A model for intracellular translocation of protein kinase C involving synergism between Ca2+ and phorbol esters

The activation of protein kinase C by diacylglycerol and by tumour promoters has implicated this enzyme in transmembrane signalling and in the regulation of the cell cycle. In vitro studies revealed that catalytic activity requires the presence of calcium and phospholipids with a preference for phosphatidylserine. Diacylglycerol and tumour promoters such as phorbol esters bind to the enzyme, leading to its activation while sharply increasing its affinity for Ca2+ and phospholipid. Addition of diacylglycerol analogues or phorbol esters to intact cells results in the phosphorylation of specific polypeptides. Several cellular processes, including hormone and neurotransmitter release and receptor down-regulation, are modulated by the activation of protein kinase C, while phorbol ester-induced stimulation of the enzyme in whole cells has been associated with its translocation from the cytoplasm to the plasma membrane. Moreover, the use of Ca2+ ionophores has revealed an apparent synergism between Ca2+ mobilization and protein kinase C activation. This synergism has recently also been found to apply to receptor down-regulation (ref. 23 and accompanying paper). Here we describe a reconstitution system in which intracellular translocation of protein kinase C and the synergism between Ca2+ and enzyme activators can be studied. The results suggest a rationale for concomitant Ca2+ mobilization and diacylglycerol formation in response to some hormones, neurotransmitters and growth factors.     

Phosphorylation of the glucose transporter in vitro and in vivo by protein kinase C

The Ca2+- and phospholipid-dependent protein kinase (protein kinase C) is present in many mammalian tissues, and its important physiological protein substrates are only now beginning to be identified. A useful advance in identifying these intracellular substrates has been the recognition that the kinase is the receptor for phorbol esters, which stimulate phosphotransferase activity. Phorbol ester-induced changes in protein phosphorylation in intact cells may thus be taken, in part, as a probable indication of protein kinase C activation. The many cellular effects of phorbol esters include the stimulation of glucose uptake, although the response of glucose uptake to phorbol esters appears to be complex, apparently varying in response time and requirement for protein synthesis. Such observations prompted us to explore one possible explanation for the alteration of glucose uptake, namely, phosphorylation of the glucose transporter by protein kinase C. We report here that incubation of purified human erythrocyte glucose transporter with rat brain protein kinase C results in the phosphorylation of a protein of relative molecular mass (Mr) 50,000-60,000 which has subsequently been identified as the glucose transporter by specific immunoprecipitation with a monoclonal antibody. Immunoprecipitation of membrane proteins from 32P-labelled human erythrocytes revealed a phorbol ester-stimulated phosphorylation of the transporter. This covalent modification of the glucose transporter may thus, in part, underlie the ability of phorbol esters and certain hormones to stimulate glucose uptake.     

Effects of protein kinase C activators on cardiac Ca2+ channels

Phorbol esters have marked effects on voltage-dependent Ca2+ channels. Inhibitory and stimulatory effects on cardiac Ca2+ channels have been attributed in both cases to activation of protein kinase C. We show that the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate stimulates dihydropyridine-sensitive 45Ca2+ influx in primary cultures of neonatal rat ventricular myocytes within 5 s, but that after a 20-min pre-incubation period the phorbol ester markedly inhibits 45Ca2+ influx. The sequence of stimulation followed by inhibition is confirmed in cell-attached patch clamp recordings of single Ca2+ channel currents. The stimulatory effect is faster at 0 mV than at -40 mV, leading to the novel conclusion that the rate of protein kinase C activation is modulated by the state of the Ca2+ channel.     

A diacylglycerol analogue reduces neuronal calcium currents independently of protein kinase C activation

Diacylglycerol analogues (for example 1,2-oleoylacetylglycerol, OAG) and phorbol esters are activators of protein kinase C, and have been widely used to study the function of this enzyme in both intact cells and cell-free preparations. Electrophysiological studies have shown that these activators can either depress or increase Ca2+ currents, or decrease K+ currents when applied outside the cell. It has been assumed that these effects are mediated by protein kinase C activation. Here we report that micromolar levels of OAG and phorbol esters depress Ca2+ currents in chick sensory neurons independently of their effect as activators of protein kinase C. The depression of the Ca2+ current is rapid and is unaffected by intracellular application of the protein kinase C inhibitors staurosporin, sphingosine and H-7. Furthermore, the activators were ineffective when applied intracellularly, indicating that their site of action is on the outside of the membrane.     

