Zinc as a possible mediator of signal transduction in T lymphocytes

Our recent findings indicate that phorbol esters, the specific activators of protein kinase C induce the translocation of heavy metals (mostly: zinc) from the nucleus and mitochondria to the cytosol and microsomes of T lymphocytes. Phorbol ester treatment impairs the action of Ca-ionophores, this effect is mediated by intracellular heavy metal ions (most probably: by zinc). Zinc activates cytosolic protein kinase C, increases its affinity towards phorbol esters and contributes to its binding to plasma membranes. These results suggest that zinc may play a role in the "cross-talk" of second messengers and hence in signal transduction in T lymphocytes.     

Zinc increases the affinity of phorbol ester receptor in T lymphocytes

In the primary structure of the major phorbol ester receptor, protein kinase C the presence of putative metal (zinc) binding sites has been suggested. We have demonstrated earlier that zinc activates protein kinase C and contributes to its binding to plasma membranes in T lymphocytes. Here we report that zinc increases the phorbol ester binding affinity of cytosolic protein kinase C. The effect of zinc on the membrane-bound enzyme is much less pronounced. Our results raise the possibility that cytosolic protein kinase C is a mixture of isoenzymes with different sensitivity towards zinc ions.     

Interaction of protein kinase C with chromaffin granule membranes: effects of Ca2+, phorbol esters and temperature reveal differences in the properties of the association and dissociation events

Interaction of protein kinase C with chromaffin granule membranes has been studied as a means of investigating the translocation of protein kinase C from cytosol to intracellular membrane surfaces, which is believed to occur during secretion. Protein kinase C in an adrenal medullary soluble fraction was found to bind reversibly to granule membranes in a Ca2+-dependent fashion. Association and dissociation events were sensitive to Ca2+ concentrations in the low micromolar range, and the Ca2+ sensitivity of both processes was increased when the membranes had been preincubated with the protein kinase C-activating phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (TPA). Binding of protein kinase C to granule membranes occurred at 0 and 37 degrees C, irrespective of whether the membranes had been preincubated with TPA. However, dissociation of protein kinase C from granule membranes that had been preincubated with TPA occurred only at 37 degrees C and not at 0 degree C, even though dissociation of the enzyme from membranes which had not been preincubated with TPA would occur at both 37 and 0 degrees C. These effects of TPA were not reproduced by 4 alpha-phorbol 12,13-didecanoate (4 alpha PDD), a phorbol ester which does not activate protein kinase C. Soluble protein kinase C activity also associated with chromaffin granules in a Ca2+-dependent manner in an adrenal medullary homogenate, indicating that granules can compete with other intracellular membranes for the binding of protein kinase C. Results obtained with this model system differ from other systems where the interaction of protein kinase C with plasma membranes has been studied and have general implications for studies performed on the translocation of protein kinase C in intact cells and for the role of protein kinase C in stimulus-secretion coupling in the chromaffin cell.     

Interaction of protein kinase C with membranes is regulated by Ca2+, phorbol esters, and ATP

Physiologic regulation of protein kinase C activity requires its interaction with cellular membranes. We have recently shown that binding of the enzyme to plasma membranes is controlled by Ca2+, whereas enzyme activators, like phorbol esters, regulate both membrane binding and enzyme activity. Here we describe the factors which control the dissociation of protein kinase C from the plasma membrane. In the absence of phorbol esters, the dissociation reaction is rapid and is determined by varying the Ca2+ concentration between 0.1 and 1 microM. However, the presence of 4-beta-phorbol 12,13-dibutyrate greatly reduces enzyme release in response to Ca2+ depletion; removal of the phorbol ester itself permits efficient membrane-enzyme dissociation. The stabilization of the membrane-protein kinase C complex by phorbol esters can be reversed by ATP with an apparent Km for the nucleotide of 6.5 microM. The ATP effect requires MgCl2 and cannot be reproduced by other nucleotides or by a nonhydrolyzable analogue, suggesting that an ATP-dependent phosphorylation reaction may be involved. 4-beta-Phorbol 12,13-dibutyrate appears to stabilize membrane-enzyme association by reducing the apparent Km for Ca2+ to about 15 nM, whereas ATP reverses the phorbol ester effect by increasing the Km for Ca2+ to about 760 nM. Furthermore, the strong degree of negative cooperativity displayed by the Ca2+-dependent enzyme-membrane dissociation is consistent with the presence of multiple interacting Ca2+-binding sites on protein kinase C.     

