Evidence That the Bradykinin-induced Activation of Phospholipase D and of the Mitogen-activated Protein Kinase Cascade Involve Different Protein Kinase C Isoforms

Abstract The effect of alkylglycerol supplementation on protein kinase C (PKC)-mediated signaling events has been studied in fibroblasts from Zellweger patients (SF 3271 cells). Western blotting analysis established that Zellweger fibroblasts express PKC α, , and . Incubation with bradykinin induced a rapid transient translocation of PKC α and a more sustained translocation of PKC to the particulate fraction; translocation of PKC was unaffected. Bradykinin-induced translocation and activation of PKC α, but not translocation of PKC , was blocked in SF 3271 cells which had been incubated with 1-O-hexadecylglycerol (1-O-HDG; 20 μg/ml) for 24 h and then incubated in the absence of 1-O-HDG and serum for a further 24 h. Supplementation with 1-O-HDG increased the mass of ether-linked phospholipid. Bradykinin initiated a transient increase in cytosolic Ca concentration in both control and 1-O-HDG supplemented cells, indicating that the initial receptor linked events were not affected by 1-O-HDG supplementation. Bradykinin also caused a rapid activation of phospholipase D (PLD), measured by phosphatidylbutanol accumulation, and mitogen-activated protein kinase (MAPK) determined by myelin basic protein phosphorylation of Mono Q fractions. Both events were blocked by preincubation of the cells with 12-O-tetradecanoylphorbol-13-acetate for 24 h to deplete PKC protein. 1-O-HDG supplementation prevented the bradykinin-induced activation of PLD, but had no effect on the stimulation of MAPK activity. These results establish that modulation of the ether lipid composition of membranes can alter PKC isozyme translocation and indicate that a PKC isozyme other than PKC α, most likely PKC , is involved in MAPK activation.