Ca(2+)-dependent and Ca(2+)-independent isozymes of protein kinase C mediate exocytosis in antigen-stimulated rat basophilic RBL-2H3 cells. Reconstitution of secretory responses with Ca2+ and purified isozymes in washed permeabilized cells.
Rat basophilic RBL-2H3 cells, which exhibit Ca(2+)-dependent secretion of granules when stimulated with antigen, contained the Ca(2+)-dependent alpha and beta and the Ca(2+)-independent delta, epsilon, and zeta isoforms of protein kinase C. These isoforms associated, to variable extents (i.e. delta the most and zeta the least), with the membrane fraction upon antigen stimulation but without external Ca2+; only the Ca(2+)-independent isoforms did so. Both types of isozymes were probably necessary for optimal responses to antigen as indicated by the following observations. All Ca(2+)-dependent isozymes were degraded in cells treated with 20 nM phorbol 12-myristate 13-acetate for 6 h, whereas the Ca(2+)-independent isozymes were not degraded and were retained when the cells were subsequently permeabilized and washed. Cells so treated still exhibited antigen-induced secretion (25-33% of normal) which was suppressed by selective inhibitors of protein kinase C (Ro31-7549 and calphostin C) thereby indicating a possible contribution of the Ca(2+)-independent isozymes in secretion. Normally, washed permeabilized cells lost all isozymes of protein kinase C and failed to secrete in response to antigen. A full secretory response to antigen could be reconstituted by the subsequent addition of nanomolar concentrations of either beta or delta isozymes of protein kinase C (other isozymes were much less effective) but only in the presence of 1 microM free Ca2+ to indicate distinct roles for Ca2+ and protein kinase C in exocytosis.
Ozawa
K
K
Laboratory of Chemical Pharmacology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Szallasi
Z
Z
Kazanietz
M G
MG
Blumberg
P M
PM
Mischak
H
H
Mushinski
J F
JF
Beaven
M A
MA
D000818:Animals; D000941:Antigens / Q000276:immunology; D016367:Baculoviridae / Q000201:enzymology; D001491:Basophils / Q000201:enzymology* / Q000276:immunology / Q000648:ultrastructure; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000494:pharmacology*; D002460:Cell Line; D002463:Cell Membrane Permeability; D003594:Cytoplasmic Granules / Q000502:physiology; D005089:Exocytosis / Q000502:physiology*; D015151:Immunoblotting; D007527:Isoenzymes / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000941:Antigens; D007527:Isoenzymes; D011994:Recombinant Proteins; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium