title: Implication of Ca(2+)-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 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.
authors.affiliation: Department of Biochemistry, Gifu University School of Medicine, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Ito
authors.forename: S
authors.initials: S
authors.lastname: Nakashima
authors.forename: H
authors.initials: H
authors.lastname: Kanoh
authors.forename: Y
authors.initials: Y
authors.lastname: Nozawa
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D002118:Calcium / Q000502:physiology*; D002217:Carbachol / Q000494:pharmacology*; D004789:Enzyme Activation; D016716:PC12 Cells / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism*; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism; D051381:Rats; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D011506:Proteins; D055765:Shc Signaling Adaptor Proteins; C528035:Shc1 protein, rat; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014443:Tyrosine; D002217:Carbachol; D010739:Phospholipase D; D002118:Calcium