Role of Ca2+ influx in bombesin-induced mitogenesis in Swiss 3T3 fibroblasts

In Swiss 3T3 fibroblasts a peptide mitogen bombesin, which acts through the phospholipase C-protein kinase C signaling pathway, stimulates DNA synthesis in a manner strictly dependent on the medium calcium concentration: [3H]thymidine incorporation into DNA in the presence of a saturating concentration of bombesin (10(-8) M) is 4-fold greater at 3.0 mM extracellular calcium as compared with a value obtained at 0.03 mM calcium. In the present study we attempted to identify the site and the mechanism of action of Ca2+ influx along the bombesin-induced mitogenic signaling pathway, by comparing bombesin effects at 0.03 and 3.0 mM of medium calcium. Bombesin induces the same extent of increases in [3H]inositol phosphates after 1 min, and comparable sustained increases in the cellular content of 1,2-diacylglycerol for up to 4 h, at either 0.03 or 3.0 mM calcium. Bombesin induces the same extent of phosphorylation of MARCKS protein, the major cellular substrate for protein kinase C, irrespective of the medium calcium concentration for at least 4 h. Moreover, diverse cellular responses elicited by bombesin, including c-fos expression, activation of microtubule-associated protein 2 kinase and S6 kinase, glucose uptake, and protein synthesis but not the release of arachidonic acid and its metabolites, are induced similarly at either 0.03 or 3.0 mM calcium. Down-regulation of cellular protein kinase C nearly completely abolishes bombesin effects on c-fos expression, S6 kinase activation, glucose uptake, and DNA synthesis. These results suggest that the target of Ca2+ influx in bombesin-induced mitogenic signaling pathway is not located along the phospholipase C-protein kinase C signal transduction system including cellular events in early G1 phase that exist downstream to protein kinase C action.     

The neurokinin A receptor activates calcium and cAMP responses through distinct conformational states.

G protein-coupled receptors are thought to mediate agonist-evoked signal transduction by interconverting between discrete conformational states endowed with different pharmacological and functional properties. In order to address the question of multiple receptor states, we monitored rapid kinetics of fluorescent neurokinin A (NKA) binding to tachykinin NK2 receptors, in parallel with intracellular calcium, using rapid mixing equipment connected to real time fluorescence detection. Cyclic AMP accumulation responses were also monitored. The naturally truncated version of neurokinin A (NKA-(4-10)) binds to the receptor with a single rapid phase and evokes only calcium responses. In contrast, full-length NKA binding exhibits both a rapid phase that correlates with calcium responses and a slow phase that correlates with cAMP accumulation. Furthermore, activators (phorbol esters and forskolin) and inhibitors (Ro 31-8220 and H89) of protein kinase C or A, respectively, exhibit differential effects on NKA binding and associated responses; activated protein kinase C facilitates a switch between calcium and cAMP responses, whereas activation of protein kinase A diminishes cAMP responses. NK2 receptors thus adopt multiple activatable, active, and desensitized conformations with low, intermediate, or high affinities and with distinct signaling specificities.     

A study on the role of protein kinase C and intracellular calcium in the activation of superoxide generation

Accumulating evidence indicates that protein kinase C plays an essential role in the activation of NADPH oxidase. In the present study, the correlation between superoxide generation, intracellular calcium, activation of purified protein kinase C and stabilized membrane-bound protein kinase C was studied. Phorbol 12-myristate 13-acetate (PMA) and 1-deacyl-2-acetyl-rac-glycerol (OAG) were found to induce equal activation of purified protein kinase C and translocation of protein kinase C to the membrane fraction, but differed significantly in their ability to induce superoxide generation. Intracellular calcium was varied using calcium ionophores and increasing the intracellular calcium concentration to more than 1 microM was found to induce increased superoxide generation in maximally OAG-stimulated cells; this contrasted to maximally PMA-stimulated leukocytes. Ionomycin and A23187 were both found to induce a translocation of protein kinase C to the membrane fraction. This translocation was highly dependent upon extracellular calcium. In contrast, PMA- and OAG-induced translocation of protein kinase C was not dependent upon extracellular calcium. In conclusion, our results indicate that although PMA, OAG and calcium ionophores seem to activate protein kinase C in human polymorphonuclear leukocytes these activators differ in their ability to induce superoxide generation.     

