Activation of MAPK cascades by GnRH: ERK and Jun N-terminal kinase are involved in basal and GnRH-stimulated activity of the glycoprotein hormone LHbeta-subunit promoter

The role of ERK and Jun N-terminal kinase (JNK) in basal- and GnRH-stimulated LHbeta-promoter activity was examined in the gonadotroph cell line LbetaT-2. GnRH agonist (GnRH-A) stimulates the MAPK cascades ERK, JNK, and p38MAPK, with a peak at 7 min for ERK and at 60 min for JNK and p38MAPK. The rat glycoprotein hormone LHbeta-subunit promoter, linked to the chloramphenicol acetyl transferase (CAT) reporter gene, was used to follow its activation. Addition of GnRH-A (10 nM) to LbetaT-2 cells resulted in a 6-fold increase in LHbeta-CAT activity at 8 h, which was markedly reduced by a GnRH antagonist. The PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA), but not the Ca(2+) ionophore ionomycin, stimulated LHbeta-CAT activity. Addition of GnRH-A and TPA together did not produce an additive response. Down-regulation of PKC, but not removal of Ca(2+), abolished the GnRH-A and the TPA response. Cotransfection of the LHbeta-promoter and the constitutively active form of Raf-1 stimulated basal and GnRH-A-induced LHbeta-CAT activity. The dominant negative forms of the ERK cascade members Ras, Raf-1, and MAPK/ERK kinase (MEK) markedly reduced basal and GnRH-A-induced LHbeta-CAT activity, Similar results were obtained with the MEK inhibitor PD 098059. Cotransfection of the LHbeta-promoter and the constitutively active CDC42 stimulated basal and GnRH-A-induced LHbeta-CAT activity. The dominant negative forms of the JNK cascade members Rac, CDC42, and SEK markedly diminished basal and GnRH-A-induced LHbeta-CAT activity. Interestingly, the constitutively active form of c-Src stimulated the basal and the GnRH-A response, whereas the dominant negative form of c-Src, or the c-Src inhibitor PP1 diminished basal and the GnRH-A response. We conclude that ERK and JNK are involved in basal and GnRH-A stimulation of LHbeta-CAT activity. c-Src participates also in LHbeta-promoter activation by a mechanism which might be linked to ERK and JNK activation.     

Activation of mitogen-activated protein kinase phosphatase 2 by gonadotropin-releasing hormone

The aim of these studies was to identify the signaling mechanism(s) that contribute to GnRH-induced expression of MAPK phosphatase (MKP)-2, a dual specificity phosphatase that selectively inactivates MAPKs. GnRH receptor activation induced MKP-2 expression in both clonal (alphaT3-1) and primary gonadotropes. Activation of PKC isozymes was sufficient and required for MKP-2 induction. Inhibition of the extracellular signal-regulated kinase (ERK) or c-Jun N-terminal kinase (JNK) but not the p38 MAPK cascade was sufficient to block GnRH-induced MKP-2 expression. Induction of MKP-2 by GnRH was dependent on elevation in intracellular Ca(2+). Inhibition of Ca(2+) influx through L-type voltage-gated calcium channels blocked GnRH-induced MKP-2 expression. Depletion of intracellular Ca(2+) stores with thapsigargin blocked MKP-2 activation by GnRH independent of ERK and JNK activity. These results support the conclusion that MKP-2 induction by GnRH occurs via MAPK-dependent and -independent pathways. One mechanism requires GnRH-induced ERK and JNK activation, while a second MAPK-independent pathway requires a thapsigargin-sensitive calcium signal.     