Control of exocytosis from adrenal chromaffin cells

1. Calcium-dependent exocytosis of catecholamines from intact and digitonin-permeabilized bovine adrenal chromaffin cells was investigated. 2. 45Ca2+ uptake and secretion induced by nicotinic stimulation or depolarization in intact cells were closely correlated. The results provide strong support for Ca2+ entry being the trigger for exocytosis. 3. Experiments in which the H+ electrochemical gradient across the intracellular secretory granule (chromaffin granule) membrane was altered indicated that the gradient does not play an important role in exocytosis. 4. Ca2+ entry into the cells is associated with activation of phospholiphase C and a rapid translocation of protein kinase C to membranes. 5. The plasma membrane of chromaffin cells was rendered permeable to Ca2+, ATP, and proteins by the detergent digitonin without disruption of the intracellular secretory granules. In this system in which the intracellular milieu can be controlled, micromolar Ca2+ directly stimulated catecholamine secretion. 6. Treatment of the cells with phorbol esters and diglyceride, which activate protein kinase C, enhanced phosphorylation and subsequent Ca2+-dependent secretion in digitonin-treated cells. 7. Phorbol ester-induced secretion could be specifically inhibited by trypsin. The experiments indicate that protein kinase C modulates but is not necessary for Ca2+-dependent secretion.     

Inhibition of phorbol ester binding and protein kinase C activity by heavy metals

Other laboratories have reported biphasic effects of heavy metals on protein kinase C activity: stimulation followed by inhibition at higher concentrations. We demonstrate that these earlier findings most likely resulted from a combination of the effect of the heavy metals to liberate Ca2+ from Ca2+-EGTA buffer systems and the direct inhibitory effects of the metals on protein kinase C. Simulations of such interactions substantiate this conclusion. When soluble protein kinase C is prepared without the addition of Ca2+ or chelator, heavy metals (Cd2+, Cu2+, Hg2+, Zn2+, in the 10 microM range) inhibit the activity of, and the binding of regulatory ligands to, protein kinase C. Heavy metals inhibit the extent of [3H]phorbol dibutyrate binding without affecting the affinity of the interaction, an inhibition that is not surmounted by excess phospholipid. Heavy metals also inhibit the phospholipid-dependent catalytic activity of protein kinase C in a manner that excess phosphatidylserine can overcome. The inhibition of enzyme activity by heavy metals cannot be surmounted by excess Ca2+ or Mg2+. The inhibitory effects of heavy metals are not confined to protein kinase C. Heavy metals also inhibit cyclic AMP binding to cyclic AMP-dependent protein kinase and the catalytic activity of that kinase, but in a distinctly different pattern.     

Factors influencing chelator-stable, detergent-extractable, phorbol diester-induced membrane association of protein kinase C. Differences between Ca2+-induced and phorbol ester-stabilized membrane bindings of protein kinase C

One of the early events associated with the treatment of cells by tumor promotor phorbol esters is the tight association of protein kinase C to the plasma membrane. To better understand the factors that regulate this process, phorbol ester-induced membrane binding of protein kinase C was studied using homogenates, as well as isolated membranes and purified enzyme. Addition of 12-O-tetradecanoylphorbol 13-acetate (TPA) to the homogenates of parietal yolk sac cells and NIH 3T3 cells in the presence of Ca2+ resulted in plasma membrane binding of protein kinase C which subsequently remained bound to the membrane independent of Ca2+. Although protein kinase C was activated by TPA in the absence of Ca2+ and by diolein in the presence of Ca2+, both these agents when added to homogenates under these respective conditions had no effect on membrane association of protein kinase C. However, under these conditions relatively weak binding of protein kinase C was found if purified protein kinase C was used with isolated membranes. Binding studies using purified protein kinase C and washed membranes showed that the binding of the TPA-kinase complex to membranes required phospholipids and reached saturation at 0.1 unit (24 ng of protein kinase C)/mg of parietal yolk sac cell membrane protein. Phorbol ester treatment of cells in media with and without Ca2+ showed that the TPA-induced increase in membrane-associated protein kinase C was regulated by Ca2+ levels even in intact cells. TPA-stabilized membrane binding of protein kinase C differs in several aspects from the previously reported Ca2+-induced reversible binding. TPA-stabilized binding of protein kinase C to isolated membranes is temperature dependent, relatively high in the plasma membrane-enriched fraction, saturable at physiological levels of protein kinase C, requires the presence of both membrane protein(s) and phospholipids, and further requires the addition of phospholipid micelles. In contrast, Ca2+-induced reversible binding is more rapid, not appreciably influenced by temperature, not selective for a particular subcellular fraction, not saturable with physiological amounts of protein kinase C, exhibits trypsin-insensitive membrane binding sites, and requires membrane phospholipids but not added phospholipid micelles.     

