CysLT1 signal transduction in differentiated U937 cells involves the activation of the small GTP-binding protein Ras

We investigated the signal transduction pathway(s) of leukotriene D(4) (LTD(4)) in the human promonocytic U937 cells, a cell line known to constitutively express CysLT(1) receptors. Herein, we demonstrate that LTD(4) specifically acts on a CysLT(1) receptor to dose-dependently increase (three to five-fold over basal) RasGTP through a G(i/o) protein. In fact, while cytosolic Ca(2+) ([Ca(2+)](i)) increase was only partially sensitive to pertussis toxin (PTx), Ras activation was almost completely inhibited by the same toxin. Furthermore, the phospholipase C (PLC) inhibitor U73122 completely inhibited both [Ca(2+)](i) and RasGTP increase, suggesting that in these cells PLC is the point of convergence for both PTx insensitive and sensitive pathways leading to [Ca(2+)](i) release and Ras activation. Indeed, chelating intracellular Ca(2+) strongly (>70%) prevented LTD(4)-induced Ras activation, indicating that this ion plays an essential role for CysLT(1)-induced downstream signaling in differentiated U937 (dU937) cells. In addition, while Src did not appear to be substantially involved in CysLT(1)-induced signaling, genistein was able to partially inhibit LTD(4)-induced [Ca(2+)](i) transient ( approximately 34%) and almost completely prevented Ras activation (>90%), suggesting a potential role for other Ca(2+)-dependent tyrosine kinases in LTD(4)-induced signaling. Finally, agonist-induced CysLT(1) stimulation was followed by a specific extracellular regulated kinase (ERK) 1/2 phosphorylation, an event with a pharmacological profile similar to that of Ras activation, partially ( approximately 40%) sensitive to Clostridium sordellii lethal toxin and totally blocked by PTx. In conclusion, LTD(4)-induced CysLT(1) receptor activation in dU937 cells leads to Ras activation and ERK phosphorylation mostly through a PTx-sensitive G(i/o) protein, PLC, and Ca(2+)-dependent tyrosine kinase(s).     

Characterization of ERK1/2 signalling pathways induced by adenosine receptor subtypes in newborn rat cardiomyocytes

1. Adenosine A(1), A(2A), and A(3) receptors (ARs) and extracellular signal-regulated kinase 1/2 (ERK1/2) play a major role in myocardium protection from ischaemic injury. In this study, we have characterized the adenosine receptor subtypes involved in ERK1/2 activation in newborn rat cardiomyocytes. 2. Adenosine (nonselective agonist), CPA (A(1)), CGS 21680 (A(2A)) or Cl-IB-MECA (A(3)), all increased ERK1/2 phosphorylation in a time- and dose-dependent manner. The combined maximal response of the selective agonists was similar to adenosine alone. Theophylline (nonselective antagonist) inhibited completely adenosine-mediated ERK1/2 activation, whereas a partial inhibition was obtained with DPCPX (A(1)), ZM 241385 (A(2A)), and MRS 1220 (A(3)). 3. PD 98059 (MEK1; ERK kinase inhibitor) abolished all agonist-mediated ERK1/2 phosphorylation. Pertussis toxin (PTX, G(i/o) blocker) inhibited completely CPA- and partially adenosine- and Cl-IB-MECA-induced ERK1/2 activation. Genistein (tyrosine kinase inhibitor) and Ro 318220 (protein kinase C, PKC inhibitor) partially reduced adenosine, CPA and Cl-IB-MECA responses, without any effect on CGS 21680-induced ERK1/2 phosphorylation. H89 (protein kinase A, PKA inhibitor) abolished completely CGS 21680 and partially adenosine and Cl-IB-MECA responses, without any effect on CPA response. 4. Cl-IB-MECA-mediated increases in cAMP accumulation suggest that A(3)AR-induced ERK1/2 phosphorylation involves adenylyl cyclase activation via phospholipase C (PLC) and PKC stimulation. 5. In summary, we have shown that ERK1/2 activation by adenosine in cardiomyocytes results from an additive stimulation of A(1), A(2A), and A(3)ARs, which involves G(i/o) proteins, PKC, and tyrosine kinase for A(1) and A(3)ARs, and Gs and PKA for A(2A)ARs. Moreover, the A(3)AR response also involves a cAMP/PKA pathway via PKC activation.     