Activation of the p38 mitogen-activated protein kinase pathway by gonadotropin-releasing hormone

Previous studies have shown that interaction of GnRH with its serpentine, G protein-coupled receptor results in activation of the extracellular signal regulated protein kinase (ERK) and the Jun N-terminal protein kinase (JNK) pathways in pituitary gonadotropes. In the present study, we examined GnRH-stimulated activation of an additional member of the mitogen-activated protein kinase (MAPK) superfamily, p38 MAPK GnRH treatment of alphaT3-1 cells resulted in tyrosine phosphorylation of several intracellular proteins. Separation of phosphorylated proteins by ion exchange chromatography suggested that GnRH receptor stimulation can activate the p38 MAPK pathway. Immunoprecipitation studies using a phospho-tyrosine antibody resulted in increased amounts of immunoprecipitable p38 MAPK from alphaT3-1 cells treated with GnRH. Immunoblot analysis of whole cell lysates using a phospho-specific antibody directed against dual phosphorylated p38 kinase revealed that GnRH-induced phosphorylation of p38 kinase was dose and time dependent and was correlated with increased p38 kinase activity in vitro. Activation of p38 kinase was blocked by chronic phorbol ester treatment, which depletes protein kinase C isozymes alpha and epsilon. Overexpression of p38 MAPK and an activated form of MAPK kinase 6 resulted in activation of c-jun and c-fos reporter genes, but did not alter the expression of the glycoprotein hormone alpha-subunit reporter. Inhibition of p38 activity with SB203580 resulted in attenuation of GnRH-induced c-fos reporter gene expression, but was not sufficient to reduce GnRH-induced c-jun or glycoprotein hormone alpha-subunit promoter activity. These studies provide evidence that the GnRH signaling pathway in alphaT3-1 cells includes protein kinase C-dependent activation of the p38 MAPK pathway. GnRH integration of c-fos promoter activity may include regulation by p38 MAPK.     

Activation of mitogen-activated protein kinase (MAPK) by GnRH is cell-context dependent

The interaction of GnRH with its cognate receptor (GnRHR) in pituitary gonadotropes includes activation of Gq/G11 and phospholipase Cbeta (PLCbeta), which generates the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), which are required for Ca2+ mobilization and PKC isoforms activation. Activation of PKC in pituitary gonadotropes leads to the activation of the major members of the mitogen-activated protein kinase superfamily (MAPK), namely: extracellular signal-regulated kinase (ERK), jun-N-terminal Kinase (JNK) and p38MAPK. The above pathways mediate GnRH-induced gonadotropin release and synthesis. Here we summarise the diverse mechanisms utilized by GnRH to activate the MAPK members and show that they depend on "cell-context".     

Peroxynitrite activates ERK via Raf-1 and MEK, independently from EGF receptor and p21Ras in H9C2 cardiomyocytes

Peroxynitrite is a potent oxidant and nitrating species proposed as a direct effector of myocardial damage in a wide range of cardiac diseases. Whether peroxynitrite also acts indirectly, by modulating cell signal transduction pathways in the myocardium, has not been investigated. Here, we examined the ability of peroxynitrite to activate extracellular signal-related kinase (ERK), a MAP kinase which has been linked with hypertrophic and anti-apoptotic responses in the heart, in cultured H9C2 cardiomyocytes. Peroxynitrite elicited a concentration- and time-dependent activation of ERK, secondary to the upstream activation of MEK 1 (ERK kinase). Activation of MEK-ERK by peroxynitrite was related to the upstream activation of Raf-1 kinase, as ERK and MEK phosphorylation were prevented by the Raf-1 inhibitor BAY43-9006. These effects of peroxynitrite were not associated with the activation of p21(Ras), known as a common signaling target of cellular oxidative stress. In contrast to ERK activation mediated by the epidermal growth factor (EGF), ERK activation by peroxynitrite was not prevented by AG1478 (EGF receptor inhibitor). Peroxynitrite acted through oxidative, but not nitrative chemistry, as ERK remained activated while nitration was prevented by the flavanol epicatechin. In addition to ERK, peroxynitrite also potently activated two additional members of the MAP kinase family of signaling proteins, JNK and p38. Thus, peroxynitrite activates ERK in cardiomyocytes through an unusual signaling cascade involving Raf-1 and MEK 1, independently from EGFR and P21(Ras), and also acts as a potent activator of JNK and p38. These results provide the novel concept that peroxynitrite may represent a previously unrecognized signaling molecule in various cardiac pathologies.     

