Different isozymes of protein kinase C mediate feedback inhibition of phospholipase C and stimulatory signals for exocytosis in rat RBL-2H3 cells.
Previous studies indicated that rat basophilic RBL-2H3 cells contained the Ca(2+)-dependent alpha and beta and the Ca(2+)-independent delta, epsilon, and zeta isoforms of protein kinase C (PKC); of these, PKC beta and delta were the most potent transducers of signals for exocytosis in antigen-stimulated permeabilized cells. Exocytosis, nevertheless, was still dependent on an elevated free Ca2+. (Ozawa, K., Szallasi, Z., Kazanietz, M. G., Blumberg, P. M., Mischak, H., Mushinski, J. F., and Beaven, M. A. (1993) J. Biol. Chem. 268, 1749-1756). We now demonstrate that PKC alpha and epsilon, exclusively, inhibit antigen-induced hydrolysis of inositol phospholipids in the same permeabilized RBL-2H3 cells. Unlike secretion, the inhibitory actions occurred at a basal concentration (0.1 microM) of free Ca2+. The inhibitory actions of the two isozymes were potentiated by 20 nM phorbol 12-myristate 13-acetate. As indicated by the effects of the phorbol ester, the probable mechanism was reduced tyrosine phosphorylation of phospholipase C gamma 1. The negative regulation of phospholipase C was apparent in intact cells, because the PKC inhibitor Ro31-7549 or down-regulation of PKC with phorbol ester enhanced antigen-induced hydrolysis of inositol phospholipids. The concentrations of the various isozymes of PKC in RBL-2H3 cells, as estimated by immunoblotting studies, were sufficient for promotion of exocytosis (i.e. beta and delta) and inhibition of phospholipid hydrolysis (i.e. alpha and epsilon).
Ozawa
K
K
Laboratory of Chemical Pharmacology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Yamada
K
K
Kazanietz
M G
MG
Blumberg
P M
PM
Beaven
M A
MA
D000818:Animals; D000941:Antigens / Q000008:administration & dosage; D001491:Basophils / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism; D005089:Exocytosis; D005246:Feedback; D007073:Immunoglobulin E / Q000502:physiology; D007211:Indoles / Q000494:pharmacology; D007295:Inositol Phosphates / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D007942:Leukemia, Experimental / Q000201:enzymology; D008301:Maleimides / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors* / Q000378:metabolism
D000941:Antigens; D007211:Indoles; D007295:Inositol Phosphates; D007527:Isoenzymes; D008301:Maleimides; D010716:Phosphatidylinositols; D007073:Immunoglobulin E; D011493:Protein Kinase C; D010738:Type C Phospholipases; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; C064755:Ro 31-7549