Calcium influx through L-type channels is required for selective activation of extracellular signal-regulated kinase by gonadotropin-releasing hormone.

The hypothalamic decapeptide gonadotropin-releasing hormone stimulates mobilization of two discrete pools of calcium in clonal (alphaT3-1) and primary pituitary gonadotropes. A multidisciplinary approach was implemented to investigate the effects of discrete calcium fluctuations on the signaling pathways linking the gonadotropin-releasing hormone receptor to activation of mitogen-activated protein kinases and immediate early genes. Blockade of calcium influx through nifedipine-sensitive voltage-gated calcium channels reduced buserelin-induced activation of extracellular signal-regulated kinase (ERK) and c-Fos while activation of c-Jun N-terminal kinase and c-Jun was unaffected. Inhibition of buserelin-stimulated ERK activity by nifedipine was also observed in rat pituitary cells in primary culture. Direct activation of alphaT3-1 cell L-type calcium channels with the agonist Bay-K 8644 resulted in phosphorylation of ERK and induction of c-Fos. However, simple voltage-induced channel activation did not produce a sufficient calcium signal, since depolarization with 35 mM KCl failed to induce activation of ERK. Depletion of intracellular calcium stores with thapsigargin did not affect buserelin-induced ERK activation. An inhibitor of protein kinase C decreased calcium influx through nifedipine-sensitive calcium channels and phosphorylation of ERK induced by buserelin. Pharmacological inhibition of protein kinase C did not block Bay-K 8644-induced ERK activation. These observations suggest that calcium influx through L-type channels is required for GnRH-induced activation of ERK and c-Fos and that the influence of calcium lies downstream of protein kinase C.     

Proteolytic degradation of protein kinase C in the phorbol ester-induced interleukin-2 secreting thymoma cells

Effects of phorbol 12-myristate 13-acetate (PMA) on the fate of protein kinase C in two mouse thymoma cell lines, which are either responsive (EL4) or unresponsive (IEL4) to PMA-induced interleukin-2 (IL-2) production, were investigated with polyclonal antibodies raised against rat brain enzyme. These antibodies immunoprecipitated completely the protein kinase C from both cell lines and detected mainly an 82-kDa protein by immunoblot analysis of the crude homogenates as well as the partially purified kinase preparations. PMA elicited a time- and dose-dependent redistribution of protein kinase C from cytosol to the particulate fraction and proteolytic degradation of the kinase from both cell lines. The dose of PMA required for half-maximum protein kinase C translocation and degradation was at least five times lower for EL4 than for IEL4. In the presence of 16 nM PMA the rates of protein kinase C translocation and degradation were faster in EL4 than in IEL4, and the half-lives of protein kinase C in EL4 and IEL4 were less than 5 min and greater than 2 h, respectively. Analysis of the tryptic fragments of the immunoprecipitated enzyme, previously phosphorylated in the presence of [gamma-32P]ATP, revealed minor structural differences between the protein kinase C from these two cell lines. In neither cell line did the PMA-induced degradation of protein kinase C result in an accumulation of the Ca2+/phospholipid-independent kinase (catalytic unit) as analyzed by immunoblotting and gel filtration chromatography. Thus, activation of protein kinase C through the proteolytic conversion to the effector-independent catalytic unit plays little role in IL-2 production. The role of protein kinase C translocation and degradation in the PMA-induced responses in EL4 cells is unknown. However, IL-2 production in EL4 cells was reduced when PMA-induced degradation of protein kinase C was retarded by exogenously added protease inhibitors.     

