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Evidence for a protein kinase C‐directed mechanism in the phorbol diester‐induced phospholipase D pathway of diacylglycerol generation from phosphatidylcholine
Author(s) -
Cabot Myles C.,
Welsh Clement J.,
Zhang Zu-chuan,
Cao Hui-ting
Publication year - 1989
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(89)80197-8
Subject(s) - staurosporine , protein kinase c , diacylglycerol kinase , phorbol , phospholipase c , diglyceride , myristic acid , phospholipase d , phosphatidate , biochemistry , phosphatidylcholine , forskolin , phospholipase , chemistry , protein kinase a , phospholipid , enzyme , in vitro , fatty acid , palmitic acid , membrane
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 [ 3 H]choline, and the formation of [ 3 H]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.