Antigen-induced secretion of histamine and the phosphorylation of myosin by protein kinase C in rat basophilic leukemia cells.
IgE-mediated stimulation of rat basophilic leukemia (RBL-2H3) cells results in the secretion of histamine. Myosin immunoprecipitated from these cells shows an increase in the amount of radioactive phosphate incorporated into its heavy (200 kDa) and light (20 kDa) chains. In unstimulated cells two-dimensional mapping of tryptic peptides of the myosin light chain reveals one phosphopeptide containing the serine residue phosphorylated by myosin light chain kinase. Following stimulation a second phosphopeptide appears containing a serine residue phosphorylated by protein kinase C. Tryptic phosphopeptide maps derived from myosin heavy chains show that unstimulated cells contain three major phosphopeptides. Following stimulation a new tryptic phosphopeptide appears containing a serine site phosphorylated by protein kinase C. The stoichiometry of phosphorylation of the myosin light and heavy chains was determined before and after antigenic stimulation. Before stimulation, myosin light chains contained 0.4 mol of phosphate/mol of light chain all confined to a serine not phosphorylated by protein kinase C. Cells that secreted 44% of their total histamine in 10 min exhibited an increase in phosphate content at sites phosphorylated by protein kinase C from 0 mol of phosphate/mol of myosin subunit to 0.7 mol of phosphate/mol of light chain and to 1 mol of phosphate/mol of heavy chain. When RBL-2H3 cells were made permeable with streptolysin O they still showed a qualitatively similar pattern of secretion and phosphorylation. Our results show that the time course of histamine secretion from stimulated RBL-2H3 cells parallels that of myosin heavy and light chain phosphorylation by protein kinase C.
Ludowyke
R I
RI
Laboratory of Chemical Pharmacology, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892.
Peleg
I
I
Beaven
M A
MA
Adelstein
R S
RS
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000941:Antigens; D002460:Cell Line; D004140:Dinitrophenols / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D006241:Haptens; D006636:Histamine Release; D007073:Immunoglobulin E; D007700:Kinetics; D015471:Leukemia, Basophilic, Acute / Q000201:enzymology / Q000503:physiopathology*; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D014357:Trypsin; D014407:Tumor Cells, Cultured / Q000201:enzymology / Q000502:physiology
D000596:Amino Acids; D000941:Antigens; D004140:Dinitrophenols; D006241:Haptens; D010446:Peptide Fragments; C034638:dinitrophenyl-bovine serum albumin; D012710:Serum Albumin, Bovine; D007073:Immunoglobulin E; D011493:Protein Kinase C; D014357:Trypsin; D009218:Myosins