Loss of mouse fibroblast cell response to phorbol esters restored by microinjected protein kinase C

The phorbol esters in addition to being among the most potent mouse skin tumour promoters profoundly affect many different biological systems. It is postulated that they act through activation of protein kinase C, but substantial heterogeneity in their pharmacological and binding behaviour in some systems has caused concern about whether this is their only target. Evidence linking protein kinase C activation with biological responses to the phorbol esters includes similarity in structure-activity relations for binding and response; in vitro phosphorylation of specific proteins by protein kinase C at the same sites at which phorbol ester treatment induces phosphorylation in intact cells; and correlation in certain cell types between down regulation of protein kinase C on chronic phorbol ester treatment and loss of cellular responsiveness to the phorbol ester. Here we report that microinjection of purified protein kinase C into Swiss 3T3 fibroblasts pretreated with the phorbol ester phorbol 12,13-dibutyrate (PDBu) restores the mitogenic response of the cells to PDBu, directly establishing the involvement of protein kinase C in this response.     

Phorbol ester facilitates 45Ca accumulation and catecholamine secretion by nicotine and excess K+ but not by muscarine in rat adrenal medulla

Several investigators have shown that tumour promoter phorbol esters mimic the effects of endogenous diacylglycerol to activate a second messenger, protein kinase C. These phorbol esters have proved to be valuable tools for exploring the role of protein kinase C in many cellular functions. We demonstrate here that secretion of catecholamines evoked from the rat adrenal gland by stimulation of splanchnic nerves, excess potassium (K+) and nicotine is facilitated by phorbol 12,13-dibutyrate. An inhibitor of protein kinase C, polymixin B, produced concentration-dependent inhibition of the evoked secretion, and the effect was reversed by the phorbol ester. Furthermore, we show that an increase in the accumulation of radioactively labelled calcium (45Ca) obtained in the adrenal medulla after stimulation with nicotinic agonists and excess K+ is further enhanced by phorbol ester. Muscarine-evoked secretion of catecholamines, which depends on mobilization of intracellularly bound Ca2+, was not associated with an increase in 45Ca2+ uptake, and phorbol ester did not facilitate either catecholamine secretion or 45Ca2+ accumulation. We suggest that protein kinase C is involved in the exocytotic secretion of catecholamines by regulating the influx of Ca2+ through voltage-sensitive and nicotine receptor-linked Ca2+ channels of rat chromaffin cells.     

Interleukin-2 stimulates association of protein kinase C with plasma membrane

Interleukin-2 (IL-2) is a regulatory peptide important for the growth and differentiation of antigen-specific T lymphocytes and large granular lymphocytes. Interaction of IL-2 with its specific receptor results in the promotion of S-phase progression as well as, in certain circumstances, the production and release of gamma-interferon (IFN-gamma). Although the binding of IL-2 with high-affinity specific receptors has been well characterized, the intracellular mechanisms by which this ligand-receptor interaction promotes growth and differentiation are unknown. Here, we present evidence that IL-2/receptor interaction produces a rapid and transient redistribution of protein kinase C (PK-C) from the cytosol to the plasma membrane. Phorbol myristate acetate (PMA) also induces PK-C transposition in an analogous manner, except that PMA-induced PK-C transposition to the plasma membrane is apparently protracted. As phorbol esters have been shown to mimic IL-2 in the regulation of cellular proliferation as well as IFN-gamma production, the activation of PK-C by either phorbol esters or IL-2/receptor interaction seems to have a crucial role in signal transduction elicited by these extracellular messengers.     

Allosteric modulation of the presynaptic Ca2+ sensor for vesicle fusion

Neurotransmitter release is triggered by an increase in the cytosolic Ca2+ concentration ([Ca2+]i), but it is unknown whether the Ca2+-sensitivity of vesicle fusion is modulated during synaptic plasticity. We investigated whether the potentiation of neurotransmitter release by phorbol esters, which target presynaptic protein kinase C (PKC)/munc-13 signalling cascades, exerts a direct effect on the Ca2+-sensitivity of vesicle fusion. Using direct presynaptic Ca2+-manipulation and Ca2+ uncaging at a giant presynaptic terminal, the calyx of Held, we show that phorbol esters potentiate transmitter release by increasing the apparent Ca2+-sensitivity of vesicle fusion. Phorbol esters potentiate Ca2+-evoked release as well as the spontaneous release rate. We explain both effects by an increased fusion 'willingness' in a new allosteric model of Ca2+-activation of vesicle fusion. In agreement with an allosteric mechanism, we observe that the classically high Ca2+ cooperativity in triggering vesicle fusion (approximately 4) is gradually reduced below 3 microM [Ca2+]i, reaching a value of <1 at basal [Ca2+]i. Our data indicate that spontaneous transmitter release close to resting [Ca2+]i is a consequence of an intrinsic property of the molecular machinery that mediates synaptic vesicle fusion.     

