Evidence for a protein kinase C-directed mechanism in the phorbol diester-induced phospholipase D pathway of diacylglycerol generation from phosphatidylcholine.
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 hydrolysis. The inhibition of phorbol diester-induced choline release by staurosporine was dose dependent with an approximate ED50 of 150 nM. Pretreatment of cells with phorbol diester inhibited subsequent phorbol diester-induced PC degradation by 78-92%. A close correlation between the ED50 for phorbol diester-stimulated choline release and the Kd 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.
Cabot
M C
MC
W. Alton Jones Cell Science Center, Inc., Lake Placid, NY 12946.
Welsh
C J
CJ
Zhang
Z C
ZC
Cao
H T
HT
D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002460:Cell Line; D002794:Choline / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D004075:Diglycerides / Q000378:metabolism*; D004622:Embryo, Mammalian; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology*; D005989:Glycerides / Q000378:metabolism*; D020404:Glycerophospholipids; D006868:Hydrolysis; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D010712:Phosphatidic Acids / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism*; D010740:Phospholipases / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000470:Alkaloids; D004075:Diglycerides; D005989:Glycerides; D020404:Glycerophospholipids; D009227:Myristic Acids; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; C051521:phosphatidylethanol; D019814:Myristic Acid; D005576:Colforsin; D015240:Phorbol 12,13-Dibutyrate; D003994:Bucladesine; D011493:Protein Kinase C; D010740:Phospholipases; D010739:Phospholipase D; D019311:Staurosporine; D002794:Choline; D013755:Tetradecanoylphorbol Acetate