Up-regulation of phospholipase D activity induced by overexpression of protein kinase C-alpha. Studies in intact Swiss/3T3 cells and in detergent-solubilized membranes in vitro.
The role of protein kinase C in the mechanism of phospholipase D activation by platelet-derived growth factor and 12-O-tetradecanoylphorbol-13-acetate was studied in Swiss/3T3 fibroblasts that overexpress protein kinase C-alpha. Production of [3H]phosphatidylpropanol (specific product of the phospholipase D-catalyzed transphosphatidylation reaction) was determined in cells which were prelabeled with [3H]oleic acid. Accumulation of [3H]phosphatidylpropanol in response to platelet-derived growth factor and 12-O-tetradecanoylphorbol-13-acetate was 2-3-fold greater in protein kinase C-alpha-overexpressing SF1.4 cells compared with the vector control cells, SC1. Basal [3H] phosphatidylpropanol production also was 2-fold higher in SF1.4 cells than in SC1 cells. Hence, -fold stimulation of basal phospholipase D activity by platelet-derived growth factor and 12-O-tetradecanoyl-phorbol-13-acetate was comparable in the two cell lines and was not significantly altered by the overexpression of protein kinase C-alpha. Similarly, overexpression of protein kinase C-alpha did not affect either the kinetics of phospholipase D activation nor its dependence on platelet-derived growth factor or 12-O-tetradecanoylphorbol-13-acetate concentration. In vitro assay of phospholipase D activity in membranes isolated from the cells, utilizing exogenous [3H]phosphatidylcholine as a substrate, revealed nearly 2-fold higher phospholipase D activity in SF1.4 cell membranes. Kinetic analysis of detergent-solubilized phospholipase D activity indicated that the apparent Vmax and Km of phospholipase D derived from SF1.4 and SF3.2 (protein kinase C-alpha-overexpressing) cells are significantly higher than those of phospholipase D from control cells. These results indicate that in Swiss/3T3 cells overexpression of protein kinase C-alpha elevates basal and agonist-stimulated phospholipase D activity in intact cells as well as phospholipase D activity in vitro. These data are consistent with the hypothesis that overexpression of protein kinase C-alpha up-regulates phospholipase D, leading to a constitutive higher level of enzyme activity. Thus, protein kinase C-alpha may play a role in regulating phospholipase D expression.
Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.
H
H
Eldar
P
P
Ben-Av
U S
US
Schmidt
E
E
Livneh
M
M
Liscovitch
D016475:3T3 Cells; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D020404:Glycerophospholipids; D007527:Isoenzymes / Q000096:biosynthesis* / Q000302:isolation & purification; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D019301:Oleic Acid; D009829:Oleic Acids / Q000378:metabolism; D010712:Phosphatidic Acids / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011493:Protein Kinase C / Q000096:biosynthesis* / Q000302:isolation & purification; D012995:Solubility; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014316:Tritium
D003902:Detergents; D020404:Glycerophospholipids; D007527:Isoenzymes; D009829:Oleic Acids; D010712:Phosphatidic Acids; D010982:Platelet-Derived Growth Factor; C051521:phosphatidylethanol; D014316:Tritium; D019301:Oleic Acid; D011493:Protein Kinase C; D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate