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).
Laboratory of Chemical Pharmacology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
K
K
Ozawa
K
K
Yamada
M G
MG
Kazanietz
P M
PM
Blumberg
M A
MA
Beaven
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