Activation of Protein Kinase C in Permeabilized Human Neuroblastoma SH-SY5Y Cells

The activation of protein kinase C was investigated in digitonin-permeabilized human neuroblastoma SH-SY5Y cells by measuring the phosphorylation of the specific protein kinase C substrate myelin basic protein4-14. The phosphorylation was inhibited by the protein kinase C inhibitory peptide PKC19-36 and was associated to a translocation of the enzyme to the membrane fractions of the SH-SY5Y cells. 1,2-Dioctanoyl-sn-glycerol had no effect on protein kinase C activity unless the calcium concentration was raised to concentrations found in stimulated cells (above 100 nM). Calcium in the absence of other activators did not stimulate protein kinase C. Phorbol 12-myristate 13-acetate was not dependent on calcium for the activation or the translocation of protein kinase C. The induced activation was sustained for 10 min, and thereafter only a small net phosphorylation of the substrate could be detected. Calcium or dioctanoylglycerol, when applied alone, only caused a minor translocation, whereas in combination a marked translocation was observed. Arachidonic acid (10 microM) enhanced protein kinase C activity in the presence of submaximal concentrations of calcium and dioctanoylglycerol. Quinacrine and p-bromophenacyl bromide did not inhibit calcium- and dioctanoylglycerol-induced protein kinase C activity at concentrations which are considered to be sufficient for phospholipase A2 inhibition.     

Activation of protein kinase C by naturally occurring ether-linked diglycerides

Recent studies have demonstrated that ether-linked diglycerides are endogenous constituents of biologic tissues and accumulate during agonist stimulation (Daniel, L. W., Waite, M., and Wykle, R. L. (1986) J. Biol. Chem. 261, 9128-9132) and myocardial ischemia (Ford, D. A., and Gross, R. W. (1989) Circ. Res. 64, 173-177). Although protein kinase C previously had been thought to specifically require 1,2-diacyl-sn-glycerol (DAG) molecular species for activation, the present study demonstrates that purified rat brain protein kinase C is activated by naturally occurring ether-linked diglycerides (e.g. 1-O-hexadec-1'-enyl-2-octa-dec-9'-enoyl-sn-glycerol and 1-O-hexadecyl-2-octa-dec-9'-enoyl-sn-glycerol) with a similar dose response curve to that for DAG molecular species. Although in vitro assays demonstrated that DAG could partially activate protein kinase C in the absence of free calcium, activation by ether-linked diglycerides required free calcium concentrations found only in stimulated cells (greater than 1 microM [Ca2+]free). To substantiate these findings the alpha and beta isoforms of protein kinase C from rat brain cortical grey matter were resolved by hydroxylapatite chromatography. Although the beta isoform of protein kinase C was substantially activated by DAG in the absence of free calcium, activation by ether-linked diglycerides had an absolute requirement for physiologic increments in free calcium ion found in stimulated cells. Since ether lipids are localized in specific subcellular membrane compartments, accumulate during several pathophysiologic perturbations and are effective activators of protein kinase C with separate and distinct calcium requirements in comparison to DAG, these results suggest that ether-linked diglycerides are important and potentially specific biologic activators of one or more isoforms of protein kinase C.     

Activation of protein kinase C potentiates cyclic AMP production and stimulates steroidogenesis in differentiated ovarian granulosa cells

Gonadotropin-stimulated steroidogenesis in the differentiating ovarian granulosa cell is mediated through the activation of cAMP-dependent protein kinase, and is also modulated by calcium-dependent mechanisms. Granulosa cells contain calcium-activated, phospholipid-dependent protein kinase (C kinase), and show an increase in phosphatidylinositol turnover in response to GnRH agonist analogs. To evaluate the role of C kinase in ovarian steroidogenesis, the potent phorbol ester, TPA, and the permeant diacylglycerol, OAG, were used to activate C kinase in granulosa cells from PMSG-treated immature rats. Both TPA and OAG caused dose-dependent stimulation of progesterone production without affecting intra- or extracellular cAMP levels. However, the maximum steroid responses to these compounds were less than those stimulated by cAMP. The ED50 for TPA-stimulated progesterone production was 3 nM, which is close to the known Km for activation of C kinase. Stimulation of steroidogenesis was only observed with biologically-active phorbol esters and permeant diacylglycerols such as OAG and DOG. Exposure of granulosa cells to phospholipase C also increased progesterone production in a dose-dependent manner without changing the cAMP content. Although TPA and OAG did not increase basal cAMP production, both agents enhanced the cAMP responses stimulated by hCG and forskolin; likewise, phospholipase C alone did not change cAMP production but caused a dose-dependent increase in the cAMP responses to hCG and forskolin. These results demonstrate that activation of C kinase promotes steroidogenesis in ovarian granulosa cells, and potentiates the activation of adenylate cyclase by hCG and forskolin. Such findings support the possibility that the calcium, phospholipid-dependent enzyme could be involved in the regulation of progesterone production by hormonal ligands such as gonadotropins and GnRH.     

