Implication of Ca2+‐Dependent Protein Tyrosine Phosphorylation in Carbachol‐Induced Phospholipase D Activation in Rat Pheochromocytoma PC12 Cells

Abstract: The mechanism for carbachol (CCh)‐induced phospholipase D (PLD) activation was investigated in [3H]palmitic acid‐labeled pheochromocytoma PC12 cells with respect to the involvement of protein tyrosine phosphorylation and Ca2+. PLD activity was assessed by measuring the formation of [3H]phosphatidylbutanol in the presence of 0.3% butanol. Pretreatment of cells with the tyrosine kinase inhibitors herbimycin A, genistein, and tyrphostin inhibited PLD activation by CCh. Western blot analysis revealed several apparent tyrosine‐phosphorylated protein bands (111, 91, 84, 74, 65–70, 44, and 42 kDa) in PC12 cells treated with CCh. Phosphorylation of the 111‐, 91‐, 84‐, and 65–70‐kDa proteins peaked within 1 min, and their time‐dependent changes seemingly correlated with that of PLD activation. Others (74, 44MAPK, and 42MAPK kDa) were phosphorylated rather slowly, and maximal tyrosine phosphorylation was observed at 2 min. Herbimycin A inhibited PLD activity and tyrosine phosphorylation of four proteins (111, 91, 84, and 65–70 kDa) in a preincubation time‐ and concentration‐dependent fashion. In Ca2+‐free buffer, CCh‐induced [3H]phosphatidylbutanol formation and protein tyrosine phosphorylation were abolished. A Ca2+ ionophore, A23187, caused PLD activation and tyrosine phosphorylation of four proteins of 111, 91, 84, and 65–70 kDa only in the presence of extracellular Ca2+. Extracellular Ca2+ dependency for CCh‐induced PLD activation was well correlated with that for tyrosine phosphorylation of the four proteins listed above, especially the 111‐kDa protein. These results suggest that Ca2+‐dependent protein tyrosine phosphorylation is closely implicated in CCh‐induced PLD activation in PC12 cells.

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