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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.
AuthorsY Ito, S Nakashima, H Kanoh, Y Nozawa
JournalJournal of neurochemistry (J Neurochem) Vol. 68 Issue 1 Pg. 419-25 (Jan 1997) ISSN: 0022-3042 [Print] England
PMID8978754 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • Adaptor Proteins, Signal Transducing
  • Adaptor Proteins, Vesicular Transport
  • Proteins
  • Shc Signaling Adaptor Proteins
  • Shc1 protein, rat
  • Src Homology 2 Domain-Containing, Transforming Protein 1
  • Tyrosine
  • Carbachol
  • Phospholipase D
  • Calcium
Topics
  • Adaptor Proteins, Signal Transducing
  • Adaptor Proteins, Vesicular Transport
  • Animals
  • Calcium (physiology)
  • Carbachol (pharmacology)
  • Enzyme Activation
  • PC12 Cells (metabolism)
  • Phospholipase D (metabolism)
  • Phosphorylation
  • Proteins (metabolism)
  • Rats
  • Shc Signaling Adaptor Proteins
  • Src Homology 2 Domain-Containing, Transforming Protein 1
  • Tyrosine (metabolism)

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