Overexpression of protein kinase C beta 1 enhances phospholipase D activity and diacylglycerol formation in phorbol ester-stimulated rat fibroblasts.
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.
Pai
J K
JK
Department of Microbiology, Schering-Plough Research, Bloomfield, NJ 07003.
Pachter
J A
JA
Weinstein
I B
IB
Bishop
W R
WR
D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D002227:Carbazoles / Q000494:pharmacology; D002460:Cell Line; D004075:Diglycerides / Q000378:metabolism*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D026121:Indole Alkaloids; D007294:Inositol / Q000378:metabolism; D007700:Kinetics; D008244:Lysophosphatidylcholines / Q000378:metabolism; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D000470:Alkaloids; D002227:Carbazoles; D004075:Diglycerides; D026121:Indole Alkaloids; D008244:Lysophosphatidylcholines; D009227:Myristic Acids; D019814:Myristic Acid; D007294:Inositol; C049985:staurosporine aglycone; D011493:Protein Kinase C; D010739:Phospholipase D; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate