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

H Eldar, P Ben-Av, U S Schmidt, E Livneh, M Liscovitch

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Abstract

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.

Original languageEnglish
Pages (from-to)12560-4
Number of pages5
JournalThe Journal of biological chemistry
Volume268
Issue number17
StatePublished - 15 Jun 1993

Keywords

  • 3T3 Cells
  • Animals
  • Cell Line
  • Cell Membrane/enzymology
  • Detergents
  • Electrophoresis, Polyacrylamide Gel
  • Gene Expression
  • Glycerophospholipids
  • Isoenzymes/biosynthesis
  • Kinetics
  • Mice
  • Molecular Weight
  • Oleic Acid
  • Oleic Acids/metabolism
  • Phosphatidic Acids/metabolism
  • Phospholipase D/metabolism
  • Platelet-Derived Growth Factor/pharmacology
  • Protein Kinase C/biosynthesis
  • Solubility
  • Tetradecanoylphorbol Acetate/pharmacology
  • Tritium

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