Cardiotoxin III suppresses MDA-MB-231 cell metastasis through the inhibition of EGF/EGFR-mediated signaling pathway

Cardiotoxin III (CTX III), a basic polypeptide isolated from Naja naja atra venom, has been shown to exhibit anticancer activity. Epidermal growth factor (EGF) and its receptor, EGFR, play roles in cancer metastasis in various tumors. We use EGF as a metastatic inducer of MDA-MB-231 cells to investigate the effect of CTX III on cell migration. CTX III inhibited the EGF-induced activation of matrix metalloproteinase-9 (MMP-9), and further suppressed cell invasion and migration without obvious cellular cytotoxicity. CTX III suppressed EGF-induced nuclear factor-kappaB (NF-κB) nuclear translocation and also abrogated the EGF-induced phosphorylation of EGFR, phosphatidylinositol 3-kinase (PI3K)/Akt, and extracellular regulated kinase (ERK)1/2. In addition, CTX III similar to wortmannin (a PI3K inhibitor) and U0126 (an up-stream kinase regulating ERK1/2 inhibitor) attenuated cell migration and invasion induced by EGF. Furthermore, the EGFR inhibitor AG1478 inhibited EGF-induced MMP-9 expression, cell migration and invasion, as well as the activation of ERK1/2 and PI3K/Akt, suggesting that ERK1/2 and PI3K/Akt activation occur downstream of EGFR activation. These findings suggest that CTX III inhibited the EGF-induced invasion and migration of MDA-MB-231 cells via EGFR-dependent PI3K/Akt, ERK1/2, and NF-κB signaling, leading to the down-regulation of MMP-9 expression. These results provide a novel mechanism to explain the role of CTX III as a potent anti-metastatic agent in MDA-MB-231 cells.     

The protein kinase C inhibitor Go6976 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole] potentiates agonist-induced mitogen-activated protein kinase activation through tyrosine phosphorylation of the epidermal growth factor receptor

Protein kinase C (PKC) isoforms are important transducers of signals from G protein-coupled receptors (GPCRs) to diverse cellular targets, including extracellular signal-regulated kinases 1 and 2 (ERK1/2). Clone 9 rat hepatocytes (C9 cells) express receptors for angiotensin II (Ang II) type 1, lysophosphatidic acid (LPA), and epidermal growth factor (EGF), and their stimulation causes transient ERK1/2 phosphorylation through transactivation of the epidermal growth factor receptor (EGF-R). Inhibition of PKC by Go6983 [2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl)maleimide], or PKC depletion by prolonged phorbol 12-myristate 13-acetate (PMA) treatment, attenuated ERK1/2 activation by Ang II and PMA, but not by LPA and EGF. In contrast, another PKC inhibitor, Go6976 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole], enhanced basal and agonist-stimulated phosphorylation of ERK1/2, which was not caused by alteration in receptor binding and internalization, stimulation of inositol phosphate production, or activation of Pyk2 and Src tyrosine kinases. However, Go6976 enhanced agonist-induced tyrosine phosphorylation of the EGF receptor, possibly through inhibition of protein tyrosine phosphatase (PTP), because the PTP inhibitor sodium orthovanadate mimicked the effects of Go6976. Selective blockade of EGF-R kinase by AG1478 [4-(3-chloroanilino)6,7-dimethoxyquinazoline] abolished the ERK1/2 activation induced by Go6976. Similar experiments were conducted in human embryonic kidney 293 cells, which express receptors for LPA and EGF but exhibit no significant cross-communication between them. Although Go6976 caused a significant increase in EGF-induced tyrosine phosphorylation of the EGF-R and subsequent ERK1/2 activation, it had no such effects on LPA-induced responses. In Chinese hamster ovary cells, which express receptors for LPA but not for EGF, Go6976 also had no significant effect on LPA-induced ERK1/2 activation. These data indicate that Go6976 potentiates agonist-induced ERK1/2 activation through stimulation of tyrosine phosphorylation of the EGF-R.     

