Overexpression of protein kinase C beta 1 enhances phospholipase D activity and diacylglycerol formation in phorbol ester-stimulated rat fibroblasts

• Published in: Proceedings of the National Academy of Sciences - PNAS 1991-01, Vol.88 (2), p.598-602
• Main Authors: Pai, J K, Pachter, J A, Weinstein, I B, Bishop, W R
• Format: Article
• Language: English
• Subjects: 550201 - Biochemistry- Tracer Techniques; Alkaloids - pharmacology; ANIMAL CELLS; Animals; BASIC BIOLOGICAL SCIENCES; BETA DECAY RADIOISOTOPES; BETA-MINUS DECAY RADIOISOTOPES; BIOLOGICAL EFFECTS; Carbazoles - pharmacology; CARBOXYLESTERASES; CARCINOGENS; Cell Line; CHEMICAL REACTIONS; CONNECTIVE TISSUE CELLS; DAYS LIVING RADIOISOTOPES; DECOMPOSITION; diacylglycerol; Diglycerides - metabolism; DOUBLE LABELLING; ENZYMATIC HYDROLYSIS; ENZYME ACTIVITY; ENZYMES; ESTERASES; ESTERS; FIBROBLASTS; Fibroblasts - drug effects; Fibroblasts - metabolism; GENE REGULATION; HYDROGEN COMPOUNDS; HYDROLASES; HYDROLYSIS; Indole Alkaloids; Inositol - metabolism; ISOENZYMES; ISOTOPES; Kinetics; LABELLING; LIGHT NUCLEI; LIPASES; LIPIDS; LYSIS; Lysophosphatidylcholines - metabolism; Myristic Acid; Myristic Acids - metabolism; NUCLEI; ODD-ODD NUCLEI; ORGANIC COMPOUNDS; ORGANIC PHOSPHORUS COMPOUNDS; phorbol 12-myristate beta -acetate diester; PHORBOL ESTERS; phospholipase D; Phospholipase D - metabolism; PHOSPHOLIPIDS; PHOSPHORUS 32; PHOSPHORUS ISOTOPES; PHOSPHORUS-GROUP TRANSFERASES; PHOSPHOTRANSFERASES; Protein Kinase C - antagonists & inhibitors; Protein Kinase C - genetics; Protein Kinase C - metabolism; RADIOISOTOPES; Rats; SOLVOLYSIS; SOMATIC CELLS; Staurosporine; Tetradecanoylphorbol Acetate - pharmacology; TRANSFERASES; TRITIUM COMPOUNDS
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.88.2.598

We are using a Rat-6 fibroblast cell line that stably overexpresses the beta 1 isozyme of protein kinase C (PKC) to study regulation of phospholipid hydrolysis by PKC. Stimulation of control (R6-C1) or overexpressing (R6-PKC3) cells with phorbol ester results in an increase in diacylglycerol (DAG) mass with no increase in inositol phosphates, indicating that DAG is not formed by inositol phospholipid breakdown. A more dramatic DAG increase occurs in R6-PKC3 cells (4.0-fold over basal) compared to R6-C1 cells (1.5-fold over basal). To further define the source of DAG, phosphatidylcholine (PC) pools were labeled with [3H]myristic acid or with [3H]- or [32P]alkyllyso-PC and formation of labeled phosphatidylethanol, an unambiguous marker of phospholipase D activation, was monitored. Phorbol ester-stimulated phosphatidylethanol formation is 5-fold greater in the R6-PKC3 cell line. Formation of radiolabeled phosphatidic acid (PA) is also enhanced by PKC overexpression. In cells double-labeled with [3H]- and [32P]-alkyl-lysoPC, the 3H/32P ratio of PA and PC are identical 15 min after stimulation, suggesting that a phospholipase D mechanism predominates. In support of this, the PA phosphohydrolase inhibitor propranolol decreased phorbol 12-myristate 13-acetate-stimulated DAG formation by 72%. Increases in DAG and phosphatidylethanol were inhibited by the PKC inhibitors K252a and staurosporine. These results indicate that phospholipase D is regulated by the action of PKC. Enhanced phospholipase D activity may contribute to the growth abnormalities seen in PKC-overexpressing cells.