Effects of a monoclonal anti-calpain antibody on responses of stimulated human neutrophils. Evidence for a role for proteolytically modified protein kinase C

A monoclonal antibody directed against the Ca2+-requiring proteinase (calpain) of human neutrophils was employed to assess the role of this proteinase in mediating the responses to stimuli such as phorbol 12-myristate 13-acetate or fMet-Leu-Phe. In the presence of either phorbol 12-myristate 13-acetate or fMet-Leu-Phe the antibody is taken up by the neutrophils, and a marked inhibition of intracellular calpain is observed. The decreased calpain activity is accompanied by (a) a significant decrease in the proteolytic conversion of native protein kinase C (Ca2+/phospholipid-dependent enzyme) to the soluble form that does not require Ca2+ or phospholipids for activity; (b) a marked increase in the production of superoxide anion; and (c) a decrease in the exocytosis of granule contents. The increase in superoxide production can be attributed to a more prolonged association of native protein kinase C with the plasma membrane, thus enhancing the phosphorylation of membrane proteins that precedes O(2-) production (Pontremoli, S., Melloni, E., Salamino, F., Sparatore, B., Michetti, M., Sacco, O., and Horecker, B. L. (1986), Biochem. Biophys. Res. Commun. 140, 1121-1126). The decreased exocytosis can be attributed to a decreased phosphorylation of certain cytoskeletal proteins, catalyzed by the soluble form of protein kinase C (Pontremoli, S., Melloni, E., Michetti, M., Sparatore, B., Salamino, F., Sacco, O., and Horecker, B. L. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 3604-3608); the subsequent reorganization of the cytoskeleton appears to be related to degranulation. These effects of the monoclonal anti-calpain provide direct evidence for an essential role for calpain in the activation of human neutrophils.     

The involvement of calpain in the activation of protein kinase C in neutrophils stimulated by phorbol myristic acid

The Ca2+/phospholipid-dependent protein kinase (protein kinase C) of human neutrophils is converted to a proteolytically modified Ca2+/phospholipid-independent form (Inoue, M., Kishimoto, A., Takai, Y.U., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7610-7616) on incubation with neutrophil membranes in the presence of micromolar concentrations of Ca2+ and an endogenous Ca2+-requiring proteinase (Melloni, E., Pontremoli, S., Michetti, M., Sacco, O., Sparatore, B., Salamino, F., and Horecker, B. L. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6435-6439). We have now demonstrated the appearance of a similar Ca2+/phospholipid-independent kinase in intact human neutrophils stimulated by phorbol 12-myristate 13-acetate (PMA). The following evidence supports the conclusion that the Ca2+/phospholipid-independent protein kinase recovered from the PMA-treated cells is a proteolytically modified form of the "native" protein kinase C. 1) In cells exposed to PMA, the rate of disappearance of Ca2+/phospholipid-dependent protein kinase C activity is correlated with the rate of appearance of the Ca2+/phospholipid-independent kinase. 2) The chromatographic behavior of the new protein kinase and its molecular size (approximately 65 kDa) are identical to those previously reported for the proteolytically modified form of protein kinase C. 3) The modified protein kinase no longer binds to the cell membrane and is recovered almost entirely in the cytosol fraction. 4) In neutrophils preloaded with inhibitors of the Ca2+-requiring proteinase, stimulation with PMA results in translocation of protein kinase C from the cytosol fraction to the particulate fraction, but the appearance of the soluble, Ca2+/phospholipid-dependent form is prevented. We conclude that binding of protein kinase C to the plasma membrane and its proteolytic conversion are related, but independent, processes both elicited by exposure of neutrophils to the phorbol ester. Proteolytic cleavage of the membrane-bound protein kinase C provides an alternative mechanism for its activation and may account for certain of the cellular responses observed in PMA-stimulated neutrophils.     