Epidermal growth factor induces vasoconstriction through the phosphatidylinositol 3-kinase-mediated mitogen-activated protein kinase pathway in hypertensive rats

We investigated whether increased contractile responsiveness to epidermal growth factor (EGF) is associated with altered activation of mitogen-activated protein kinase (MAPK) in the aortic smooth muscle of deoxycorticosterone acetate (DOCA)-salt hypertensive rats. EGF induced contraction and MAPK activity in aortic smooth muscle strips, which were significantly increased in tissues from the DOCA-salt hypertensive rats compared with those from sham-operated rats. AG1478, PD98059, and LY294002, inhibitors of EGF receptor (EGFR) tyrosine kinase, MAPK/extracellular signal-regulated kinase (ERK) kinase, and phosphatidylinositol 3-kinase (PI3K), respectively, inhibited the contraction and the activity of ERK1/2 that were elevated by EGF. Y27632 and GF109203X, inhibitors of Rho kinase and protein kinase C, respectively, attenuated EGF-induced contraction, with no diminution of ERK1/2 activity. Although EGF also elevated the activity of EGFR tyrosine kinase in both sham-operated and DOCA-salt hypertensive rats, the expression and the magnitude of activation did not differ between strips. These results strongly suggest that EGF induces contraction by the activation of ERK1/2, which is regulated by the PI3K pathway in the aortic smooth muscle of DOCA-salt hypertensive rats.     

Effect of cholecystokinin octapeptide on diacylglycerol-PKC signaling pathway in rat pulmonary interstitial macrophages stimulated by lipopolysaccharide

AIM: To investigate the effect of cholecystokinin octapeptide (CCK-8) on the diacylglycerol-protein kinase C (DAG-PKC) signaling pathway in rat pulmonary interstitial macrophages (PIM) stimulated by lipopolysaccaride (LPS). METHODS: The PIM from rat lung tissues were isolated using the collagenase digestion method combined with alveolar lavage and pulmonary vessel perfusion. DAG content and PKC activity were measured by radioenzymatic assay. The translocation of PKCzeta was determined by semi-quantitative immunoblot analysis. RESULTS: CCK-8, at high concentrations (1 x 10(-6) - 1 x 10(-5) mol/L), decreased DAG content and inhibited PKC activity and PKCzeta translocation compared with that in rat resting PIM of a control group (P< 0.01). LPS increased DAG content, and promoted PKC activity and PKCzeta translocation (P< 0.01). CCK-8 decreased LPS-induced DAG content and inhibited LPS-induced PKC activity and PKCzeta translocation significantly at 1 x 10(-8) - 1 x 10(-5) mol/L (P< 0.01). This inhibitory effect of CCK-8 could be abrogated partly by proglumide (non-selective CCK receptor antagonist), CR-1409 (selective CCK-A receptor antagonist), and CR-2945 (selective CCK-B receptor antagonist) in a concentration-dependent manner (P< 0.01). CONCLUSION: CCK-8 was a negative modulator of the DAG-PKC signaling pathway in rat resting PIM, which is very important for maintaining body homeostasis. It significantly inhibited LPS-induced DAG content, PKC activity and PKCzeta translocation in a concentration-dependent manner. The CCK receptor, especially the CCK-A receptor, might play a major role in this process.     

Opioid control of MAP kinase cascade

Activation of G protein-coupled receptors (GPCRs) may result in phosphorylation of extracellular signal-regulated kinases 1/2 (ERK 1/2). The signaling pathway involves ectodomain shedding, generating epidermal growth factor (EGF)-like ligands, which in turn stimulate the mitogen-activated protein kinase (MAPK) via EGF receptors. The present study investigates into the control of MAPKs by opioidergic GPCRs in human embryonic kidney cells (HEK 293). Experiments were conducted with cells expressing opioid receptors, G protein-coupled receptor kinases, and ERKs. The outcome of our studies let us suggest that EGF-like ligands released by opioid receptor stimulation utilize different EGF receptors to phosphorylate ERKs, while EGF utilizes type 1 receptors. Differences between multiple opioid receptors are apparent with respect to the activation of ERKs. EGF rapidly triggers internalization of the fluorescent EGF receptor type 1, but we failed to observe any sequestration of this receptor type upon exposure of cells to an opioid, since opioids most likely trigger stimulation of a different EGF receptor type. In conclusion, G protein-coupled opioid receptors control the MAPK cascade in a similar fashion as described for non-opioid GPCRs, although distinct differences exist between μ-, δ- and κ-receptors. EGF-induced ERK activation is mediated by EGF receptor type 1 while opioid receptor activation seems to brings about stimulation via EGF receptor type.     