Phorbol esters regulate CD2- and CD3-mediated calcium responses in peripheral blood-derived human T cells

The purpose of the present study was to examine the effect of protein kinase C (pkC) activation on calcium responses generated through the CD3 and CD2 Ag in both normal peripheral blood-derived T lymphocytes and the leukemic T cell line Jurkat. The data reveal a major difference with respect to the regulation of receptor-mediated calcium responses in these two cells. Thus, the pkC activator phorbol-12,13-dibutyrate (Pdbu) enhances calcium responses induced via CD3 and CD2 molecules in normal T cells by accelerating the rate of elevation of intracellular calcium levels and increasing the maximum change in calcium concentration achieved. In contrast, Pdbu inhibits both CD3- and CD2-induced calcium responses in Jurkat cells. Pdbu does not influence calcium responses generated by the guanine nucleotide-binding protein activator, aluminium fluoride, indicating that the effect of pkC occurs at a point proximal to a guanine nucleotide-binding protein regulation of T cell calcium responses.     

Prolactin stimulation of ornithine decarboxylase activity in the mammary gland may involve an activation of protein kinase C

Phorbol myristate acetate (TPA), a protein kinase C activator, stimulates ornithine decarboxylase (ODC) activity in mammary gland explants derived from 12-14 day pregnant mice. The calcium ionophore A23187 similarly stimulates ODC activity. Maximally stimulatory concentrations of TPA and A-23187 produce additive responses. The prolactin (PRL) stimulation of ODC activity is nonadditive to that caused by TPA, A23187 or TPA plus A23187. These observations are compatible with the thesis that the stimulation of ODC activity by PRL may occur via an activation of protein kinase C.     

Intracellular pH on Protein Kinase C and Ionomycin Potentiation of Isoproterenol-Stimulated Cyclic AMP and Cyclic GMP Production in Rat Pinealocytes

In rat pinealocytes, alpha 1-adrenergic activation, which leads to cytoplasmic alkalinization, also potentiates the beta-adrenergic stimulated cyclic AMP (cAMP) and cyclic GMP (cGMP) responses. Both elevation of intracellular calcium ([Ca2+]i) and activation of protein kinase C are involved in the potentiation mechanism. Recently, intracellular pH has also been found to modulate the adrenergic-stimulated cyclic nucleotide responses, suggesting intracellular pH may also affect the potentiation mechanism. This possibility was examined in the present study. Cytoplasmic alkalinization by ammonium chloride had an enhancing effect on the isoproterenol and ionomycin-stimulated cAMP and cGMP accumulation. In comparison, cytoplasmic acidification by sodium propionate reduced the isoproterenol and ionomycin-stimulated cAMP and cGMP responses. Direct measurement of [Ca2+]i indicated that neither ammonium chloride nor sodium propionate had an effect on the ionomycin-stimulated elevation of [Ca2+]i, suggesting their effects on cyclic nucleotide responses may be independent of [Ca2+]i. In cells stimulated by isoproterenol and an activator of protein kinase C, ammonium chloride had an enhancing effect on both cAMP and cGMP responses, whereas sodium propionate had no effect. Taken together, these results suggest that a site distal to elevation of [Ca2+]i and activation of protein kinase C, of importance to the potentiation mechanism, is modulated by intracellular pH.     

