Activation by phorbol esters of protein kinase C in MCF-7 human breast cancer cells

Exposure of MCF-7 human breast cancer cells to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) leads to the inhibition of cell proliferation. We investigate here the short-term effects of TPA on subcellular distribution of protein kinase C, and on protein phosphorylation in cultured MCF-7 cells. We report a rapid and dramatic decrease in cytosolic protein kinase C activity after TPA treatment. Only 30% of the enzymatic activity lost in the cytosol was recovered in the particulate fraction. These data suggest that subcellular translocation of protein kinase C is accompanied by a rapid down-regulation of the enzyme (70%). Furthermore, TPA and other protein kinase C activators rapidly induce the phosphorylation of a 28 kDa protein in intact MCF-7 cells. Phorbol esters devoid of tumor-promoting activity are ineffective both for inducing these early biochemical events and for inhibiting cell proliferation.     

TPA induces subcellular translocation and subsequent down-regulation of both phorbol ester binding and protein kinase C activities in MCF-7 cells

Phorbol ester TPA has been previously shown to induce a rapid translocation, followed by a progressive decline of protein kinase C activity in MCF-7 cells (J.M. Darbon et al, 1986, Biochem. Biophys. Res. Comm. 137: 1159-1166). We show now a parallel TPA-induced movement of phorbol ester binding sites from the cytosolic to the particulate fraction with no change in the binding affinities for the (3H) PDBu probe (KD congruent to 2 nM). The subcellular redistribution process is followed by a rapid decrease of the phorbol ester binding capacity at the membrane level. The concomitant decline in both phorbol ester binding and protein kinase C activities that we observed during the course of TPA treatment strongly argues for a real down-regulation of the enzyme in phorbol ester-treated MCF-7 cells. The molecular mechanisms of these events and their relations to the inhibition of cell growth remain to be clarified.     

1,2-Dioctanoyl-glycerol induces a discrete but transient translocation of protein kinase C as well as the inhibition of MCF-7 cell proliferation

Exposure of MCF-7 human breast cancer cells to phorbol esters such as 12-0-tetradecanoylphorbol-13-acetate (TPA) results in a dose-dependent inhibition of cell proliferation. One of the earliest biochemical events induced by TPA is the translocation of protein kinase C from the cytosolic to the particulate compartment. We have investigated the effects of permeant diacylglycerol 1,2-dioctanoyl-glycerol (diC8) on both protein kinase C activity and MCF-7 cell proliferation. DiC8 induces a discrete but significant translocation of protein kinase C within the first minutes of MCF-7 cell treatment (26 +/- 6%, mean +/- SD of 5 different experiments, upon 5 min incubation in the presence of 43 micrograms/ml diC8). However, this effect is only transient as the enzymatic activity returns to the control value after 60 min. DiC8 mimics the effect of TPA on MCF-7 cell proliferation. The dose-response curves for both protein kinase C translocation and cell growth inhibition show that diC8 exerts its effects on both parameters in the same range of concentrations, despite some discrepancies at the lowest doses. We also report that long-term treatment of the cells with diC8 does not lead to the protein kinase C disappearance observed during prolonged exposure to TPA. All together, our results reinforce the hypothesis of a negative modulatory role of protein kinase C in MCF-7 cell proliferation and suggest that the enzyme translocation but not its down-regulation could be a pre-requisite in the biological cell response.     

Phorbol ester prevents the thyroid-stimulating-hormone-induced but not the forskolin-induced decrease of cAMP-dependent protein kinase activity in thyroid cell cultures