Activation of extracellular-signal regulated kinase by epidermal growth factor is potentiated by cAMP-elevating agents in primary cultures of adult rat hepatocytes

We investigated the effects of α- and β-adrenergic agonists on epidermal growth factor (EGF)-stimulated extracellular-signal regulated kinase (ERK) isoforms in primary cultures of adult rat hepatocytes. Hepatocytes were isolated and cultured with EGF (20 ng/ml) and/or α(1)-, α(2)- and β(2)-adrenergic agonists. Phosphorylated ERK isoforms (ERK1; p44 mitogen-activated protein kinase (MAPK) and ERK2; p42 MAPK) were detected by Western blotting analysis using anti-phospho-ERK1/2 antibody. The results show that EGF induced a 2.5-fold increase in ERK2-, but not ERK1-, phosphorylation within 3 min. This EGF-induced ERK2 activation was abolished by treatment with the EGF-receptor kinase inhibitor AG1478 (10(-7) M) or the MEK (MAPK kinase) inhibitor PD98059 (10(-6) M). The α(2)-adrenergic and β(2)-adrenergic agonists, UK14304 (10(-6) M) and metaproterenol (10(-6) M), respectively, had no effect in the absence of EGF, but metaproterenol significantly potentiated EGF-induced ERK2 phosphorylation. Moreover, the cell-permeable cAMP analog 8-bromo cAMP (10(-7) M), also potentiated EGF-induced ERK2 phosphorylation. The effects of these analogs were antagonized by the protein kinase A (PKA) inhibitor H-89 (10(-7) M). These results suggest that direct or indirect activation of PKA represents a positive regulatory mechanism for EGF stimulation of ERK2 induction.     

Activated human hydroxy-carboxylic acid receptor-3 signals to MAP kinase cascades via the PLC-dependent PKC and MMP-mediated EGFR pathways

BACKGROUND AND PURPOSE 3-Hydroxy-octanoate, recently identified as a ligand for, the orphan GPCR, HCA(3), is of particular interest given its ability to treat lipid disorders and atherosclerosis. Here we demonstrate the pathway of HCA(3)-mediated activation of ERK1/2. EXPERIMENTAL APPROACH Using CHO-K1 cells stably expressing HCA(3) receptors and A431 cells, a human epidermoid cell line with high levels of endogenous expression of functional HCA(3) receptors, HCA(3)-mediated activation of ERK1/2 was measured by Western blot. KEY RESULTS HCA(3)-mediated activation of ERK1/2 was rapid, peaking at 5 min, and was Pertussis toxin sensitive. Our data, obtained by time course analyses in combination with different kinase inhibitors, demonstrated that on agonist stimulation, HCA(3) receptors evoked ERK1/2 activation via two distinct pathways, the PLC/PKC pathway at early time points (≤ 2 min) and the MMP/ epidermal growth factor receptor (EGFR) transactivation pathway with a maximum response at 5 min. Furthermore, our present results also indicated that the βγ-subunits of the G(i) protein play a critical role in HCA(3)-activated ERK1/2 phosphorylation, whereas β-arrestins and Src were not required for ERK1/2 activation. CONCLUSIONS AND IMPLICATIONS We have described the molecular mechanisms underlying the coupling of human HCA(3) receptors to the ERK1/2 MAP kinase pathway in CHO-K1 and A431 cells, which implicate the G(i) protein-initiated, PLC/PKC -and platelet-derived growth factor receptor/EGFR transactivation-dependent pathways. These observations may provide new insights into the pharmacological effects and the physiological functions modulated by the HCA(3)-mediated activation of ERK1/2.     

