Evidence for a protein kinase C-directed mechanism in the phorbol diester-induced phospholipase D pathway of diacylglycerol generation from phosphatidylcholine

• Published in: FEBS letters 1989-03, Vol.245 (1), p.85-90
• Main Authors: Cabot, Myles C., Welsh, Clement J., Zhang, Zu-chuan, Cao, Hui-ting
• Format: Article
• Language: English
• Subjects: Alkaloids - pharmacology; Animals; BSA, bovine serum albumin; Bu2cAMP, 2′-O-dibutyryladenosine 3′,5′-cyclic monophosphate; Bucladesine - pharmacology; Cell Line; Choline - metabolism; Colforsin - pharmacology; DG, diacylglycerol; Diacylglycerol; DiC8, dioctanoyl glycerol; Diglycerides - metabolism; Embryo, Mammalian; Fibroblasts - drug effects; Fibroblasts - enzymology; Glycerides - metabolism; Glycerophospholipids; Hydrolysis; IP3, myo-inositol 1,4,5-trisphosphate; Me2SO, dimethyl sulfoxide; Myristic Acid; Myristic Acids - metabolism; PBS, phosphate-buffered saline; PC, phosphatidylcholine; PDBu, phorbol dibutyrate; PE, phosphatidylethanol. In the naming of PC, PA, DG and PE, we do not distinguish between the type of aliphatic linkage to glycerol (ester, ether); Phorbol 12,13-Dibutyrate - pharmacology; Phorbol diester; Phosphatidic Acids - metabolism; Phosphatidylcholine; Phosphatidylcholines - metabolism; Phospholipase D; Phospholipase D - metabolism; Phospholipases - metabolism; PKC, Ca2+-activated, phospholipid-dependent protein kinase C; Protein kinase C; Protein Kinase C - metabolism; Rats; Staurosporine; Tetradecanoylphorbol Acetate - pharmacology; TLC, thin-layer chromatography; TPA, 12-O-tetradecanoyl phorbol-13-acetate
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(89)80197-8

In this study we provide evidence for the involvement of protein kinase C (PKC) in phorbol diester-induced phosphatidylcholine (PC) hydrolysis by the phospholipase D pathway. Rat embryo fibroblasts (REF52) were prelabeled with either tritiated choline or myristic acid; these compounds are preferentially incorporated into cellular PC. Phorbol diester-induced PC degradation was determined by measuring the release of [ 3H]choline, and the formation of [ 3H]myristoyl-containing phosphatidate (PA), diacylglycerol (DG), and phosphatidylethanol (PE). Staurosporine, a PKC inhibitor, blocked from 73 to 90% of the phorbol diester-induced PC hyrolysis. The inhibition of phorbol diester-induced choline release by staurosporine was dose dependent with an approximate ED 50 of 150 nM. Pretreatment of cells with phorbol diester inhibited subsequent phorbol diester-induced PC degradation by 78–92%. A close correlation between the ED 50 for phorbol diester-stimulated choline release and the K d for phorbol diester binding was demonstrated. Neither forskolin nor dibutyryl cAMP elicited cellular PC degradation. In vitro experiments using phospholipase D from Streptomyces chromofuscus showed that staurosporine did not inhibit and TPA did not stimulate enzyme activity.