The potent tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate (TPA) affects several thyroid cell functions and interacts with thyroid-stimulating hormone (TSH) either by inhibiting or potentiating its action on different cellular parameters. Since phorbol ester acts mainly through the activation of protein kinase C, which is its receptor, we studied this activation and its interaction with TSH and forskolin in suspension cultures of porcine thyroid cells. In thyroid cell cultures, TPA has a dual effect on protein kinase C activity: immediately (2-5 min) after exposure of cells to TPA, it began to be translocated from the cytosol to the particulate fraction. The transfer of the cytosolic enzyme was total and could occur with or without a loss of activity. The translocated enzyme still needed Ca2+ and phospholipids for its activation. The basal activity increased transiently (2-4 h) in both the cytosol and particulate fractions during translocation. The peak activity in the particulate fraction was reached 10-30 min after exposure of cells to TPA, and was followed by down-regulation of protein kinase C and almost complete disappearance of its activity. The residual activity was about 13% of control after a 2-day exposure to TPA. It was unequally distributed between cytosol (4%) and particulate fraction (9%). Prolonged exposure of cells to TPA did not affect either the activity or the subcellular distribution of the cAMP-dependent protein kinase activity. TPA interacted with TSH and prevented the decrease of this activity induced by prolonged exposure of cells to the hormone not only when it was introduced simultaneously with TSH, but also when it was added 24 h after TSH. However, the forskolin-induced decrease in cAMP-dependent protein kinase activity was not prevented by the presence of TPA. TPA also affected the increases in cAMP accumulation mediated by TSH and forskolin. The TSH-induced increase was significantly stimulated by TPA after short contacts (5-15 min), while longer preincubations of cells with TPA provoked a very strong inhibition of the TSH action. However, the forskolin-induced stimulation of the cAMP accumulation was maintained and even further increased in the presence of TPA. Consequently, the actions of TSH and TPA are apparently interdependent, while those of forskolin and TPA seem to be parallel and independent. Neither TSH nor forskolin prevented the TPA-induced down regulation of protein kinase C. The biologically inactive phorbol ester analogue 4 alpha-phorbol 12,13-didecanoate had no effect on protein kinase C activity, and did not interact with either TSH or forskolin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Gonadotropin release and redistribution of calcium-activated, phospholipid-dependent protein kinase in phorbol-stimulated rat pituitary cells

The effect of phorbol esters on calcium-activated, phospholipid-dependent kinase (protein kinase C) and luteinizing hormone (LH) secretion was examined in cultured rat anterior pituitary cells. The potent tumor promoter 12-O-tetra-decanoylphorbol-13-acetate (TPA) stimulated LH secretion and activated pituitary protein kinase C in the presence of calcium and phosphatidylserine. The enzyme activity present in cytosol and particulate fractions was eluted at about 0.05 M NaCl during DE52-cellulose chromatography. Preincubation of pituitary cells with TPA markedly decreased cytosolic protein kinase C activity and increased enzyme activity in the particulate fraction. The maximal TPA-induced change in enzyme activity, with a 76% decrease in cytosol and a 4.3-fold increase in the particulate fraction, occurred within 10 min. The dose-dependent changes in protein kinase C redistribution in TPA-treated cells were correlated with the stimulation of LH release by the phorbol ester. These results suggest that activation of protein kinase C by TPA is associated with intracellular redistribution of the enzyme and is related to the process of secretory granule release from gonadotrophs.     

Thyrotropin-releasing hormone and phorbol esters induce phosphatidylcholine synthesis in GH3 pituitary cells. Evidence for stimulation via protein kinase C

Phorbol esters have been shown to stimulate phosphatidylcholine synthesis via the CDP-choline pathway. The present study compares the effects of phorbol esters and thyrotropin-releasing hormone (TRH) on phosphatidylcholine metabolism in GH3 pituitary cells. In a previous study (Kolesnick, R.N., and Paley, A.E. (1987) J. Biol. Chem. 262, 9204-9210), the potent phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) induced time- and concentration-dependent incorporation of 32Pi and [3H]choline into phosphatidylcholine in short-term labeling experiments. In this study, TPA is shown to activate choline-phosphate cytidylyltransferase (EC 2.7.7.15), the regulatory enzyme of the CDP-choline pathway, by stimulating redistribution of the inactive cytosolic form of the enzyme to the membrane. Redistribution was quantitative. TPA reduced cytosolic activity from 3.5 +/- 0.4 to 1.5 +/- 0.3 nmol . min-1 x 10(7) cells-1 and enhanced particulate activity from 2.5 +/- 0.4 to 4.9 +/- 0.6 nmol . min-1 x 10(7) cells-1. TRH also stimulated time- and concentration-dependent 32Pi and [3H]choline incorporation into phosphatidylcholine. An increase was detectable after 5 min; and after 30 min, the levels were 164 +/- 9 and 150 +/- 11% of control, respectively; EC50 congruent to 2 X 10(-10) M TRH. These events correlated directly with TRH-induced 32Pi incorporation into phosphatidylcholine. TRH also stimulated redistribution of cytidylyl-transferase specific activity. TRH reduced cytosolic activity 45% and enhanced particulate activity 51%. Neither TRH nor TPA stimulated phosphatidylcholine degradation. In cells down-modulated for protein kinase C (Ca2+/phospholipid-dependent protein kinase), the effects of TPA and TRH on 32Pi incorporation into phosphatidylcholine were abolished. However, TRH-induced incorporation into phosphatidylinositol still occurred. These studies provide evidence that hormones may regulate phosphatidylcholine metabolism via the protein kinase C pathway.     