Mu opioid transactivation and down-regulation of the epidermal growth factor receptor in astrocytes: implications for mitogen-activated protein kinase signaling

Astroglia are a principal target of long-term mu antiproliferative actions. The mitogen-activated protein (MAP) kinase known as extracellular signal-regulated kinase (ERK), is a key mediator of cell proliferation. In studies on the mechanism of short- and long-term mu opioid regulation of the ERK signaling pathway, we show that the mu opioid agonist [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO), acting via the endogenous mu opioid receptor (MOR), induced sequential epidermal growth factor receptor (EGF) receptor (EGFR) Tyr phosphorylation, Ser phosphorylation, and down-regulation in immortalized rat cortical astrocytes. The short-term action of DAMGO resulted in the stimulation of ERK phosphorylation. 4(3-Chlorophenylamino)-6,7-dimethoxyquinazoline (Tyrphostin AG1478), a selective inhibitor of EGFR Tyr kinase activity, blocked EGFR and ERK activation by short-term DAMGO administration, implicating EGFR transactivation in its stimulation of ERK activity. Inhibitors of matrix metalloproteinases attenuated MOR-mediated ERK phosphorylation, suggesting that shedding of EGF-like ligands from the plasma membrane may be involved in the EGFR transactivation process. Prolonged DAMGO exposure induced EGFR internalization/down-regulation, did not activate ERK, and inhibited exogenous EGF-stimulated ERK phosphorylation. MOR-mediated EGFR down-regulation seems to be MAP kinase-dependent, because it was inhibited by the ERK kinase inhibitor 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio) butadiene (U0126), and tyrphostin AG1478. The kappa opioid agonist (5alpha,7alpha,8beta)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl) benzeneacetamide (U69,593) induced Tyr and Ser phosphorylation of EGFR and activation of ERK. However, long-term application of U69,593 neither down-regulated EGFR nor inhibited EGF-induced ERK activation. Instead, it engendered a sustained activation of ERK. Collectively, our data suggest that long-term application of DAMGO initiates heterologous down-regulation of EGFR via a mechanism involving ERK in astrocytes.     

Signaling mechanisms of sphingosine 1-phosphate-induced ERK1/2 activation in cultured feline esophageal smooth muscle cells

Sphingosine 1-phosphate (S1P) is a bioactive lipid, stored and released from activated platelets, macrophages, and other mammalian cells. We previously reported that S1P induces esophageal smooth muscle contraction in freshly isolated intact cells. Here, we measured S1P-induced ERK1/2 activation and upstream signaling in cultured feline esophageal smooth muscle cells. Activation of ERK1/2 by S1P peaked at 5 min, was sustained up to 30 min, and was blocked by PTX. In contrast, S1P did not activate p38 MAPK or JNK. PTX inhibited S1P-induced ERK1/2 activation. We then used phospholipase inhibitors, DEDA for PLA₂, U73122 for PLC, and ρCMB for PLD, to determine that ERK1/2 activation was downstream of PLC activation. The PKC inhibitors, GF109203X and chelerythrine, also suppressed ERK1/2 activation. Whereas the PTK inhibitor, genistein, partially inhibited ERK1/2 activation, the EGFR tyrosine kinase inhibitor, tyrphostin 51, had no effect. Taken together, S1P-induced ERK1/2 activation in cultured ESMCs requires a PTX-sensitive G protein, stimulation of the PLC pathway, and subsequent activation of the PKC and PTK pathways.     

Evidence for cross-talk between M2 and M3 muscarinic acetylcholine receptors in the regulation of second messenger and extracellular signal-regulated kinase signalling pathways in Chinese hamster ovary cells

