Involvement of tyrosine phosphorylation and protein kinase C in the activation of phospholipase D by H₂O₂ in swiss 3T3 fibroblasts

We have investigated the mechanisms involved in H₂O₂-mediated phospholipase D (PLD) activation in Swiss 3T3 fibroblasts. In the presence of vanadate, H₂O₂ induced tyrosine phosphorylation of PLD as well as the platelet-derived growth (PDGF) factor receptor, protein kinase Cα (PKCα), and a 62-kDa protein in rat brain PLD1 (rPLD1) immune complexes. PDGF also induced tyrosine phosphorylation of PLD, but this was abolished by catalase, indicating that it was mediated by H₂O₂ generation. Interestingly, PLD was found to be constitutively associated with the PDGF receptor and PKCα. Stimulation by H₂O₂ showed a concentration- and time-dependent tyrosine phosphorylation of the proteins in rPLD1 immunoprecipitates and activation of PLD in the cells. Pretreatment of the cells with the protein-tyrosine kinase inhibitors genistein and herbimycin A resulted in a concentration-dependent inhibition of H₂O₂-induced tyrosine phosphorylation and PLD activation. Activation of PLD by H₂O₂ was also inhibited dose-dependently by the PKC inhibitors Ro 31-8220 and calphostin C. Down-regulation of PKC by prolonged treatment with 4β-phorbol 12-myristate 13-acetate also abolished H₂O₂- stimulated PLD activity. H₂O₂ or vanadate alone did not induce tyrosine phosphorylation of proteins in the rPLD1 immune complex or PLD activation. Reduction of intracellular H₂O₂ levels by pretreatment of the cells with catalase dramatically abrogated tyrosine phosphorylation of proteins in the rPLD1 immune complex and PLD activation, suggesting the potential role of intracellular H₂O₂ in H₂O₂-mediated PLD signaling. Taken together, these results suggest that both protein-tyrosine kinase(s) and protein kinase C participate in H₂O₂-induced PLD activation in Swiss 3T3 cells.