Antiproliferative effect of phorbol esters on MCF-7 human breast adenocarcinoma cells: relationship with enhanced expression of transforming growth-factor-beta 1.

In human breast carcinoma MCF-7 cells, phorbol diesters inhibit proliferation and induce cell maturation. We have recently reported that exogenous TGF-beta 1 reverses the resistance of a breast adenocarcinoma MCF-7 subline (MCF-7:RPh-4) to these phorbol ester effects. Here, we investigated the involvement of TGF-beta 1 in the PKC-mediated inhibition of breast-cancer cell proliferation. Parental MCF-7-conditioned medium contained a 20-fold higher transforming activity on NRK-49F fibroblasts than the TPA-resistant subline. TPA increased TGF-beta activity in MCF-7 conditioned medium. MCF-7 cells also expressed more TGF-beta 1 mRNA than the resistant subline. TPA induced a dose-dependent increase in TGF-beta 1 mRNA levels that paralleled the inhibitory effect on MCF-7 proliferation. The lower level of TGF-beta mRNA expression in TPA resistant subline was not modified after addition of TPA, but was significantly increased in the presence of exogenous TGF-beta 1. These data argue in favor of a role of endogenous TGF-beta 1 in the maturation process induced by protein kinase C activation.     

Changes in protein kinase C and cAMP-dependent kinase in lymphocytes after treatment with 12-O-tetradecanoylphorbol-13-acetate or concanavalin A: quantitation of activities with an in situ gel assay

Primary lymphocytes can be stimulated to proliferate by mitogenic lectins such as concanavalin A (Con A). While the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) alone is not mitogenic for these cells, it can enhance the response to Con A. Previously, protein kinases and phosphorylation have been reported to be important in lymphocyte proliferation. More recently TPA has been found to bind and activate protein kinase C. Therefore, we examined kinase activity in lymphocytes stimulated with the complete mitogen Con A and the comitogen TPA. In order to monitor more than one kinase we used an in situ gel assay and developed the system to compare both protein kinase C and cAMP-dependent kinases. When total cell extracts were assayed in the presence of histone five major bands of activity were detected by autoradiography of the gel. The bands corresponding to protein kinase C and to cAMP-dependent kinases were identified by partial purification of the enzymes, by binding of [20-3H(N)]7-phorbol-12, 13-dibutyrate (3H-PDBU), and by photoaffinity labelling with 8-azidoadenosine-3':5'-cyclic monophosphate (8-N3-[32P]cAMP). Differential extraction of cell lysate allowed comparison of soluble and particulate kinases. We found that when the preparations from either TPA- or Con A-treated lymphocytes were assayed, protein kinase C activity increased three- to four-fold in the particulate fraction within 5 min after treatment. A concurrent decrease of 30-50% occurred in the cytosol. In contrast, cytosolic cAMP-dependent protein kinase II increased 1.4-fold in the same period with Con A. PKI and PKII showed the most significant changes after 24 h of stimulation by Con A when the activity of the holoenzyme decreased to half that of the unstimulated cells. Therefore, although TPA and Con A separately can affect protein kinase C this alone is not sufficient for proliferation to occur.     

Inhibition of DNA synthesis by protein kinase C-activating phorbol esters in NIH/3T3 cells

Fibroblast growth factor (FGF) plus insulin induced DNA synthesis in and proliferation of NIH/3T3 cells. The protein kinase C-activating phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), inhibited both the DNA synthesis and cell proliferation induced by FGF plus insulin. The concentration of TPA required for 50% inhibition of the DNA synthesis was about 5 nM. Phorbol-12,13-dibutyrate, another protein kinase C-activating phorbol ester, also inhibited the DNA synthesis but 4 alpha-phorbol-12,13-didecanoate, known to be inactive for this enzyme, was ineffective. DNA synthesis started at about 12 h after the addition of FGF plus insulin. The inhibitory action of TPA on the DNA synthesis was observed when it was added within 12 h after the addition of FGF plus insulin. These results suggest that phorbol esters exhibit an antiproliferative action through protein kinase C activation in NIH/3T3 cells, and that this action of phorbol esters is due to inhibition of the progression from the late G1 to the S phase of the cell cycle.     

