Analysis of protein kinase C requirement for exocytosis in permeabilized rat basophilic leukaemia RBL-2H3 cells: A GTP-binding protein(s) as a potential target for protein kinase C

R. Buccione, G. Di Tullio, M. Caretta, M. R. Marinetti, C. Bizarri, S. Francavilla, A. Luini, M. A. De Matteis

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Abstract

The role of protein kinase C in calcium-dependent exocytosis was investigated in permeabilized rat basophilic leukaemia cells. When protein kinase C was down-regulated by phorbol myristate acetate (1 μM for 3-6 h) or inhibited by pharmacological agents such as calphostin C (1 μM) or a protein kinase C-specific pseudo-substrate peptide inhibitor (100-200 μM), cells lost the ability to secrete in response to 10 μM free Ca 2+. In contrast, a short treatment (15 min) with phorbol myristate acetate, which maximally activates protein kinase C, potentiated the effects of calcium. Biochemical analysis of protein kinase C-deprived cells indicated that loss of the Ca 2+-induced secretory response correlated with disappearance of protein kinase C-α. In addition, at the concentrations effective for exocytosis, calcium caused translocation of protein kinase C-α to the membrane fraction and stimulated phospholipase C, suggesting that, in permeabilized cells, protein kinase C can be activated by calcium through generation of the phospholipase C metabolite diacylglycerol. The δ, ε and ζ Ca 2+-independent protein kinase C isoenzymes were insensitive to phorbol myristate acetate-induced down-regulation and did not, as expected, translocate to the particulate fraction in response to calcium. Interestingly, secretory competence was restored in cells depleted of protein kinase C or in which protein kinase C itself was inhibited by non-hydrolysable GTP analogues, but not by GTP, suggesting that protein kinase C might regulate the ability of a G protein(s) directly controlling the exocytotic machinery to be activated by endogenous GTP.

Original languageEnglish
Pages (from-to)149-156
Number of pages8
JournalBiochemical Journal
Volume298
Issue number1
Publication statusPublished - 1994

ASJC Scopus subject areas

  • Biochemistry

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    Buccione, R., Di Tullio, G., Caretta, M., Marinetti, M. R., Bizarri, C., Francavilla, S., Luini, A., & De Matteis, M. A. (1994). Analysis of protein kinase C requirement for exocytosis in permeabilized rat basophilic leukaemia RBL-2H3 cells: A GTP-binding protein(s) as a potential target for protein kinase C. Biochemical Journal, 298(1), 149-156.