EVIDENCE THAT THE BRADYKININ-INDUCED ACTIVATION OF PHOSPHOLIPASE-D AND OF THE MITOGEN-ACTIVATED PROTEIN-KINASE CASCADE INVOLVE DIFFERENT PROTEIN-KINASE-C ISOFORMS

The effect of alkylglycerol supplementation on protein kinase C (PKC) mediated signaling events has been studied in fibroblasts from Zellweg er patients (SF 3271 cells), Western blotting analysis established tha t Zellweger fibroblasts express PKC alpha, epsilon, and zeta, Incubati on with bradykinin induced a rapid transient translocation of PKC alph a and a more sustained translocation of PKC epsilon to the particulate fraction; translocation of PKC zeta was unaffected, Bradykinin-induce d translocation and activation of PKC alpha, but not translocation of PKC epsilon, was blocked in SF 3271 cells which had been incubated wit h 1-O-hexadecylglycerol (1-O-HDG; 20 mu g/ml) for 24 h and then incuba ted in the absence of 1-O-HDG and serum for a further 24 h, Supplement ation with 1-O-HDG increased the mass of ether-linked phospholipid. Br adykinin initiated a transient increase in cytosolic Ca2+ concentratio n in both control and 1-O-HDG supplemented cells, indicating that the initial receptor linked events were not affected by 1-O-HDG supplement ation. Bradykinin also caused a rapid activation of phospholipase D (P LD), measured by phosphatidylbutanol accumulation, and mitogen-activat ed protein kinase (MAPK) determined by myelin basic protein phosphoryl ation of Mono Q fractions, Both events were blocked by preincubation o f the cells with 12-O-tetradecanoylphorbol-13-acetate for 24 h to depl ete PKC protein, 1-O-HDG supplementation prevented the bradykinin indu ced activation of PLD, but had no effect on the stimulation of MAPK ac tivity, These results establish that modulation of the ether lipid com position of membranes can alter PKC isozyme translocation and indicate that a PKC isozyme other than PKC alpha, most likely PKC epsilon, is involved in MAPK activation.