Activation of protein kinase C augments evoked transmitter release

In view of the emerging role of the phosphoinositide system in cellular communication we examined its involvement in quantal-transmitter release, which is a key element in synaptic transmission. Transmitter release is normally activated by an increase in intracellular calcium, achieved either by entry of calcium ions through the presynaptic membrane or by intracellular calcium liberation. One of the targets of the phosphoinositide signalling system is the enzyme protein kinase C (PKC), which can be activated experimentally by tumour promoting phorbol esters, including 12-O-tetradecanoylphorbol-13-acetate (TPA). Such activation of PKC may be implicated in transmitter release in two ways. First, phorbol esters were found to increase secretion and enhance calcium currents; it might therefore be expected that they would increase synaptic transmitter release. But phorbol esters also inhibit the calcium current in dorsal root ganglion neurones. We report that the phorbol ester TPA augments synaptic transmission at the neuromuscular junction by increasing transmitter liberation. Activation of PKC also depends synaptic depression.     

Positive feedback of glutamate exocytosis by metabotropic presynaptic receptor stimulation.

Glutamate is important in several forms of synaptic plasticity such as long-term potentiation, and in neuronal cell degeneration. Glutamate activates several types of receptors, including a metabotropic receptor that is sensitive to trans-1-amino-cyclopenthyl-1,3-dicarboxylate, coupled to G protein(s) and linked to inositol phospholipid metabolism. The activation of the metabotropic receptor in neurons generates inositol 1,4,5-trisphosphate, which causes the release of Ca2+ from intracellular stores and diacylglycerol, which activates protein kinase C. In nerve terminals, the activation of presynaptic protein kinase C with phorbol esters enhances glutamate release. But the presynaptic receptor involved in this protein kinase C-mediated increase in the release of glutamate has not yet been identified. Here we demonstrate the presence of a presynaptic glutamate receptor of the metabotropic type that mediates an enhancement of glutamate exocytosis in cerebrocortical nerve terminals. Interestingly, this potentiation of glutamate release is observed only in the presence of arachidonic acid, which may reflect that this positive feedback control of glutamate exocytosis operates in concert with other pre- or post-synaptic events of the glutamatergic neurotransmission that generate arachidonic acid. This presynaptic glutamate receptor may have a physiological role in the maintenance of long-term potentiation where there is an increase in glutamate release mediated by postsynaptically generated arachidonic acid.     

Protein kinase C regulation of the receptor-coupled calcium signal in histamine-secreting rat basophilic leukaemia cells

It has been proposed that protein kinase C mediates cellular responses evoked by external stimuli, leading to alterations in internal free calcium concentrations. We have shown previously that histamine-secreting rat basophilic leukaemia cells (RBL-2H3), which degranulate on aggregation of the receptors for immunoglobulin IgE, contain a Ca2+- and phospholipid-dependent protein kinase (kinase C). The partially purified enzyme is activated directly by the tumour-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). In intact RBL cells, TPA potentiates histamine release induced by the Ca2+-ionophore A23187 (similar to the synergy reported for platelets, neutrophils and rat peritoneal mast cells). Although TPA at concentrations below 15 nM synergizes with the antigen, higher TPA concentrations inhibit secretion. This selective inhibition suggested that kinase C is involved in both the activation and termination of the secretory process. To examine this possibility, we have determined the effect of TPA on changes in free cytosolic Ca2+ concentration during antigen-induced release. We report here that TPA completely blocks the increase in Ca2+ concentration induced by antigen. Our results strongly suggest that protein kinase C is involved in the regulation of receptor-dependent Ca2+ signalling.     

Application of phorbol ester to mouse skin causes a rapid and sustained loss of protein kinase C

It is now widely accepted that tumour-promoting phorbol esters activate a Ca2+- and phospholipid-dependent protein kinase (protein kinase C) both in vitro and in intact cells, and that the kinase represents a major cellular phorbol ester-binding protein. The phorbol esters act as analogues of diacylglycerol, a natural regulator of protein kinase C, and stabilize the membrane-association of the kinase. Although other molecular targets may exist, protein kinase C activation is probably important in mediating the diverse responses of cultured cells to phorbol esters and in promoting in vivo tumours. The enzyme comprises a family of closely related proteins and has been detected in extracts from mouse epidermal cells, the likely targets for two-stage carcinogenesis in mouse skin. In this report we show that application of a single dose of TPA (12-O-tetradecanoyl phorbol-13-acetate) to mouse skin results in a rapid and complete loss of protein kinase C activity which is maintained for 3-4 days. This is associated with a loss of immunologically detectable protein kinase C and the accumulation of a smaller protein detectable by antibodies recognizing the regulatory domain of protein kinase C.     