Protein kinase Czeta mediated Raf-1/extracellular-regulated kinase activation by daunorubicin

In light of the emerging concept of a protective function of the mitogen-activated protein kinase (MAPK) pathway under stress conditions, we investigated the influence of the anthracycline daunorubicin (DNR) on MAPK signaling and its possible contribution to DNR-induced cytotoxicity. We show that DNR increased phosphorylation of extracellular-regulated kinases (ERKs) and stimulated activities of both Raf-1 and extracellular-regulated kinase 1 (ERK1) within 10 to 30 minutes in U937 cells. ERK1 stimulation was completely blocked by either the mitogen-induced extracellular kinase (MEK) inhibitor PD98059 or the Raf-1 inhibitor 8-bromo-cAMP (cyclic adenosine monophosphate). However, only partial inhibition of Raf-1 and ERK1 stimulation was observed with the antioxidant N-acetylcysteine (N-Ac). Moreover, the xanthogenate compound D609 that inhibits DNR-induced phosphatidylcholine (PC) hydrolysis and subsequent diacylglycerol (DAG) production, as well as wortmannin that blocks phosphoinositide-3 kinase (PI3K) stimulation, only partially inhibited Raf-1 and ERK1 stimulation. We also observed that DNR stimulated protein kinase C zeta (PKCzeta), an atypical PKC isoform, and that both D609 and wortmannin significantly inhibited DNR-triggered PKCzeta activation. Finally, we found that the expression of PKCzeta kinase-defective mutant resulted in the abrogation of DNR-induced ERK phosphorylation. Altogether, these results demonstrate that DNR activates the classical Raf-1/MEK/ERK pathway and that Raf-1 activation is mediated through complex signaling pathways that involve at least 2 contributors: PC-derived DAG and PI3K products that converge toward PKCzeta. Moreover, we show that both Raf-1 and MEK inhibitors, as well as PKCzeta inhibition, sensitized cells to DNR-induced cytotoxicity.     

Calcium has a permissive role in interleukin-1beta-induced c-jun N-terminal kinase activation in insulin-secreting cells

The c-jun N-terminal kinase (JNK) signaling pathway mediates IL-1beta-induced apoptosis in insulin-secreting cells, a mechanism relevant to the destruction of pancreatic beta-cells in type 1 and 2 diabetes. However, the mechanisms that contribute to IL-1beta activation of JNK in beta-cells are largely unknown. In this study, we investigated whether Ca(2+) plays a role for IL-1beta-induced JNK activation. In insulin-secreting rat INS-1 cells cultured in the presence of 11 mm glucose, combined pharmacological blockade of L- and T-type Ca(2+) channels suppressed IL-1beta-induced in vitro phosphorylation of the JNK substrate c-jun and reduced IL-1beta-stimulated activation of JNK1/2 as assessed by immunoblotting. Inhibition of IL-1beta-induced in vitro kinase activity toward c-jun after collective L- and T-type Ca(2+) channel blockade was confirmed in primary rat and ob/ob mouse islets and in mouse betaTC3 cells. Ca(2+) influx, specifically via L-type but not T-type channels, contributed to IL-1beta activation of JNK. Activation of p38 and ERK in response to IL-1beta was also dependent on L-type Ca(2+) influx. Membrane depolarization by KCl, exposure to high glucose, treatment with Ca(2+) ionophore A23187, or exposure to thapsigargin, an inhibitor of sarco(endo)plasmic reticulum Ca(2+) ATPase, all caused an amplification of IL-1beta-induced JNK activation in INS-1 cells. Finally, a chelator of intracellular free Ca(2+) [bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-acetoxymethyl], an inhibitor of calmodulin (W7), and inhibitors of Ca(2+)/calmodulin-dependent kinase (KN62 and KN93) partially reduced IL-1beta-stimulated c-jun phosphorylation in INS-1 or betaTC3 cells. Our data suggest that Ca(2+) plays a permissive role in IL-1beta activation of the JNK signaling pathway in insulin-secreting cells.     