Norepinephrine- and phorbol ester-induced phosphorylation of alpha(1a)-adrenergic receptors. Functional aspects

Maximal adrenergic responses in Rat-1 fibroblasts expressing alpha(1a)-adrenergic receptors are not blocked by activation of protein kinase C. In contrast, activation of protein kinase C induces the phosphorylation of alpha(1b)-adrenoreceptors and blocks their actions. The effect of norepinephrine and phorbol esters on alpha(1a)-adrenoreceptor phosphorylation and coupling to G proteins were studied. Both stimuli lead to dose-dependent receptor phosphorylation. Interestingly, protein kinase C activation affected to a much lesser extent the actions of alpha(1a)-adrenergic receptors than those of the alpha(1b) subtype (norepinephrine elicited increases in calcium in whole cells and [(35)S]GTPgammaS binding to membranes). Basal phosphorylation of alpha(1a)-adrenergic receptors was much less than that observed with the alpha(1b) subtype. The carboxyl terminus seems to be the main domain for receptor phosphorylation. Therefore, chimeric receptors, where the carboxyl-terminal tails of alpha(1a) and alpha(1b) adrenergic receptors were exchanged, were constructed and expressed. alpha(1a)-Adrenoreceptors wearing the carboxyl tail of the alpha(1b) subtype had a high basal phosphorylation and displayed a strong phosphorylation in response to norepinephrine and phorbol esters. Our results demonstrate that stimulation of alpha(1a)-adrenergic receptor, or activation of protein kinase C, leads to alpha(1a)-adrenergic receptor phosphorylation. alpha(1a)-Adrenoreceptors are affected to a much lesser extent than alpha(1b)-adrenoreceptors by protein kinase C activation.     

The antioxidant butylated hydroxytoluene (BHT) inhibits the dioctanoylglycerol-evoked platelet response but potentiates that elicited by ionomycin.

Preincubation of aspirin-treated human platelets with butylated hydroxytoluene (BHT) inhibits secretion, aggregation, and protein phosphorylation induced by dioctanoylglycerol or phorbol 12-myristate 13-acetate (PMA). BHT alone elicits a rapid and transient phosphorylation of a 47-kDa protein, which is indistinguishable from the well-recognized major substrate of protein kinase C (PKC). Inhibition of diacylglycerol- or PMA-induced platelet activation is also observed after decay to the basal level of the BHT-evoked phosphorylation of the 47-kDa protein. By contrast BHT potentiates platelet responses elicited by the calcium ionophore ionomycin. In the presence of the PKC inhibitor staurosporine BHT fails to increase the ionomycin-promoted platelet aggregation, indicating that its effect occurs through a PKC activation, even if no correlation with the 47-kDa protein phosphorylation is observed. BHT does not significantly modify the affinity of protein kinase C purified from calf brain for Ca2+ or dioctanoylglycerol. It is concluded that: (a) a short exposure of platelets to BHT induces an activation, whereas a long exposure an inhibition of PKC, (b) at variance with diacylglycerols BHT decreases the platelet responses promoted by subsequent challenge with PKC activators themselves, and (c) similarly to other PKC activators BHT potentiates the cellular response elicited by calcium ionophores most likely by activating the phospholipase A2.     

Gastrin-induced DNA synthesis requires p38-MAPK activation via PKC/Ca(2+) and Src-dependent mechanisms

We present evidence that gastrin, binding to a G protein-coupled receptor, activates the p38-mitogen-activated protein kinase (MAPK) pathway. Blockage of protein kinase C (PKC) by GF109203X, depletion of intracellular calcium by thapsigargin or inhibition of Src family kinases by PP2 prevented p38-MAPK activation and the Src kinase activity stimulated by gastrin. Inhibition of the PI 3-kinase by wortmannin or LY294002 did not affect these responses. In addition, the p38-MAPK inhibitor, SB203580, repressed gastrin-induced [(3)H]thymidine incorporation, indicating a major role of p38-MAPK in the growth-promoting effect of gastrin. Our results demonstrate that gastrin-induced DNA synthesis requires p38-MAPK activation through mechanisms that involve calcium mobilization, PKC and Src family kinases.     

