Abstract
The role of phosphatidylcholine (PC) hydrolysis in activation of the mitogen-activated protein kinase (MAPK) pathway by platelet-derived growth factor (PDGF) was studied in Rat-1 fibroblasts. PDGF induced the transient formation of phosphatidic acid, choline, diacylglycerol (DG), and phosphocholine, the respective products of phospholipase D (PLD) and phospholipase C (PC-PLC) activity, with peak levels at 5-10 min. PLD-catalyzed transphosphatidylation (with n-butyl alcohol) diminished DG formation at 5 min but not at later stages of PDGF stimulation. Phorbol ester-induced down-regulation of protein kinase C (PKC) completely blocked PLD activation but not the formation of DG and phosphocholine at 10 min of PDGF stimulation. Collectively, these data indicate that PDGF activates both PLD and PC-PLC. In contrast, epidermal growth factor did not activate PC-PLC in these cells, and it activated PLD only weakly. DG formation by itself, through Bacillus cereus PC-PLC treatment of cells, was sufficient to mimic PDGF in activation of MAPK independent of phorbol ester-sensitive PKC. Since PKC down-regulation blocked PDGF-induced PLD but not MAPK activation, we conclude that PLD is not involved in MAPK signaling. In contrast, MAPK activation by exogenous (bacterial) PLD was not affected by PKC down-regulation, indicating that signals evoked by exogenous PLD differ from endogenous PLD. D609 (2-10 microg/ml), an inhibitor of PC-PLC, blocked PDGF- but not epidermal growth factor-induced MAPK activation. However, D609 should be used with caution since it also affects PLD activity. The results suggest that PC-PLC rather than PLD plays a critical role in the PDGF-activated MAPK pathway.
Highlights
Activation of receptor tyrosine kinases leads to receptor autophosphorylation and subsequent recruitment and activation of many signaling proteins that interact in cascades of enzymatic reactions
We demonstrate that platelet-derived growth factor (PDGF) transiently activates both phospholipase D (PLD) and PC-PLC and present evidence that the latter phospholipase activity is necessary for PDGF-induced mitogen-activated protein kinase (MAPK) activation
At later time points, DG formation is no longer decreased by n-butyl alcohol, supporting the notion that at least part of the DG formation is due to a PC-PLC
Summary
Activation of receptor tyrosine kinases leads to receptor autophosphorylation and subsequent recruitment and activation of many signaling proteins that interact in cascades of enzymatic reactions (for review, see Refs. 1–3). This completely blocked PDGF-induced PLD activation, as demonstrated by the lack of PA and choline formation and the absence of PBut formation in the presence of n-butyl alcohol These results indicate that PDGF activates both PLD and PC-PLC in Rat-1 cells.
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