Identification of ERK and JNK as signaling mediators on protein kinase C activation in cultured granulosa cells

PKC signaling is critical for follicular development and the induction of ovulatory genes including Pgr, Prkg2, and Cyp11a1 (SCC). We investigated PKC signaling mechanisms in the JC-410 porcine granulosa cell line stably expressing an SCC-luciferase reporter gene containing 2kb of the porcine SCC promoter. Addition of phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C, induced the promoter approximately 6-fold over the basal levels in 4h. This effect was predominantly mediated by the PKC beta and delta isoforms. PMA-mediated induction of the SCC promoter was sensitive to inhibition of ERK1/2 or JNK. Inhibition of p38 MAP kinase or Src tyrosine kinase did not alter the PMA-mediated inducibility of the promoter. SCC promoter induction in response to PMA treatment required basal EGF-receptor activity, but did not involve ectodomain shedding. Western blot analyses using phospho-specific antibodies showed that PMA treatment of JC-410 cells induced phosphorylation of MEK1/2, ERK1/2, and its downstream target p90 RSK at 15min. We also documented the rapid phosphorylation of JNK1/2 in response to PMA treatment. Phosphorylation of ERK and JNK was robust and sustained in contrast to activation of PKA and EGF-receptor signaling in these cells. Pretreatment of JC-410 granulosa cells with IGF-1 had a synergistic effect on PMA-mediated induction of the SCC promoter. We demonstrated the importance of PMA activation of ERK signaling and the synergism with IGF-1 by showing similar responses for Prkg2 expression in primary granulosa cells. In conclusion, our studies demonstrated PMA activation of ERK and JNK signaling which is relevant in the regulation of gene expression during follicular development, ovulation, and luteinization.     

Gonadotropin-releasing hormone-desensitized LbetaT2 gonadotrope cells are refractory to acute protein kinase C,cyclic AMP,and calcium-dependent signaling

Sustained exposure of gonadotropes to GnRH causes a pronounced desensitization of gonadotropin release, but the mechanisms involved are poorly understood. It is known that desensitization is associated with decreased GnRH receptor and Gq/11 levels in alphaT3-1 cells, but it is not known whether downstream signaling is impaired. We have shown previously that chronic stimulation of signaling via expression of an active form of Galphaq causes GnRH resistance in LbetaT2 cells. In this study we investigated whether chronic GnRH treatment could down-regulate protein kinase C (PKC), cAMP, or Ca2+-dependent signaling in LbetaT2 cells. We found that chronic GnRH treatment desensitizes cells to acute GnRH stimulation not only by reducing GnRH receptor and Gq/11 expression but also by down-regulating PKC, cAMP, and calcium-dependent signaling. Desensitization was observed for activation of ERK and p38 MAPK and induction of c-fos and LHbeta protein expression. Activation of individual signaling pathways was able to partially mimic the desensitizing effect of GnRH on ERK, p38 MAPK, c-fos, and LHbeta but not on Gq/11. Chronic stimulation with phorbol esters reduced GnRH receptor expression to the same extent as chronic GnRH. Sustained GnRH also desensitized PKC signaling by down-regulating the delta, epsilon, and theta isoforms of PKC. We further show that chronic GnRH treatment causes heterologous desensitization of other Gq-coupled receptors.     

