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.     

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.     

Role of intracellular calcium mobilization in the regulation of protein kinase C-mediated membrane processes

Phorbol esters are potent tumour-promoting agents that exert pleiotropic effects on cells. Among these are the control of growth, stimulation of release of stored bioactive constituents and regulation of growth-factor surface receptors. Phorbol esters bind to and activate protein kinase C, leading to the phosphorylation of specific protein substrates presumed to be necessary for eliciting the full response. Strong evidence exists that specific binding of tumour promoter occurs at the membrane level in intact cells, resulting in activation of protein kinase C. Recent evidence concerning the release of bioactive constituents from platelets and neutrophils has linked agonist-induced protein kinase C activation and Ca2+ mobilization in a synergistic mechanism. Here we present a novel model of synergism between Ca2+ and phorbol esters that leads to transferrin receptor phosphorylation and down-regulation in HL-60 human leukaemic cells. Raising intracellular Ca2+, although ineffective by itself, increases the potency and rate of action of phorbol ester for activating protein kinase C and mediating transferrin receptor phosphorylation and down-regulation. We propose a molecular model in which increased intracellular Ca2+ recruits protein kinase C to the plasma membrane, thus "priming' the system for activation by phorbol ester.     

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.     

Cholecystokinin induces a decrease in Ca2+ current in snail neurons that appears to be mediated by protein kinase C

Three distinct classes of protein kinases have been shown to regulate Ca2+ current in excitable tissues. Cyclic AMP-dependent protein kinase mediates the action of noradrenaline on the Ca2+ current of cardiac muscle cells. Cyclic GMP-dependent protein kinase mediates the serotonin-induced modulation of the Ca2+ current in identified snail neurons. The Ca2+/diacylglycerol-dependent protein kinase (protein kinase C) has also been found to regulate Ca2+ currents of neurons. However, no neurotransmitter has yet been shown to regulate Ca2+ current through the activation of protein kinase C. We now report that cholecystokinin, a widely occurring neuropeptide which is present in molluscan neuron, modulates the Ca2+ current in identified neurons of the snail Helix aspersa, and that this effect appears to be mediated by protein kinase C. Specifically, sulphated cholecystokinin octapeptide 26-33 (CCK8), activators of protein kinase C, and intracellular injection of protein kinase C, all shorten the Ca2+-dependent action potential and decrease the amplitude of the Ca2+ current in these cells. All these effects are not reversible within the duration of the experiments. Moreover, intracellular injections of low concentrations of protein kinase C, which are ineffective by themselves, enhance the effectiveness of low concentrations of CCK8 on the Ca2+ current.     

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.     

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.     

Cyclic nucleotides control a system which regulates Ca2+ sensitivity of platelet secretion

Cellular responses to extracellular signals are mediated by changes in the intracellular concentrations of one or more second messengers. In platelets, inhibitory agonists increase intracellular cyclic-3',5'-AMP [( cyclic AMP]i (refs 2, 3] whereas excitatory agonists increase [Ca2+]i and/or [1,2-diacylglycerol]i (refs 4-9), and in some cases decrease [cyclic AMP]i (refs 10, 11). Both activation and inhibition of platelet responses have been attributed to an increase in [cyclic-3',5'-GMP]i (refs 8, 12). The activity of protein kinase C, which is associated with the platelet secretory response, is increased by both 1,2-diacylglycerol and Ca2+ (refs 4, 7, 8). The role of cyclic AMP may involve either inhibition of Ca2+ mobilization to the cytosol or stimulation of intracellular Ca2+ uptake, and in addition inhibition of 1,2-diacylglycerol formation. The relationship between cyclic-3',5'-GMP (cyclic GMP) and other second messengers in platelet activation has not been defined. Using platelets made permeable by exposure to an intense electric field, we demonstrate here modulation of the Ca2+ sensitivity of platelet secretion by thrombin, and by 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1-oleyl-2- acetylglycerol ( OAG ), both potent activators of protein kinase C. The effect of thrombin is selectively modified by cyclic GMP and cyclic AMP. The response to OAG and TPA is also modulated by cyclic AMP but to a much lesser extent.     

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.     

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.     

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.     

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.     

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.     

Phorbol ester and diacylglycerol mimic growth factors in raising cytoplasmic pH

There is now good evidence that cytoplasmic pH (pHi) may have an important role in the metabolic activation of quiescent cells. In particular, growth stimulation of mammalian fibroblasts leads to a rapid increase in pHi (refs 3-6), due to activation of a Na+/H+ exchanger in the plasma membrane, and this alkalinization is necessary for the initiation of DNA synthesis. However, the mechanism by which mitogens activate the Na+/H+ exchanger to raise pHi is not known, although an increase in cytoplasmic free Ca2+ ([Ca2+]i) has been postulated as the primary trigger. We now present data suggesting that the Na+/H+ exchanger is set in motion through protein kinase C, a phospholipid- and Ca2+-dependent enzyme normally activated by diacylglycerol produced from inositol phospholipids in response to external stimuli. Using newly developed pH microelectrodes and fluorimetric techniques, we show that a tumour promoting phorbol ester and synthetic diacylglycerol, both potent activators of kinase C (refs 12-15), mimic the action of mitogens in rapidly elevating pHi in different cell types. Furthermore, we demonstrate that, contrary to previous views, an early rise in [Ca2+]i is not essential for the activation of Na+/H+ exchange and the resultant increase in pHi. Finally, we suggest that an alkaline pHi shift, mediated by Na+/H+ exchange, may be a common signal in the action of those hormones which elicit the breakdown of inositol phospholipids.     

