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J. Biol. Chem., Vol. 269, Issue 45, 27823-27826, Nov, 1994
EJ Yeo, A Kazlauskas and JH Exton
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.
Activation of phospholipase C-gamma is necessary for stimulation of phospholipase D by platelet-derived growth factor
Howard Hughes Medical Institute, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0295.
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