Protein Kinase C in Primary Astrocyte Cultures: Cytoplasmic Localization and Translocation by a Phorbol Ester

The distribution of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) in supernatant and particulate fractions of primary cultures of rat astrocytes and its translocation by a phorbol ester were studied. We observed that 91% of protein kinase C activity in astrocytes was in the supernatant fraction, as measured by lysine-rich histone phosphorylation assay. Attempts to uncover latent activity in the particulate fraction were unsuccessful. Approximately 75% of the supernatant protein kinase C activity could be translocated to the particulate fraction by prior treatment (30-60 min) of the cultures with 100 nM 12-O-tetradecanoyl-phorbol 13-acetate (TPA), but not with 4 alpha-phorbol, an inactive phorbol ester. Investigation of endogenous substrates for protein kinase C showed that TPA treatment brought about an increase in phosphorylation in membrane proteins and a decrease in phosphorylation of supernatant proteins. These findings indicate that the distribution of protein kinase C in astrocytes differs substantially from that in whole brain tissue, where approximately two-thirds of the protein kinase C activity is associated with the particulate fraction. Because protein kinase C is concentrated in the cytosol of astrocytes and most of this activity can be translocated to membranes, astrocytes may be particularly well-suited to respond to signals that activate phosphoinositide-linked receptors in brain.     

Survival of Chick Embryonic Sensory Neurons in Culture Is Supported by Phorbol Esters

Sensory neurons of the chick embryo are supported in culture by several neurotrophic factors, including the phorbol esters. Because phorbol esters are known to activate one of the second messengers, namely, protein kinase C, it was of interest to see if the neurotrophic action of phorbol 12,13-dibutyrate (PDB) was related to the activation of protein kinase C in sensory neurons. Sensory neurons were obtained from dorsal root ganglia of 10-day-old chick embryos and maintained in a serum-free medium for several days to quantify survival and analyze protein kinase C activity. PDB (30 nM) supported the survival of approximately 50% of the total number of neurons plated. This value was comparable to that supported by nerve growth factor (NGF; 40 ng/ml). If PDB and NGF were added together, there was no additive effect on the survival. The protein kinase C activity of the particulate and cytosolic fractions of sensory neurons supported by NGF for 3 days was 1.26 +/- 0.1 and 2.9 +/- 0.32 pmol/min/mg of protein, respectively. In contrast, neurons supported by PDB showed an approximately 500% increase in enzyme activity in their particulate fraction. The enzyme activity of the cytosolic fraction was decreased by approximately 40%. If NGF-supported neurons were treated with PDB (30 nM) for 15 min, protein kinase C activity increased greater than 400% in the particulate fraction, whereas an approximately 50% decrease was observed in the cytosolic fraction. The protein kinase C value, expressed as a ratio of the activities in the particulate to cytosol fractions, showed large increases after phorbol treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

1,2-Diacylglycerols, but not phorbol esters, activate a potential inhibitory pathway for protein kinase C in GH3 pituitary cells. Evidence for involvement of a sphingomyelinase

