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 Ca(2+)-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 Ca(2+)-dependent
protein tyrosine phosphorylation is closely implicated in CCh-induced
PLD activation in PC12 cells.