Calmodulin- and protein phosphorylation-independent release of catecholamines from PC-12 cells

Catecholamine secretion from PC-12 cells can be triggered by agents that increase intracellular Ca2+ and is enhanced by phorbol esters and agents that elevate intracellular cAMP concentrations. In mutant PC-12 cells lacking cAMP-dependent protein kinase (PK-A) in which protein kinase C (PK-C) was down-regulated, Ca2+-dependent secretion occurred normally but was no longer enhanced by cAMP or phorbol esters. In digitonin-permeabilized PC-12 cells that lacked PK-C and PK-A, a range of calmodulin (CaM) inhibitors failed to block Ca2+-triggered catecholamine release. Moreover, Mn2+, a CaM activator, failed to trigger catecholamine release whereas Ba2+, which does not activate CaM, supported secretion. These results indicate that the basic mechanism of stimulus/secretion coupling in PC-12 cells does not absolutely require a regulated protein phosphorylation- or calmodulin-dependent step.     

Chelation of cytoplasmic Ca2+ increases plasma membrane permeability in murine macrophages

Cytoplasmic free Ca2+ (Ca2+i) was chelated to 10-20 nM in the macrophage cell line J774 either by incubation with quin2 acetoxymethyl ester in the absence of external Ca2+ (Di Virgilio, F., Lew, P.D., and Pozzan, T. (1984) Nature 310, 691-693) or by loading [ethyl-enebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) into the cytoplasm via reversible permeabilization of the plasma membrane with extracellular ATP (Steinberg, T.H., Newman, A.S., Swanson, J.A., and Silverstein, SS.C. (1987) J. Biol. Chem. 262, 8884-8888; Di Virgilio, F., Meyer, B.C., Greenberg, S., and Silverstein, S.C. (1988) J. Cell Biol. 106, 657-666). After removal of ATP from the incubation medium, ATP-permeabilized Ca2+i-depleted macrophages recovered a near-normal plasma membrane potential which slowly depolarized over a 2-4 h incubation at low [Ca2+]i. In both ATP-treated and quin2-loaded cells, depolarization of plasma membrane potential was paralleled by an increase in plasma membrane permeability to low molecular weight aqueous solutes such as eosin yellowish (Mr 692), ethidium bromide (Mr 394), and lucifer yellow (Mr 463). This increased plasma membrane permeability was not accompanied by release of the cytoplasmic marker lactic dehydrogenase for incubations up to 4 h and was likely a specific effect of Ca2+i depletion since it was not caused by: (i) the mere incubation of macrophages with extracellular EGTA, i.e. at near-normal [Ca2+]i; and (ii) loading into the cytoplasm of diethylenetriaminepentaacetic acid, a specific chelator of heavy metals with low affinity for Ca2+. Treatment of Ca2+i-depleted cells with direct (phorbol 12-myristate 13-acetate) or indirect (platelet-activating factor) activators of protein kinase C prevented the increase in plasma membrane permeability. Down-regulation of protein kinase C rendered Ca2+i-depleted macrophages refractory to the protective effect of phorbol 12-myristate 13-acetate. This report suggests a role for Ca2+i and possibly protein kinase C in the regulation of plasma membrane permeability to low molecular weight aqueous solutes.     

Zinc stimulates the activity of the insulin- and nutrient-regulated protein kinase mTOR

Recent studies indicate that zinc activates p70 S6 kinase (p70(S6k)) by a mechanism involving phosphatidylinositol 3-kinase (PI 3-kinase) and Akt (protein kinase B). Here it is shown that phenanthroline, a zinc and heavy metal chelator, inhibited both amino acid- and insulin-stimulated phosphorylation of p70(S6k). Both amino acid and insulin activations of p70(S6k) involve a rapamycin-sensitive step that involves the mammalian target of rapamycin (mTOR, also known as FRAP and RAFT). However, in contrast to insulin, amino acids activate p70(S6k) by an unknown PI 3-kinase- and Akt-independent mechanism. Thus the effects of chelator on amino acid activation of p70(S6k) were surprising. For this reason, we tested the hypothesis that zinc directly regulates mTOR activity, independently of PI 3-kinase activation. In support of this, basal and amino acid stimulation of p70(S6k) phosphorylation was increased by zinc addition to the incubation media. Furthermore, the protein kinase activities of mTOR immunoprecipitated from rat brain lysates were stimulated two- to fivefold by 10-300 microM Zn2+ in the presence of an excess of either Mn2+ or Mg2+, whereas incubation with 1,10-phenanthroline had no effect. These findings indicate that Zn2+ regulates, but is not absolutely required for, mTOR protein kinase activity. Zinc also stimulated a recombinant human form of mTOR. The stimulatory effects of Zn2+ were maximal at approximately 100 microM but decreased and became inhibitory at higher physiologically irrelevant concentrations. Micromolar concentrations of other divalent cations, Ca2+, Fe2+, and Mn2+, had no effect on the protein kinase activity of mTOR in the presence of excess Mg2+. Our results and the results of others suggest that zinc acts at multiple steps in amino acid- and insulin cell-signaling pathways, including mTOR, and that the additive effects of Zn2+ on these steps may thereby promote insulin and nutritional signaling.     