It has been suggested that sphingoid bases may serve as physiologic inhibitors of protein kinase C. Because 1,2-diacylglycerols, but not phorbol esters, enhance sphingomyelin degradation via a sphingomyelinase in GH3 pituitary cells (Kolesnick, R. N. (1987) J. Biol. Chem. 262, 16759-16762), the effects of phorbol esters, 1,2-diacylglycerols, and sphingomyelinase on protein kinase C activation were assessed. Under basal conditions, the inactive cytosolic form of protein kinase C predominated. 1,2-Diacylglycerols stimulated transient protein kinase C redistribution to the membrane. 1,2-Dioctanoylglycerol (200 micrograms/ml) reduced cytosolic protein kinase C activity to 67% of control from 72 to 48 pmol.min-1.10(6) cells-1 and enhanced membrane-bound activity to 430% of control from 6 to 25 pmol.min-1.10(6) cells-1 after 4 min of stimulation. Thereafter, protein kinase C activity returned to the cytosol. In contrast, the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulated redistribution to the membrane without return to the cytosol. Exogenous sphingomyelinase reduced membrane-bound protein kinase C activity to 30% of control, yet did not alter cytosolic activity. Sphingomyelinase, added after phorbol ester-induced redistribution was completed, restored activity to the cytosol. In these studies, TPA (10(-8) M) reduced cytosolic activity to 62% of control and elevated membrane-bound protein kinase C activity to 650% of control. Sphingomyelinase restored cytosolic activity to 84% of control and reduced membrane-bound activity to 297% of control. Similarly, the free sphingoid bases, sphingosine, sphinganine, and phytosphingosine, reversed phorbol ester-induced protein kinase C redistribution. Since 1,2-diacylglycerols activate a sphingomyelinase and sphingomyelinase action can reverse protein kinase C activation, these studies suggest that a pathway involving a sphingomyelinase might comprise a physiologic negative effector system for protein kinase C. Further, the failure of phorbol esters to activate this system might account for some differences between these agents.     

Effects of phorbol ester and cholecystokinin on the intracellular distribution of protein kinase C in rabbit pancreatic acini.

Treatment of rabbit pancreatic acini with the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), resulted in a time- and dose-dependent decrease of soluble protein kinase C activity coinciding with an increase of protein kinase C activity in the particulate fraction. After 5 min, soluble protein kinase C activity had decreased to almost 10% of the corresponding control. Total extractable protein kinase C activity, however, remained unchanged, indicating that the decrease of soluble protein kinase C activity was not due to TPA-induced inactivation of the enzyme. The biologically inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate, did not induce such a translocation of protein kinase C. The half-maximal concentration for TPA-induced translocation of protein kinase C was 40 nM, and was equal to that for TPA-induced amylase secretion from isolated acini. This suggests that translocation of protein kinase C to the particulate fraction is an important step in TPA-induced activation of protein kinase C and enzyme secretion. On the other hand, cholecystokinin, a secretagogue of the calcium-mobilizing type, whose secretory action is thought to be mediated, at least in part, by protein kinase C, did not change the subcellular distribution of protein kinase C. In the presence of R59022 6-(2-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl ) ethyl-7-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one, an inhibitor of diacylglycerol kinase activity, cholecystokinin produced a small but significant translocation of protein kinase C, suggesting that the inability of the hormone to induce translocation is not due to a rapid conversion of the diacylglycerol formed into phosphatidic acid.     

The involvement of protein kinase C in exocytosis in mast cells.

Diacylglycerol generated from inositolphospholipid hydrolysis and tumor-promoting phorbol esters stimulate protein kinase C. The synthetic diacylglycerol 1-oleoyl-2-acetyl-rac-glycerol and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) have been used in pure rat peritoneal mast cells. Both caused histamine release associated with exocytosis. The release by the stimulation of protein kinase C alone in the absence of secretagogues was slow although up to 50% of the histamine content was released by TPA in 120 min. Remarkable potentiation of histamine release was observed when the mast cells were preincubated with TPA before exposure to the calcium ionophore A23187. The potentiation of histamine release corresponded with an intensification of exocytosis. The potentiation is consistent with a participation of protein kinase C in the secretory process. An inhibitory effect due to protein kinase C activity was also demonstrated using TPA and mast cells from sensitized rats. When sensitized mast cells preincubated with 50 nM TPA for 5 min were exposed to the antigen, the histamine release was substantially reduced compared to the sum of the release by the antigen and TPA or by the antigen alone. There was a corresponding decrease in exocytosis. The inhibition of exocytosis and histamine release seems to reflect a regulatory function of protein kinase C for the termination of the response, as demonstrated in other types of cells apparently acting through an inhibition of inositolphospholipid hydrolysis.     

