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

The mechanism for carbachol (CCh)-induced phospholipase D (PLD) activation was investigated in [ 3 H] palmitic acid-labeled pheochromocytoma PC12 cells with respect to the involvement of protein tyrosine phosphorylation and Ca 2+ . 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, 44 MAPK , and 42 MAPK 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 Ca 2+ -free buffer, CCh-induced [ 3 H]phosphatidylbutanol formation and protein tyrosine phosphorylation were abolished. A Ca 2+ 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 Ca 2+ . Extracellular Ca 2+ 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 Ca 2+ -dependent protein tyrosine phosphorylation is closely implicated in CCh-induced PLD activation in PC12 cells.