Differential down-regulation of protein kinase C isozymes

Types I, II, and III protein kinase C have been shown to be products of, respectively, gamma, beta, and alpha genes of this enzyme family (Huang, F. L., Yoshida, Y., Nakabayashi, H., Knopf, J. L., Young, W. S., III, and Huang, K.-P. (1987) Biochem. Biophys. Res. Commun. 149, 946-952). Incubation of the highly purified rat brain protein kinase C isozymes with trypsin (kinase/trypsin (w/w) = 100) under identical conditions results in a preferential degradation of types I and II enzymes, whereas the type III enzyme was relatively resistant to tryptic proteolysis. Degradation of the type III enzyme by trypsin could be facilitated with the addition of Ca2+, phosphatidylserine, and dioleoylglycerol; none of these components alone was effective. Limited proteolysis of the three protein kinase C isozymes generated distinctive fragments for each isozyme, indicating that each isozyme has different trypsin-sensitive sites. Tryptic digestion of the type III protein kinase C was used as a model to determine the effects of various modulators on protein kinase C degradation. While Ca2+ and phosphatidylserine together were sufficient to convert the type III protein kinase C from a trypsin-insensitive to a -sensitive form, addition of dioleoylglycerol greatly reduced the Ca2+ requirement for such a conversion. Among the various phospholipids tested, in the presence of either dioleoylglycerol or phorbol ester, phosphatidylserine, cardiolipin, and phosphatidic acid were the most effective, and phosphatidylcholine and phosphatidylethanolamine were the least effective in supporting the digestion of type III protein kinase. Other acidic phospholipids, such as lysophosphatidylserine and phosphatidylinositol, were also effective in supporting the degradation in the presence of phorbol ester but not in the presence of dioleoylglycerol. The relevance of these proteolytic reactions to physiological responses was assessed with phorbol ester on rat basophilic leukemia RBL-2H3 cells, which contained both types II and III protein kinase C. Immunoblot analysis with the isozyme-specific antibodies revealed that phorbol ester induced a faster degradation of type II than that of type III isozyme in these cells. The results demonstrate that the various protein kinase C isozymes have different susceptibilities to proteolysis in vitro, when tested with trypsin, as well as to endogenous proteases in intact cells.     

Regulation of protein kinase C activity by cooperative interaction of Zn2+ and Ca2+

cis-Fatty acids such as oleic acid or linoleic acid have been previously shown to induce full activation of protein kinase C in the absence of Ca2+ and phospholipids (Murakami, K., and Routtenberg, A. (1985) FEBS Lett. 192, 189-193; Murakami, K., Chan, S.Y., and Routtenberg, A. (1986) J. Biol. Chem. 261, 15424-15429). In this study, we have investigated the effects of various metal ions on protein kinase C activity without the interference of Ca2+ since cis-fatty acid requires no Ca2+ for protein kinase C activation. Here we report a specific interaction of Zn2+ with protein kinase C in either a positive or negative cooperative fashion in concert with Ca2+. At low concentrations (approximately 5 microM) of Ca2+, Zn2+ enhances protein kinase C activity induced by both oleic acid and phosphatidylserine/diolein. In contrast, Zn2+ inhibits the activity at higher concentrations (over 50 microM) of Ca2+. In the absence of Ca2+, Zn2+ shows no effect on protein kinase C activity. Our results suggest that Zn2+ does not recognize or interact with protein kinase C in the absence of Ca2+, that protein kinase C possesses high and low affinity Ca2+-binding sites, and that at least one Zn2+-binding site exists which is distinct from Ca2+-binding sites.     

