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.
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 microM for 3-6 h) or inhibited by pharmacological agents such as calphostin C (1 microM) or a protein kinase C-specific pseudo-substrate peptide inhibitor (100-200 microM), cells lost the ability to secrete in response to 10 microM free Ca2+. 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-alpha. In addition, at the concentrations effective for exocytosis, calcium caused translocation of protein kinase C-alpha 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 delta, epsilon and zeta 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.
Buccione
R
R
Laboratory of Molecular Neurobiology, Istituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, S. Maria Imbara (Chieti), Italy.
Di Tullio
G
G
Caretta
M
M
Marinetti
M R
MR
Bizzarri
C
C
Francavilla
S
S
Luini
A
A
De Matteis
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002463:Cell Membrane Permeability; D015536:Down-Regulation; D004789:Enzyme Activation; D005089:Exocytosis; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D008969:Molecular Sequence Data; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000378:metabolism
D007527:Isoenzymes; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D011493:Protein Kinase C; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D002118:Calcium