Regulation of protein kinase C by cyclic adenosine 3':5'-monophosphate and a tumor promoter in skeletal myoblasts

The specific activity of protein kinase C in rat skeletal myoblasts decreased when they were exposed for very short periods to isoproterenol, forskolin, dibutyryl cyclic AMP (Bt2cAMP), or the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). In the presence of Bt2cAMP or forskolin only the cytosolic but not the membrane-bound kinase activity was found to decrease. Treatment with TPA, however, led to a decrease in the activity of the enzyme both in the cytosolic as well as the membrane fractions. The effects observed in vivo could be duplicated in crude extracts of myoblasts incubated with cAMP analogues or TPA. In the presence of ATP, protein kinase C activity decreased considerably in crude cytosolic fractions treated with the cAMP analogues, but a requirement for ATP was not evident for the decrease in activity brought about by TPA. For the cAMP analogues the decrease in protein kinase C was also prevented by incubation of the extracts with an inhibitor of cAMP-dependent protein kinase. The regulation of protein kinase C by Bt2cAMP (but not by TPA) was altered in Rous sarcoma virus-transformed myoblasts. It is considered likely that a component affected by cAMP (probably a substrate for cAMP-dependent protein kinase) participates in the regulation of protein kinase C activity, and it is altered in unknown ways in transformed myoblasts.     

Bombesin and phorbol ester stimulate phosphatidylcholine hydrolysis by phospholipase C: evidence for a role of protein kinase C

Bombesin caused a marked stimulation of 32Pi into phosphatidylinositol (PI), with no apparent lag, and into phosphatidylcholine (PC), after a lag of about 20 min. Stimulation was blocked by the bombesin receptor antagonist, [D-Arg1, D-Pro2, D-Trp7,9, Leu11] substance P, indicating that the effects on both PI and PC were mediated through the same receptor. The tumor-promoting phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) and dioctanoylglycerol (diC8) both directly activate protein kinase C and in this report were shown to stimulate 32Pi incorporation into PC but not into Pl. In addition, TPA stimulated the release of [3H]choline and [3H]phosphocholine and the accumulation of [3H]diacyglycerol from prelabelled cells. These results strongly suggest that TPA activates a phospholipase C specific for PC. Pretreatment of cells with phorbol-12, 13-dibutyrate (PDBu) for 24 h depleted cellular protein kinase C activity and inhibited the ability of TPA to induce these effects suggesting a direct involvement of protein kinase C. Similarly the bombesin stimulation of 32Pi into PC and of [3H]choline and [3H]phosphocholine release was inhibited by PDBu pretreatment. DiC8 and, to a lesser extent, TPA stimulated the translocation of CTP:phosphocholine cytidylytransferase from the cytosolic to the particulate fraction. DiC8 also stimulated this translocation in cells depleted of protein kinase C. It was concluded that both bombesin and TPA activated protein kinase C leading to activation of a phospholipase C specific for PC.     

Phorbol ester-induced translocation of diacylglycerol kinase from the cytosol to the membrane in Swiss 3T3 fibroblasts

The tumor-promoting phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate, causes a rapid, partial redistribution of 1,2-sn-diacylglycerol kinase from the cytosol to the particulate fraction of quiescent, starved Swiss 3T3 fibroblasts. We utilized exogenous dioleoylglycerol as substrate for the kinase. The inactive alpha form of the phorbol ester does not cause any change in diacylglycerol kinase localization, and depletion of protein kinase C (Ca2+/phospholipid-dependent enzyme) by chronic administration of phorbol ester blocks the redistribution. Phorbol ester has no direct effect on Swiss 3T3 membrane-bound diacylglycerol kinase nor does it directly effect cytosolic diacylglycerol kinase. When phorbol ester is added to Swiss 3T3 membranes in the presence of ATP, magnesium, and calcium, there is no activation of membrane-bound kinase, indicating that phorbol ester does not activate membrane-bound kinase through phosphorylation by protein kinase C. Reconstitution studies show that the soluble rat brain diacylglycerol kinase binds to diacylglycerol-enriched membranes, produced by treatment of red cell ghosts with phospholipase C or calcium, suggesting that cytosolic diacylglycerol kinase may be capable of translocation to the membrane in response to elevated substrate concentration in the intact cell. Stimulation of the cells with phorbol ester increases the total mass of diacylglycerol. In protein kinase C-depleted cells, addition of a cell-permeable synthetic diacylglycerol, dioctanoylglycerol, results in a partial redistribution of cytosolic diacylglycerol kinase to the membrane, by 5 min, also suggesting that the translocation of diacylglycerol kinase activity is regulated primarily by substrate concentration.     