1. We have examined possible mechanisms of cross-talk between the G(q/11)-linked M(3) muscarinic acetylcholine (mACh) receptor and the G(i/o)-linked M(2) mACh receptor by stable receptor coexpression in Chinese hamster ovary (CHO) cells. A number of second messenger (cyclic AMP, Ins(1,4,5)P(3)) and mitogen-activated protein kinase (ERK and JNK) responses stimulated by the mACh receptor agonist methacholine were examined in CHO-m2m3 cells and compared to those stimulated in CHO-m2 and CHO-m3 cell-lines, expressing comparable levels of M(2) or M(3) mACh receptors. 2. Based on comparisons between cell-lines and pertussis toxin (PTx) pretreatment to eliminate receptor-G(i/o) coupling, evidence was obtained for (i) an M(2) mACh receptor-mediated contribution to the predominantly M(3) mACh receptor-mediated Ins(1,4,5)P(3) response and (ii) a facilitation of the inhibitory effect of M(2) mACh receptor on forskolin-stimulated cyclic AMP accumulation by M(3) mACh receptor coactivation at low agonist concentrations (MCh 10(-9)-10(-6) M). 3. The most profound cross-talk effects were observed with respect to ERK activation. Thus, while MCh stimulated ERK activation in both CHO-m2 and CHO-m3 cells (pEC(50) values: 5.64+/-0.09 and 5.57+/-0.16, respectively), the concentration-effect relation was approx 50-fold left-shifted in CHO-m2m3 cells (pEC(50): 7.17+/-0.07). In addition, the ERK response was greater and more sustained in CHO-m2m3 cells. In contrast, only minor differences were seen in the time-courses and concentration-dependencies of JNK activation in CHO-m3 and CHO-m2m3 cells. 4. Costimulation of endogenous P2Y(2) purinoceptors also caused an approx 10-fold left-shift in the MCh-stimulated ERK response in CHO-m2 cells, suggesting that the G(q/11)/G(i/o) interaction to affect ERK activation is not specific to muscarinic receptors. 5. PTx pretreatment of cells had unexpected effects on ERK activation by MCh in both CHO-m2m3 and CHO-m3 cells. Thus, in CHO-m3 cells PTx pretreatment caused a marked left-shift in the MCh concentration-effect curve, while in PTx-treated CHO-m2m3 cells the maximal responsiveness was decreased, but the potency of MCh was only slightly affected. 6. The data presented here strongly suggest that cross-talk between M(2) and M(3) mACh receptors occurs at the level of both second messenger and ERK regulation. Further, these data provide novel insights into the involvement of G(i/o) proteins in both positive and negative modulation of ERK responses evoked by G protein-coupled receptors.     

Diadenosine polyphosphates Ap3A and Ap4A, but not Ap5A or Ap6A, induce proliferation of vascular smooth muscle cells

Depending on the number of phosphate groups, diadenosine polyphosphates (ApnA, Ap3A, Ap4A, Ap5A and Ap6A) differ in properties such as proliferation, apoptosis, vasoconstriction and vasodilatation of vascular smooth muscle cells (VSMCs). Possible signaling pathways leading to effects such as proliferation are still unknown. This study examined the proliferative effects of diadenosine polyphosphates on VSMCs and their intracellular pathways. Proliferation of VSMCs was measured by the cell count and [(3)H] thymidine incorporation. Phosphorylation of the MAP kinases ERK1/2 was determined by Western blotting. Single-cell [Ca(2+)](i) measurements were done to determine the influence of [Ca(2+)](i) on intracellular signaling. Stress fiber formation was assessed by fluorescence microscopy to detect an influence of G alpha(12). Ap3A and Ap4A, but not Ap5A or Ap6A, were shown to increase proliferation of VSMCs by activating P2Y receptors, which leads to stimulation of the Ras-Raf-MEK-ERK1/2 cascade. Ap3A- and Ap4A-induced activation of the MAP kinases ERK1/2 was dependent on a signaling pathway that included the EGF receptor, PKC, PLCbeta and the increase of [Ca(2+)](i). In conclusion, Ap3A and Ap4A, but not Ap5A or Ap6A, induce proliferation of VSMCs by a signaling pathway that begins with activation of P2Y receptors and leads to stimulation of the MAP kinases ERK1/2.     

A dual role of protein kinase C in insulin signal transduction via adenylyl cyclase signaling system in muscle tissues of vertebrates and invertebrates

Further decoding of a novel adenylyl cyclase signaling mechanism (ACSM) of the action of insulin and related peptides detected earlier (Pertseva et al. Comp Biochem Physiol B Biochem Mol Biol 1995;112:689-95 and Pertseva et al. Biochem Pharmacol 1996;52:1867-74) was carried out with special attention given to the role of protein kinase C (PKC) in the ACSM. It was shown for the first time that transduction of the insulin signal via the ACSM followed by adenylyl cyclase (AC, EC 4.6.1.1) activation was blocked in the muscle tissues of rat and mollusc Anodonta cygnea in the presence of pertussis toxin, inducing the impairment of G(i)-protein function, wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), and calphostin C, a blocker of PKC. The cholera toxin treatment of muscle membranes led to an increase in basal AC activity and a decrease in enzyme insulin reactivity. Phorbol ester and diacylglycerol activation of PKC (acute treatment) induced the inhibition of the insulin AC activating effect. This negative influence was also observed in the case of the AC system activated by biogenic amines. It was first concluded that the ACSM of insulin action involves the following signaling chain: receptor tyrosine kinase => G(i) (betagamma) => PI3-K => PKCzeta (?) => G(s) => AC => adenosine 3',5'-cyclic monophosphate. It was also concluded that the PKC system has a dual role in the ACSM: (1) a regulatory role (PKC sensitive to phorbol esters) that is manifested as a negative feedback modulation of insulin signal transduction via the ACSM; (2) a transductory role, which consists in direct participation of atypical PKC (PKCzeta) in the process of insulin signal transduction via the ACSM.     

