Competitive inhibition by diacylglycerol of specific phorbol ester binding.

Abstract

Emerging evidence suggests that the phorbol ester receptor in brain may be the same as the Ca2+-phospholipid-dependent protein kinase (protein kinase C). Since protein kinase C activity is stimulated by unsaturated diacylglycerol and the phorbol esters can substitute for diacylglycerol in this stimulation, we have examined the effect of diacylglycerols on phorbol ester binding. Assays were carried out with the mouse brain cytosolic phorbol ester aporeceptor, which requires phospholipids for activity. In the presence of phosphatidylserine at 0.96 mg/ml, diolein inhibited specific binding of[3H]phorbol 12,13-dibutyrate ([3H]PBt2) in a dose-dependent fashion to <10% of control levels. The inhibition curves fit the curve expected for a competitive inhibitor and yielded a Ki of 3.6 ± 0.8 aug/ml (n = 5) [0.38% (wt/wt) the concentration of phosphatidylserine]. Scatchard analysis confirmed the competitive nature of the inhibition. At constant phospholipid concentrations, the K1 determined for diolein was independent of the diolein concentrations over the range of 1.5-80 pug/ml, suggesting that the inhibition did not arise simply by perturbation of the phospholipid bilayers. The K; of diolein was approximately proportional to the absolute phospholipid concentration. With phosphatidylserine at 4.8 ,ug/ml, for example, the K; was 52 ng/ml (1.1% of phosphatidylserine). In addition to diolein, the short-chain saturated diacylglycerol derivatives dicaprylin and dicaproin also inhibited [3H]PBt2 binding, whereas the long-chain saturated derivatives dipalmitin and distearin were much less active. Our results suggest (i) that diacylglycerol may act as an endogenous ligand for the phorbol ester receptor and (it) that variation in lipid composition provides a mechanism for modulating phorbol ester receptor affinity.The phorbol esters are the most intensively studied class of highly potent mouse skin tumor promoters (1). In addition to their activity as tumor promoters, the phorbol esters have profound biological effects in many systems (2-5). They cause partial mimicry of the transformed phenotype in normal cells; they synergize with growth factors; and they affect cellular differentiation, inhibiting differentiation in some systems and inducing it in others. This laboratory (6) and others to demonstrate and characterize phorbol ester receptors in tissue preparations and intact cells. Structure-activity analysis strongly argues that these receptors mediate biological responses to the phorbol esters (9).The tissue distribution (10, 11), evolutionary conservation (10), and phospholipid requirements (12) for the phorbol ester receptor markedly resembled those of the Ca2+-phospholipid dependent. protein kinase (protein kinase C) described by Nishizuka and co-workers (13) and by Kuo and co-workers (14, 15). Nishizuka's group, together with M. Castagna, have examined the effect of the phorbol esters directly on this kinase (16); they observed that the phorbol esters caused activation of kinase activity, apparently by shifting th...

Suppressive Effect of Maslinic Acid on PMA-induced Protein Kinase C in Human B-Lymphoblastoid Cells

“…Studies have shown that PMA substitute its binding to PKC by increasing the affinity of PKC to Ca 2+ and phosphatidylserine, thereby causing the translocation of PKC from cytosol to plasma membrane and its activation (Nishizuka, 1984;Sharkey et al, 1984 DOI:http://dx.doi.org/10.7314/APJCP.2012.13.4.1177 the membrane to activate PKC may inhibit its activity. Pentacyclic triterpenes are structurally similar to sterols and interaction with the cell membrane is thought to be the molecular mechanism underlying their biological effects.…”

Regulation of Human Involucrin Promoter Activity by Novel Protein Kinase C Isoforms

“…TPA is a stable diacylglycerol analog that mimics the effects of diacylglycerol and strongly activates PKC (43)(44)(45) and is a potent inducer of keratinocyte differentiation (46). Treating cultured keratinocytes with TPA increases cell differentiation, and this change is correlated with an increase in hINV mRNA and protein (7,9,47,48).…”

Modulation of cell communication and carcinogenesis.

“…i) Dual regulation of gap junctional communication by cAMP-dependent and Cat+/phospholipid dependent protein kinases Tumor promoters of the phorbol ester family have been shown to bind to and directly activate Ca2+/phospholipid-dependent kinase (protein kinase C) which is activated by diacylglycerols in the presence of phospholipid and physiological concentrations of Ca2+ [7]. Recent studies have shown that homogeneous preparations of protein kinase C from rat brain bind to [3H]-phorbol-12,13-dibutyrate [30] and that diacylglycerols compete with this radioactive ligand for common binding sites [50]. Moreover, diacylglycerol has been shown to exert stimulation on DNA synthesis [49], induction of differentiation of HL-60 cells [36], and inhibition of intercellular communication [15,19], which are also induced by phorbol ester tumor promoters.…”