Antagonistic effects of calcium ionophores and phorbol esters on T cell receptor mRNA levels in human thymocytes

Levels of mRNA of the T cell antigen receptors (TcR) in human thymocytes are differentially regulated in response to distinct intracellular signals. Activation of protein kinase C by the phorbol ester, tetradecanoyl phorbol acetate or other phorbol esters increases the levels of the alpha and beta T cell receptor (TcR-alpha, TcR-beta) mRNA, whereas an increase in cytosolic free Ca2+, induced by ionomycin or other Ca2+ ionophores, results in a decrease of alpha and beta TcR mRNA levels. In contrast, ionomycin increases the expression TcR-gamma mRNA whereas tetradecanoyl phorbol acetate prevents this induction. Our results suggest the existence of two opposing intracellular pathways that control expression of TcR-alpha and TcR-beta mRNA levels, on the one hand and TcR-gamma mRNA, on the other. These results provide the first evidence for antagonistic actions of protein kinase C and cytosolic-free Ca2+ on gene expression.     

Lipopolysaccharide and phorbol esters induce differentiation but have opposite effects on phosphatidylinositol turnover and Ca2+ mobilization in 70Z/3 pre-B lymphocytes

We have recently shown that both lipopolysaccharide (LPS) and the phorbol ester, 12-O-tetradecanoyl phorbol 13-acetate (TPA) induce differentiation in the transformed murine pre-B lymphocyte cell line 70Z/3 by enhancing Na+-H+ exchange across the plasma membrane through an amiloride-sensitive transport system (Rosoff, P.M., Stein, L.F., and Cantley, L.C. (1984) J. Biol. Chem. 259, 7056-7060). These data suggested that the activation of protein kinase C indirectly by LPS and directly by TPA was the critical step in the initiation of differentiation in these cells. We extend these observations to show that LPS rapidly stimulates an increase in phosphatidylinositol turnover, leading to a rise in the levels of diacylglycerol and inositol 1,4,5-trisphosphate and a concomitant decrease in the amount of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. There is also a rapid elevation of intracellular free [Ca2+] which is independent of the presence of extracellular Ca2+ or Na+. These results suggest that the increase in cytosolic [Ca2+] is due to release of cation from internal stores. TPA, which also causes differentiation in these cells, and the synthetic diacylglycerol, 1-oleoyl-2-acetylglycerol, have opposite effects from LPS on both phosphatidylinositol turnover and cellular Ca+ mobilization. These data suggest that protein kinase C inhibits the activity of phospholipase C. Thus protein kinase C plays a pivotal role in the regulation of mitogen-induced differentiation in these cells by both transducing a positive stimulus to the Na+-H+ exchange system as well as feedback regulating its own stimulatory pathway.     

Phorbol ester treatment of intact rabbit platelets greatly enhances both the basal and guanosine 5''-[gamma-thio]triphosphate-stimulated phospholipase D activities of isolated platelet membranes. Physiological activation of phospholipase D may be secondary to activation of phospholipase C.

Rabbit platelets were labelled with [3H]glycerol and incubated with or without phorbol 12-myristate 13-acetate (PMA). Membranes were then isolated and assayed for phospholipase D (PLD) activity by monitoring [3H]phosphatidylethanol formation in the presence of 300 mM-ethanol. At a [Ca2+free] of 1 microM, PLD activity was detected in control membranes, but was 5.4 +/- 0.8-fold (mean +/- S.E.M.) greater in membranes from PMA-treated platelets. Under the same conditions, 10 microM-guanosine 5'-[gamma-thio]triphosphate (GTP[S]) stimulated PLD by 18 +/- 3-fold in control membranes, whereas PMA treatment and GTP[S] interacted synergistically to increase PLD activity by 62 +/- 12-fold. GTP[S]-stimulated PLD activity was observed in the absence of Ca2+, but was increased by 1 microM-Ca2+ (3.5 +/- 0.2-fold and 1.8 +/- 0.1-fold in membranes from control and PMA-treated platelets respectively). GTP exerted effects almost as great as those of GTP[S], but 20-30-fold higher concentrations were required. Guanosine 5'-[beta-thio]diphosphate inhibited the effects of GTP[S] or GTP, suggesting a role for a GTP-binding protein in activation of PLD. Thrombin (2 units/ml) stimulated the PLD activity of platelet membranes only very weakly and in a GTP-independent manner. The actions of PMA and analogues on PLD activity correlated with their ability to stimulate protein kinase C in intact platelets. Staurosporine, a potent protein kinase inhibitor, had both inhibitory and, at higher concentrations, stimulatory effects on the activation of PLD by PMA. The results suggest that PMA not only stimulates PLD via activation of protein kinase C but can also activate the enzyme by a phosphorylation-independent mechanism in the presence of staurosporine. However, under physiological conditions, full activation of platelet PLD may require the interplay of protein kinase C, increased Ca2+ and a GTP-binding protein, and may occur as a secondary effect of the activation of phospholipase C.     