Discrepancy of protein kinase C translocation and platelet aggregation--immunocytobiochemical evidence

We searched for possible relationships between platelet aggregation induced by 12-O-tetradecanoyl phorbol 13-acetate (TPA) or thrombin and the translocation of protein kinase C. Using monoclonal antibodies against subspecies of protein kinase C, we noted a predominant expression of the isozyme, type II, in human platelets (M. Watanabe, M. Hagiwara, K. Onoda, and H. Hidaka, 1988, Biochem. Biophys. Res. Commun. 152, 642). Analysis of the subcellular distribution of protein kinase C revealed that 65% of the kinase activity was present in the cytosolic fraction in unstimulated platelets, with the remaining activity in the membrane fraction. Treatment of platelets with 100 nM TPA resulted in a greater than 60% decrease in protein kinase C activity in the cytosolic fraction and a greater than 200% increase in the activity in the membrane fraction, within 10 min after treatment. Translocation of the enzyme was also found after treatment of platelets with thrombin, although the response was of lower magnitude than that induced by TPA. Similar results were obtained by immunoblotting using MC-2a, an anti-type II protein kinase C monoclonal antibody. We also examined localization of the enzyme, by electron microscopic immunocytochemistry. The presence of type II protein kinase C seemed to be localized mostly in hyaluromeres and not in granulomeres. When platelets were fixed just after the addition of TPA (within 1 min), protein kinase C was localized at the submembranal region with no remarkable change in shape but there was a decrease in the number of granules in the cytoplasma and the open canalicular system was dilated. We then investigated the effects of cytochalasin B, W-7, ML-9, and H-7 on TPA-induced platelet aggregation and the translocation of protein kinase C. W-7 and ML-9 potently inhibited platelet aggregation but none of these compounds hampered the translocation. Thus, activation of protein kinase C may not be a complete requirement for the initiation of platelet aggregation.     

Rapid down-regulation of protein kinase C and membrane association in phorbol ester-treated leukemia cells

Peripheral blood lymphocytes from patients with chronic lymphocytic leukemia (CLL) acquire after several days of exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA) several morphological, immunological and histochemical features of hairy cell leukemia. We have investigated the short term effects of TPA treatment on protein kinase C and its subcellular distribution. Within minutes of addition of TPA to CLL cells 20% of the cytosolic protein kinase C had associated with the particulate fraction. The remaining 80% of protein kinase C activity was down-regulated. The association with the membrane dramatically increased the resistance of the enzyme to inhibition by the non-ionic detergent, Triton X-100. These results suggest that activation of protein kinase C causes multiple biological changes in CLL cells.     

Evidence for a role of protein kinase C in luteinizing hormone synthesis and secretion. Impaired responses to gonadotropin-releasing hormone in protein kinase C-depleted pituitary cells

The role of protein kinase C in luteinizing hormone (LH) release was analyzed in studies on the actions of phorbol esters and gonadotropin-releasing hormone (GnRH) in normal and protein kinase C (Ca2+/phospholipid-dependent enzyme)-depleted pituitary cell cultures. LH secretory responses of normal pituitary cells to GnRH were reduced but not abolished in Ca2+-deficient medium, consistent with the existence of extracellular Ca2+-dependent and -independent components of GnRH action. Both of these components could be elicited by treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). The LH secretory responses to TPA and GnRH were additive only at low doses and converged to a common maximum at high concentrations of the agonists in the presence or absence of extracellular Ca2+. The release of stored LH by GnRH and TPA was accompanied by secretion of newly synthesized LH from 2 to 5 h during stimulation by either of the agonists. LH synthesis was increased in a progressive and dose-dependent manner by GnRH and TPA, and the ratio between newly synthesized and released hormone was near 1:2. TPA caused rapid and complete translocation of cytosolic protein kinase C to the particulate fraction of pituitary cells, followed by a progressive decrease in total enzyme content to approximately 10% after 6 h. Partial recovery of the cytosolic enzyme (to 20%) occurred after washing and reincubation for 15 h. Such kinase C-depleted cells showed prominent, dose-dependent reductions in the actions of GnRH and TPA on LH release and synthesis in both normal and Ca2+-deficient media. These observations support the hypothesis that protein kinase C participates in LH biosynthesis and secretion in pituitary gonadotrophs and is involved in the actions of GnRH upon these processes.