Neutrophil stimulation: receptor, membrane, and metabolic events

In the neutrophil, binding of ligands to their appropriate receptors initiates a sequence of events culminating in the physiological responses of aggregation, degranulation, and superoxide anion generation. Calcium has been proposed as a second messenger in the activation sequence of the neutrophil. Increments in cytosolic free calcium are one of the first measurable events subsequent to receptor occupancy, followed by enhanced plasmalemmal permeability to calcium, a process that may serve to enhance the physiological responses. In contrast to calcium, cyclic AMP (cAMP) does not act as a signal in the activation sequence of the neutrophil. Increments in cAMP that are triggered by complete secretagogues may act as an inhibitory feedback mechanism. Protein kinases, both cAMP- and calcium/phospholipid-sensitive enzymes, may play a role in the activation sequence. Phosphorylation of proteins occurs during neutrophil activation. A role for phosphatidylinositol/phosphatidic acid turnover in calcium gating has been proposed. In addition, modulation of phospholipids could serve to activate a protein kinase C. Finally, phospholipids can serve as a source for arachidonic acid, which is metabolized by a 5-lipoxygenase pathway in the neutrophil. Products of this pathway, such as leukotriene B4, may serve to mediate or modulate the activation sequence.     

Process of re-establishment of beta-adrenergic induced protein secretion after cytochalasin D inhibition in rat parotid gland. Effect of cholinergic agonist, phorbol ester and calcium.

In rat parotid gland, 3H-protein secretion is stimulated by beta-adrenergic receptor activation (via cAMP) and also by cholinergic receptor activation (via IP3, calcium and diacylglycerol). The disorganization of microfilament system by cytochalasin D induced an inhibition of beta-adrenergic induced 3H-protein secretion whereas it did not modify the cholinergic muscarinic one. Cytochalasin D induced the formation of vacuoles in the parotid cell. In this work we show that the activation of muscarinic receptors (with carbachol) partially abolished the inhibitory effect of cytochalasin D on beta-adrenergic induced secretion. Since carbachol induced both intracellular calcium increase and protein kinase C activation, we decided to test separately the effect of calcium (using the calcium ionophore A23187) and protein kinase C activation (using phorbol ester) on the inhibitory effect of cytochalasin D on beta-adrenergic induced secretion. A23187, in the presence of calcium in the external medium was able to partially abolish cytochalasin D effect (ie re-establishing protein secretion) whereas activation of protein kinase C by phorbol 12-13 di-butyrate had no effect. These results suggest that protein kinase C is not involved in re-establishing a 'normal' secretion phenomenon whereas calcium does interfere. Furthermore, our fluorescence study shows that, when cytochalasin D is present in the incubation medium, the actin network is disturbed even in the presence of carbachol. This indicates that a calcium entry in the cell is not sufficient to restore a 'normal' actin network.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phorbol myristate acetate mediates redistribution of protein kinase C in human neutrophils: potential role in the activation of the respiratory burst enzyme

Protein kinase C may be important in leukocyte function, because it is activated by phorbol myristate acetate (PMA), a potent stimulus of the respiratory burst in neutrophils. The localization of protein kinase C was compared in unstimulated and PMA-stimulated human neutrophils. Protein kinase C was primarily cytosolic in unstimulated cells but became associated with the particulate fraction after treatment of cells with PMA. The particulate-associated kinase activity did not require added calcium and lipids, but when extracted by Triton X-100 (greater than or equal to 0.2%), calcium and phospholipid dependence could be demonstrated. The EC50 of PMA for stimulating kinase redistribution and activation of NADPH oxidase, the respiratory burst enzyme, were similar (30 to 40 nM). Redistribution of protein kinase C occurred rapidly (no lag) and preceded NADPH oxidase activation (30 sec lag). These results suggest that redistribution of protein kinase C is linked to activation of the respiratory burst in human neutrophils.     

