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.
|Number of pages||8|
|Publication status||Published - 1994|
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