Elevated levels of diacylglycerol and decreased phorbol ester sensitivity in ras-transformed fibroblasts

Diacylglycerol (DG) plays a central role in phospholipid metabolism and is an endogenous activator of protein kinase C. We have suggested that constitutive activation of this kinase is one mechanism by which oncogenes transform cells. The ras-encoded proteins are similar to regulatory G-proteins and are candidates for the unknown G-protein that modulates phosphatidylinositol (PI) turnover. Differences in polyphosphoinositide metabolism have been reported for ras-transformed cells. But because these experiments were performed on confluent cultures of established cell lines, the differences are difficult to attribute to ras transformation. Here we show that exponentially growing NIH 3T3 fibroblasts recently transformed by Ha-ras or Ki-ras possess elevated DG concentrations without significant alterations in the levels of other polyphosphoinositide metabolites. The basal phosphorylation of protein kinase C substrate of relative molecular mass (Mr) 80,000 (80K) is significantly increased in all the ras-transformed cell lines. Surprisingly, however, further phosphorylation of this protein on addition of phorbol ester was greatly reduced. Ha-ras cells also show less binding of phorbol ester than control cells, suggesting that elevation of DG causes partial down-regulation in addition to activation of protein kinase C.     

Two roles for guanine nucleotides in the stimulus-secretion sequence of neutrophils

The term 'stimulus-secretion coupling' has, since first enunciated, been held to involve the mobilization of cytosol Ca2+, which in turn is sufficient to trigger exocytotic secretory processes in metabolically competent cells. However, recent studies on a wide range of secretory cell types indicate that a role for Ca2+ can be obviated: examples are stimulation with phorbol ester (phorbol myristate acetate, PMA) which causes the activation of protein kinase C or the stimulation of platelets with collagen. Ca2+-independent exocytosis also occurs when analogues of GTP are injected through the lumen of patch pipettes directly into the cytosol of mass cells. The results presented here suggest that GTP analogues can activate secretory processes by actions at two distinct locations: one may be at the level of the receptor involving the activation of polyphosphoinositide (PPI) phosphodiesterase with consequent liberation of diacylglycerol (DG); the other involves direct activation of the exocytotic mechanism. These conclusions are based on measurements of exocytotic secretion from permeabilized neutrophils into which we have been able to introduce, individually and in combination, Ca2+ chelators (EGTA and BAPTA), Ca2+ (buffered at micromolar concentrations with EGTA), analogues of GTP and GDP and the direct activator of protein kinase C, PMA.     

Bidirectional control of cytosolic free calcium by thyrotropin-releasing hormone in pituitary cells

It is now established that a key step in the action of calcium-mobilizing agonists is stimulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) to 1,2-diacylglycerol and inositol 1,4,5-trisphosphate (InsP3). The latter substance acts as a second messenger, controlling the release of calcium from intracellular stores (see ref. 3 for review). The bifurcating nature of the signalling system is exemplified by the fact that the other product of PtdIns(4,5)P2 hydrolysis, 1,2-diacylglycerol, can alter cellular function by activating protein kinase C, the cellular target for several tumour-promoting agents such as the phorbol esters. In various tissues, including GH3 pituitary tumour cells, a synergistic interaction between calcium ions and protein kinase C underlies agonist-induced changes in cell activity. The data presented here suggest that when GH3 cells are stimulated by thyrotropin-releasing hormone (TRH), an agonist inducing PtdIns(4,5)P2 hydrolysis, the two limbs of the inositol lipid signalling system interact to control free cytosolic calcium levels [( Ca2+]i). At low levels of TRH receptor occupancy, [Ca2+]i increases rapidly, then declines relatively slowly. As receptor occupancy increases, the calcium signal becomes more short-lived due to the appearance of a second, inhibitory, component. This latter component, which is enhanced when [Ca2+]i is elevated by high potassium depolarization, is mimicked by active phorbol esters and by bacterial phospholipase C. It seems likely that protein kinase C subserves a negative feedback role in agonist-induced calcium mobilization.     

Activation of chloride channels in normal and cystic fibrosis airway epithelial cells by multifunctional calcium/calmodulin-dependent protein kinase.