Pancreatic amylase secretion and cytoplasmic free calcium. Effects of ionomycin, phorbol dibutyrate and diacylglycerols alone and in combination.

Both protein kinase C and Ca2+ may act in concert to bring about activation of secretion. This study examined the actions on pancreatic acini of ionomycin and phorbol dibutyrate, which selectively stimulate one or the other of these pathways; their stimulatory effects were compared with those of receptor agonists, such as carbachol and caerulein, which activate phospholipase C. The Ca2+ ionophore ionomycin produced a dose-dependent increase in amylase secretion and intracellular free Ca2+ (as measured by quin-2). The increase in amylase secretion elicited by carbachol or caerulein was accompanied by a small sustained increase in intracellular free Ca2+, following an initial peak. However, the elevation in intracellular free Ca2+ produced by these receptor agonists for a given level of amylase secretion was less than that observed with ionomycin. Phorbol dibutyrate stimulated amylase secretion by a mechanism that was independent of extracellular Ca2+, and no change in intracellular free Ca2+ was observed. Synergistic stimulatory effects of phorbol dibutyrate and ionomycin were observed, whether the phorbol ester was present before, or in combination with, ionomycin. Diacylglycerols containing unsaturated fatty acids (1,2-dioleoylglycerol and 1,3-dioleoylglycerol) also stimulated amylase secretion and exhibited synergistic effects on secretion with ionomycin. These findings suggest that complete activation of amylase secretion from the pancreas requires stimulation of both Ca2+-dependent and protein kinase C-activated pathways.     

Translocation of diacylglycerol kinase from the cytosol to the membrane in phorbol ester-treated Swiss 3T3 fibroblasts

The tumor-promoting phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate, causes a rapid, partial redistribution of 1,2-diacylglycerol kinase from the cytosol to the particulate fraction of quiescent Swiss 3T3 fibroblasts. The inactive alpha form of the phorbol ester does not cause any change in diacylglycerol kinase localization, and depletion of protein kinase C by chronic administration of phorbol ester blocks the redistribution. Phorbol ester has no direct effect on membrane-bound diacylglycerol kinase in 3T3 cells. When phorbol ester is added to 3T3 membranes in the presence of ATP, Mg2+, and Ca2+, there is no activation of membrane-bound kinase, indicating that phorbol ester does not activate membrane-bound kinase through phosphorylation by protein kinase C. 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, also suggesting that the translocation of DAG kinase is regulated primarily by substrate concentration.     

Phorbol ester-induced actin cytoskeletal reorganization requires a heavy metal ion.

The cell-permeant heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine(TPEN) was found to counteract phorbol ester-induced actin reorganization in PTK2 and Swiss 3T3 cells. By using fluorescence and the higher resolution technique of photoelectron microscopy to monitor actin patterns, 15-min pretreatment with 25-50 microM TPEN was found to dramatically reduce actin alterations resulting from subsequent phorbol ester treatment in PTK2 cells. Similar results were obtained with Swiss 3T3 cells using 50 microM TPEN for 1.5 h. Phorbol ester-induced actin alterations are thought to depend on activation of protein kinase C (PKC). In contrast to the phorbol ester effect, the PKC-independent actin cytoskeletal disruption caused by staurosporine and cytochalasin B was unaffected by TPEN pretreatment. TPEN did not block phorbol ester-induced activation of PKC in Swiss 3T3 cells, as observed by the phosphorylation of the 80K PKC substrate protein (MARCKS protein). TPEN also did not inhibit partially purified PKC from Swiss 3T3 cells in an in vitro PKC-specific commercial assay. To establish that the effect of TPEN is the removal of metal ions and not some other nonspecific effect of TPEN, a series of transition metal ions was added at the end of the TPEN pretreatment. The results indicate that the transient but dramatic phorbol ester-induced reorganization of the actin cytoskeleton in cultured cells depends on an interaction of PKC with a heavy metal, probably zinc.