Epidermal growth factor differentially augments G(i)-mediated stimulation of c-Jun N-terminal kinase activity

1. Signaling networks involving different receptor systems allow extracellular signals to be integrated and transformed into various biological activities. In this report, we studied the activity of the c-Jun N-terminal kinase (JNK) subgroup of mitogen-activated protein kinases (MAPKs), in response to stimulation by G protein-coupled receptors (GPCRs) and co-activation with epithermal growth factor receptor (EGFR). 2. Stimulation of exogenous GPCRs in Cos-7 cells induced JNK activation of different magnitudes depending on their G-protein coupling specificities (G(q)>G(i)>G(s)), and a moderate JNK activation was linked to stimulation of endogenous EGFR by EGF. 3. Co-stimulation with GPCR agonists and EGF resulted in differential augmentation of JNK activities, with G(i)-coupled receptors associated with a synergistic JNK activation upon co-stimulation with EGF, while G(q)- and G(s)-coupled receptors were incapable of triggering this effect. 4. This G(i)/EGF-induced synergistic JNK activation was inhibited by pertussis toxin and AG1478, and may involve Src family tyrosine kinases, PI3 K, Ca(2+)/calmodulin and small GTPases as important intermediates, while Ca(2+) mobilization was triggered by the stimulation of G(q)-coupled receptor or EGF treatment, but not by the G(i)- or G(s)-coupled receptors. 5. Transient expression of Gbetagamma subunits with EGF treatment, or co-activation of exogenous G(i)-coupled receptor with thapsigargin also resulted in a synergistic JNK activation. Activation of G(i)-coupled receptor accompanied with EGF treatment enhanced the expression level and activity of MAPK phosphatase type I, which occurred after the maximal synergistic JNK activation. 6. Our results support a mechanistic model where EGF signaling may differentially regulate the JNK activities triggered by GPCRs of different coupling specificities.     

Differential regulation of P2Y(11) receptor-mediated signalling to phospholipase C and adenylyl cyclase by protein kinase C in HL-60 promyelocytes

The regulatory mode of the P2Y(11) purinoceptor-mediated signalling cascades towards phospholipase C and adenylyl cyclase was studied in HL-60 promyelocytes. Treatment with the potent P2Y(11) receptor activator dATP evoked an elevated intracellular Ca(2+) concentration ([Ca(2+)](i)) and inositol 1,4,5-trisphosphate (IP(3)) production that was sustained for longer than 30 min. However, the dATP-induced responses were significantly inhibited by the activation of protein kinase C after a short exposure to phorbol 12-myristate 13-acetate (PMA). dATP also potently stimulated cyclic AMP production with half maximum effect seen at 23+/-7 microM dATP. In addition, a 5-min pretreatment with PMA enhanced the dATP-stimulated cyclic AMP accumulation. PMA potentiated the cyclic AMP production when adenylyl cyclase was activated directly by forskolin or indirectly by G protein activation after cholera toxin treatment. dATP also enhanced the forskolin-mediated cyclic AMP generation. Treatment of the cells with 10 microM U-73122, which almost completely blocked the dATP-stimulated IP(3) production and [Ca(2+)](i) rise, had no effect on cyclic AMP accumulation, while 10 microM 9-(tetrahydro-2-furyl)adenine (SQ 22536), which inhibited the adenylyl cyclase activation, did not effect the dATP-stimulated phosphoinositide turnover. Taken together, the results indicate that P2Y(11) receptor-mediated activation of phospholipase C and adenylyl cyclase occurs through independent pathways and is differentially regulated by protein kinase C in HL-60 cells.