Cystic fibrosis is associated with defective regulation of apical membrane chloride channels in airway epithelial cells. These channels in normal cells are activated by cyclic AMP-dependent protein kinase and protein kinase C. In cystic fibrosis these kinases fail to activate otherwise normal Cl- channels. But Cl- flux in cystic fibrosis cells, as in normal cells, can be activated by raising intracellular Ca2+ (refs 5-10). We report here whole-cell patch clamp studies of normal and cystic fibrosis-derived airway epithelial cells showing that Cl- channel activation by Ca2+ is mediated by multifunctional Ca2+/calmodulin-dependent protein kinase. We find that intracellular application of activated kinase and ATP activates a Cl- current similar to that activated by a Ca2+ ionophore, that peptide inhibitors of either the kinase or calmodulin block Ca2(+)-dependent activation of Cl- channels, and that a peptide inhibitor of protein kinase C does not block Ca2(+)-dependent activation. Ca2+/calmodulin activation of Cl- channels presents a pathway with therapeutic potential for circumventing defective regulation of Cl- channels in cystic fibrosis.     

Activation of Ca2+ entry into acinar cells by a non-phosphorylatable inositol trisphosphate.

In many cell types, receptor activation of phosphoinositidase C results in an initial release of intracellular Ca2+ stores followed by sustained Ca2+ entry across the plasma membrane. Inositol 1,4,5-trisphosphate is the mediator of the initial Ca2+ release, although its role in the mechanism underlying Ca2+ entry remains controversial. We have now used two techniques to introduce inositol phosphates into mouse lacrimal acinar cells and measure their effects on Ca2+ entry: microinjection into cells loaded with Fura-2, a fluorescent dye which allows the measurement of intracellular free calcium concentration by microspectrofluorimetry, and perfusion of patch clamp pipettes in the whole-cell configuration while monitoring the activity of Ca(2+)-activated K+ channels as an indicator of intracellular Ca2+. We report here that inositol 1,4,5-trisphosphate serves as a signal that is both necessary and sufficient for receptor activation of Ca2+ entry across the plasma membrane in these cells.     

Cross-talk between cellular signalling pathways suggested by phorbol-ester-induced adenylate cyclase phosphorylation

Receptor-mediated activation of both adenylate cyclase and phosphatidylinositide hydrolysis systems occurs through guanine nucleotide regulatory proteins and ultimately leads to specific activation of either cyclic AMP-dependent protein kinase A or Ca2+/phospholipid-dependent protein kinase C. Given the remarkable diversity of agents that influence cellular metabolism through these pathways and the similarities of their components, interactions between the two signalling systems could occur. In fact, stimulation of cells with 12-O-tetradecanoyl phorbol-13-acetate (TPA), a phorbol ester that activates protein kinase C, influences hormone-sensitive adenylate cyclase. In some cells TPA induces desensitization of receptor-mediated stimulation of adenylate cyclase, whereas in others, such as frog erythrocytes, phorbol ester treatment results in increased agonist-stimulated as well as basal, guanine nucleotide- and fluoride ion-stimulated adenylate cyclase activities. We show here that TPA produces phosphorylation of the catalytic unit of adenylate cyclase in frog erythrocytes. Moreover, purified protein kinase C can directly phosphorylate in vitro the catalytic unit of adenylate cyclase purified from bovine brain. These results suggest that phosphorylation of the catalytic unit of adenylate cyclase by protein kinase C may be involved in the phorbol ester-induced enhancement of adenylate cyclase activity. In addition to providing the first direct demonstration of a covalent modification of the catalytic unit of adenylate cyclase, these results provide a potential biochemical mechanism for a regulatory link between the two major transmembrane signalling systems.     

Insulin stimulation of glucose uptake can be mediated by diacylglycerol in adipocytes

An early effect of insulin in adipocytes is to stimulate glucose uptake. The increased uptake appears to be due to mobilization of glucose transporters from an intracellular location to the plasma membrane and to enhanced intrinsic activity of the transporters. Little is known about the insulin-generated signals causing these changes. Phorbol esters have been shown to mimic the insulin effect, but phosphorylation of the transporter does not seem to be involved. A phospho-oligosaccharide was recently shown to mimic the effects of insulin on protein phosphorylation, suggesting that it could be a mediator for some intracellular metabolic effects of the hormone, but it did not affect glucose uptake. A diacyglycerol is produced in the plasma membrane in conjunction with the generation of the phospho-oligosaccharide. Here I show that added 1,2-diacylglycerols potently increase glucose transporter-mediated uptake of glucose in rat adipocytes, but without activation of protein kinase C.