Calcium-sensing receptor induces proliferation through p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase but not extracellularly regulated kinase in a model of humoral hypercalcemia of malignancy

Using H-500 rat Leydig cancer cells as a model of humoral hypercalcemia of malignancy (HHM), we previously showed that high Ca(2+) induces PTH-related peptide (PTHrP) secretion via the calcium-sensing receptor (CaR) and mitogen- and stress-activated kinases, e.g. MAPK kinase 1 (MEK1), p38 MAPK, and stress-activated protein kinase 1/c-Jun N-terminal kinase. Because cellular proliferation is a hallmark of malignancy, we studied the role of the CaR in regulating the proliferation of H-500 cells. Elevated Ca(2+) has a mitogenic effect on these cells that is mediated by the CaR, because the calcimimetic NPS R-467 also induced proliferation. Inhibition of phosphatidylinositol 3-kinase (PI3K) and p38 MAPK but not MEK1 abolished the mitogenic effect. Activation of PI3K by elevated Ca(2+) was documented by phosphorylation of its downstream kinase, protein kinase B. Because protein kinase B activation promotes cell survival, we speculated that elevated Ca(2+) might protect H-500 cells against apoptosis. Using terminal uridine deoxynucleotidyl nick end labeling staining, we demonstrated that high Ca(2+) (7.5 mM) and NPS R-467 indeed protect cells against apoptosis induced by serum withdrawal compared with low Ca(2+) (0.5 mM). Because the CaR induces PTHrP secretion, it is possible that the mitogenic and antiapoptotic effects of elevated Ca(2+) could be indirect and mediated via PTHrP. However, blocking the type 1 PTH receptor with PTH (7-34) peptide did not alter either high Ca(2+)-induced proliferation or protection against apoptosis. Taken together, our data show that activation of PI3K and p38 MAPK but not of MEK1/ERK by the CaR promotes proliferation of H-500 cells as well as affords protection against apoptosis. These effects are likely direct without the involvement of PTHrP in an autocrine mode.     

Protein kinase C modulates ecdysteroidogenesis in the prothoracic gland of the tobacco hornworm, Manduca sexta

The prothoracic gland is the primary source of ecdysteroid hormones in the immature insect. Ecdysteroids coordinate gene expression necessary for growth, molting and metamorphosis. Prothoracicotropic hormone (PTTH), a brain neuropeptide, regulates ecdysteroid synthesis in the prothoracic gland. PTTH stimulates ecdysteroid synthesis through a signal transduction cascade that involves at least four protein kinases: protein kinase A (PKA), p70 S6 kinase, an unidentified tyrosine kinase, and the extracellular signal-regulated kinase (ERK). In this report, the participation of protein kinase C (PKC) in PTTH signalling is demonstrated and characterized. PTTH stimulates PKC activity through a PLC and Ca(2+)-dependent pathway that is not cAMP regulated. Inhibition of PKC inhibits PTTH-stimulated ecdysteroidogenesis as well as PTTH-stimulated phosphorylation of ERK and its upstream regulator, MAP/ERK kinase (MEK). These observations reveal that the acute regulation of prothoracic gland steroidogenesis is dependent on a web of interacting kinase pathways, which probably converge on factors that regulate translation.     

Role of EGF Receptor and Pyk2 in endothelin-1-induced ERK activation in rat cardiomyocytes

