphatidylcholine hydrolysis via phospholipase D ( PLD ) in several tissues .
To determine whether PLD activation is dependent on phosphoinositide hydrolysis by phospholipase C ( PLC ) , we measured the formation of phosphatidylbutanol ( PtdBut ) , in TRMP cells overexpressing wild type or various mutant PDGF receptors .
Both PLC and PLD were stimulated by PDGF in cells expressing wild type receptors whereas they were not in cells expressing kinase-deficient ( R634 ) receptors .
These data indicate that tyrosine phosphorylation is required for activation of both PLC and PLD .
Mutation of Tyr-1021 of the PDGF receptor to Phe caused loss of PDGF stimulation of both PLC and PLD .
On the other hand , a mutant PDGF receptor that was able to bind PLC gamma 1 but not other signaling proteins ( including the Ras GTPase-activating protein , phosphatidylinositol 3-kinase , and a SH2-containing phosphotyrosine phosphatase ( Syp ) ) restored the stimulatory effect of PDGF on PLC and PLD .
Furthermore , receptors in which association with the GTPase-activating protein , phosphatidylinositol 3-kinase , or Syp was individually restored were unable to mediate PDGF stimulation of PLC or PLD .
These data indicate that these other signal transduction proteins are not involved in the activation of PLD by PDGF .
Treatment of the cells with the protein kinase C inhibitor , Ro-31-8220 , and depletion of cellular protein kinase C by pretreatment with 4 beta-phorbol 12-myristate 13-acetate resulted in loss of PLD activation by PDGF indicating a PKC-dependent mechanism .
In summary , these results indicate that activation of PLC gamma 1 and protein kinase C are necessary for the stimulation of PLD by PDGF and provide no evidence for alternative mechanisms .