Regulation of platelet protein kinase C by oleic acid. Kinetic analysis of allosteric regulation and effects on autophosphorylation, phorbol ester binding, and susceptibility to inhibition

The regulation of protein kinase C by oleic acid was studied, and parameters that characterize the activation of protein kinase C by oleic acid and distinguish its effects from those of diacylglycerol (DAG) and phosphatidylserine (PS) were delineated. Activation of protein kinase C by sodium oleate required the presence of calcium and showed mild cooperative behavior (Hill number of 1.25) suggesting that Ca(oleate)2 is the active species. Kinetic analysis of the interaction of sodium oleate with substrates indicated that sodium oleate acted to increase the activity of the enzyme without modulating the KM for either MgATP or histone substrates. In this respect, sodium oleate action resembled that of DAG but not PS. However, multiple parameters distinguished the effects of sodium oleate from those of DAG. Unlike DAG, sodium oleate was unable to inhibit phorbol dibutyrate binding to protein kinase C. Sodium oleate also failed to interact with micelle-bound protein kinase C and preferentially activated "soluble" protein kinase C. The addition of histone caused protein/lipid aggregation in the presence of DAG but not in the presence of oleate. Activation of protein kinase C by sodium oleate or by PS/DAG demonstrated differential susceptibility to the action of inhibitors. Sphingosine and NaCl were more potent in inhibiting activation of protein kinase C by PS/DAG than by sodium oleate. Sodium oleate also expressed PS-like activity in that calcium and oleate acted as cofactors in activation of protein kinase C by DAG. Similar to PS, the ability of oleate to act in synergy with DAG resulted from "competitive" activation with a decrease in KM(app) of protein kinase C for DAG. Finally, sodium oleate was unable to induce autophosphorylation of protein kinase C. These studies demonstrate that oleate activates protein kinase C by a mechanism that is distinct from PS/DAG but partially overlaps the kinetic effects of both PS and DAG. The significance of these studies is discussed in relation to mechanisms of protein kinase C activation and to the possible physiological relevance of activation of protein kinase C by fatty acids.     

Divergent signaling pathways requiring discrete calcium signals mediate concurrent activation of two mitogen-activated protein kinases by gonadotropin-releasing hormone

Receptors coupled to heterotrimeric G proteins are linked to activation of mitogen-activated protein kinases (MAPKs) via receptor- and cell-specific mechanisms. We have demonstrated recently that gonadotropin-releasing hormone (GnRH) receptor occupancy results in activation of extracellular signal-regulated kinase (ERK) through a mechanism requiring calcium influx through L-type calcium channels in alphaT3-1 cells and primary rat gonadotropes. Further studies were undertaken to explore the signaling mechanisms by which the GnRH receptor is coupled to activation of another member of the MAPK family, c-Jun N-terminal kinase (JNK). GnRH induces activation of the JNK cascade in a dose-, time-, and receptor-dependent manner in clonal alphaT3-1 cells and primary rat pituitary gonadotrophs. Coexpression of dominant negative Cdc42 and kinase-defective p21-activated kinase 1 and MAPK kinase 7 with JNK and ERK indicated that specific activation of JNK by GnRH appears to involve these signaling molecules. Unlike ERK activation, GnRH-stimulated JNK activity does not require activation of protein kinase C and is not blocked after chelation of extracellular calcium with EGTA. GnRH-induced JNK activity was reduced after treatment with the intracellular calcium chelator BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester), whereas activation of ERK was not affected. Chelation of intracellular calcium also reduced GnRH-induced activation of JNK in rat pituitary cells in primary culture. GnRH-induced induction and activation of the JNK target c-Jun was inhibited after chelation of intracellular calcium, whereas induction of c-Fos, a known target of ERK, was unaffected. Therefore, although activation of ERK by GnRH requires a specific influx of calcium through L-type calcium channels, JNK activation is independent of extracellular calcium but sensitive to chelation of intracellular calcium. Our results provide novel evidence that GnRH activates two MAPK superfamily members via strikingly divergent signaling pathways with differential sensitivity to activation of protein kinase C and mobilization of discrete pools of calcium.