Phorbol esters block a voltage-sensitive chloride current in hippocampal pyramidal cells

The importance of second-messenger systems in controlling the excitability of neurones and other cells, through modulation of voltage- and calcium-dependent ionic conductances, has become increasingly clear. Cyclic AMP, acting via protein kinase A, has been identified as the second messenger for several neurotransmitters, and recent studies have suggested that activation of protein kinase C may have similar modulatory actions on neurones. Calcium and potassium currents have so far been shown to be the major ionic conductances modified by kinase activation. We now report that hippocampal pyramidal cells contain a previously undescribed voltage-dependent chloride current which is active at resting potential and is turned off either by membrane depolarization or by activation of protein kinase C by phorbol esters. We propose that this current may reside predominantly in the cell's dendritic membrane and thereby may regulate dendritic excitability.     

Multiple intracellular signallings involved in regulation of on channels by GH releasing or inhibitory hormones in pituitary somatotropes

Influx of Ca2-via Ca2+ channels is the major step triggering exocytosis of pituitary somatotropes to release growth hormone (GH). Voltage-gated Ca2+ and K+ channels, the primary determinants of the influx of Ca2+ in somatotropes, are regulated by GH-releasing hornone (GHRH) and somatostatin (SRIF) through G protein-coupled signalling systems. Using whole-cell patch-clamp techniques, the changes of the Ca2+ and K+ currents in primary cultured somatotropes were recorded and signalling systems were studied using pharmacological reagents and intracellular dialysis of non-permeable molecules including antibodies and antisense oligonucleotides. GHRH increased both L-and T-types Ca2+ currents and decreased transient (I4) and delayed rectified (Ik) K+ currents. The increase in Ca2+ currents by GHRH was mediated by cAMP/protein kinase A system but the decrease in K+ currents required normal function of protein kinase C system. The GHRH-induced alteration of Ca2+ and K+ currents augments the influx of Ca2+ , leading to an increase in the [ Ca2+ ]I and the GH secretion. In contrary, a significant reduction in Ca2+ currents and increase in K currents were obtained in response to SRIF. The ion channel response to SRIF was demonstrated as a membrane delimited pathway and can be recorded by classic whole-cell configuration, Intracellular dialysis of anti-αi3 antibodies attenuated the increase in K + currents by SRIF whereas anti-αo antibodies blocked the reduction in the Ca2+ current by SRIF. Dialysis of antisense oligonucleotides specific for αo2 sub-units also attenuated the inhibition of SRIF on the Ca2+current. The Gi3 protein mediated the increase in K + currents and the Go2 protein mediated the reduction in the Ca2 +current by SRIF. The SRIF-induced alteration of Ca2 + and K + currents diminished the influx of Ca2+ , leading to a decrease in the [ Ca2+ ]I and the GH secretion. It is therefore concluded that multiple signalling systems are employed in the ion channel response to GHRH or SRIF in somatotropes, which leads to an increase or decrease in the GH secretion.     

Voltage-dependent phosphorylation may recruit Ca2+ current facilitation in chromaffin cells.

Bovine chromaffin cells have two components of whole-cell Ca2+ current: 'standard' Ca2+ currents that are activated by brief depolarizations, and 'facilitation' Ca2+ currents, which are normally quiescent but can be activated by large pre-depolarizations or by repetitive depolarizations to physiological potentials. The activation of protein kinase A can also stimulate Ca2+ current facilitation, indicating that phosphorylation can play a part in facilitation. Here we investigate the role of protein phosphorylation in the recruitment of facilitation Ca2+ currents by pre-pulses or repetitive depolarizations. We find that recruitment of facilitation by depolarization is a rapid first-order process which is suppressed by inhibitors of protein phosphorylation or by injection of phosphatase 2A into cells. Recruitment of facilitation Ca2+ current by voltage is normally reversible but phosphatase inhibitors render it irreversible. Our results indicate that recruitment of these Ca2+ currents by pre-pulses or repetitive depolarizations involves voltage-dependent phosphorylation of the facilitation Ca2+ channel or a closely associated regulatory protein. Voltage-dependent phosphorylation may therefore be a mechanism by which membrane potential can modulate ion channel activity.     

Protein kinase C activation induces conductance changes in Hermissenda photoreceptors like those seen in associative learning

Phosphorylation of ion channels has been suggested as one molecular mechanism responsible for learning-produced long-term changes in neuronal excitability. Persistent training-produced changes in two distinct K+ currents (IA (ref. 2), IK-Ca (refs 3,4)) and a voltage-dependent calcium current (ICa; refs 3,4) have previously been shown to occur in type B photoreceptors of Hermissenda, as a result of associative learning. But the identity of the phosphorylation pathway(s) responsible for these changes has not as yet been determined. Injections of cyclic AMP-dependent protein kinase reduce a K+ current (IK) in B cells which is different from those changed by training, but fails to reduce IA and IK-Ca. Phosphorylase b kinase (an exogenous calcium/calmodulin-dependent kinase) reduces IA, but whether IK-Ca and ICa are changed in the manner of associative training is not yet known. Another protein kinase present in high concentrations in both mammalian brain and molluscan nervous systems is protein kinase C, which is both calcium- and phospholipid-sensitive. We now present evidence that activation of protein kinase C by the tumour promoter phorbol ester (PDB) and intracellular injection of the enzyme induce conductance changes similar to those caused by associative training in Hermissenda B cells (that is a reduction of IA and IK-Ca, and enhancement of ICa). These results represent the first direct demonstration that protein kinase C affects membrane K+ ion conductance mechanisms.