G protein-coupled receptor (GPCR)-evoked signal transduction pathways leading to the activation of extracellular signal-regulated kinases (ERK) are quite different among cell types. In cardiomyocytes, much attention has been focused on the activation of protein kinase C (PKC) or mobilization of intracellular Ca(2+) ([Ca(2+)](i)), however, the contributions of tyrosine kinases are controversial. In the present study, we characterized the signaling pathways involving tyrosine kinases, Pyk2 and epidermal growth factor receptor (EGFR), and their contribution to ERK activation in cultured cardiomyocytes. We initially investigated the potential involvement of [Ca(2+)](i) and PKC on the activation of these kinases in endothelin-stimulated cardiomyocytes. Interestingly, activation of Pyk2 was abrogated by chelating [Ca(2+)](i) or by downregulation of PKC, whereas transactivation of EGFR was solely dependent on PKC. By using a compound that selectively interferes with EGFR (AG1478), c-Src (PP1), or disrupts actin cytoskeleton (cytochalasin D), we demonstrated that cytochalasin D completely inhibited the activation of Pyk2, but not that of EGFR, whereas AG1478 did not inhibit the activation of Pyk2, indicating that transactivation of EGFR and signaling pathways involving Pyk2 were distinct pathways. Furthermore, activation of ERK and Shc, and c- fos gene expression were significantly inhibited by AG1478 but not by cytochalasin D or PP1. Overexpression of deletion mutant of EGFR attenuated the activation of ERK. These facts demonstrated the existence of two distinct tyrosine kinase pathways requiring Pyk2 or EGFR downstream from GPCR in cardiomyocytes. EGFR was Ca(2+)-independently activated and predominantly contributed to Shc/ERK/c- fos activation, while Pyk2 or c-Src contributed less to it.     

Induction of c-fos and c-jun messenger ribonucleic acid expression by prostaglandin F2alpha is mediated by a protein kinase C-dependent extracellular signal-regulated kinase mitogen-activated protein kinase pathway in bovine luteal cells

PGF2alpha triggers the demise of the corpus luteum whereby progesterone synthesis is inhibited, the luteal structure regresses, and the estrus cycle resumes. Upon binding to its heterotrimeric G-protein-coupled receptors, PGF2alpha initiates the phospholipase C/diacylglycerol and inositol-1,4,5-trisphosphate/Ca(2+)-protein kinase C (PKC) signaling pathway. More recently, we have demonstrated that PGF2alpha activates extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase signaling through a Raf-dependent mechanism in bovine luteal cells. However, the relationship between PKC and ERK activation in PGF2alpha signaling has not been clearly defined. Moreover, the signaling pathway that PGF2alpha uses to regulate gene expression is unknown. In this report, primary cultures of bovine luteal cells were used to address the role of PKC in ERK activation and the signaling pathway for induction of c-fos and c-jun messenger RNA (mRNA) expression in response to PGF2alpha. By using a PKC inhibitor and a PKC-deficient luteal cell model, we observed that phorbol ester-responsive isoforms of PKC were required for ERK phosphorylation and activation by PGF2alpha (1 microM) or phorbol 12-myristate 13-acetate (PMA) (20 nM). In PGF2alpha- and PMA-treated cells, active ERK MAP kinase was localized in the nucleus. PGF2alpha-induced ERK phosphorylation was dose-dependently inhibited by the MEK1 inhibitor PD098059 (1-50 microM). The expression of c-fos and c-jun mRNA in luteal cells was markedly increased by treatment with PGF2alpha (1 microM) or PMA (20 nM) for 30 min. We also observed that activation of ERK MAP kinase was required for the expression of c-fos and c-jun mRNA in response to PGF2alpha and PMA because it was abrogated by blocking the ERK pathway with PD098059. In addition, PGF2alpha and PMA-induced c-fos and c-jun mRNA expression was abolished in the PKC-deficient cells. Taken together, our data demonstrate that a PKC-dependent ERK MAP kinase pathway mediates the expression of c-fos and c-jun mRNA in PGF2alpha-treated bovine luteal cells.     

Noradrenaline-induced paxillin phosphorylation, ERK activation and MEK-regulated contraction in intact rat mesenteric arteries

In rat mesenteric arteries, noradrenaline (NA) induces a time-dependent increase in tyrosine phosphorylation of a number of proteins, one of which was identified as paxillin. NA-induced protein tyrosine phosphorylation was ablated by tyrosine kinase inhibition, virtually unaffected by protein kinase C (PKC) inhibition or PKC downregulation and was mimicked by KCl. NA also caused a time-dependent activation of the extracellular signal-regulated kinases (ERK)1 and ERK2. These responses were blocked by the ERK-activating kinase (MEK) inhibitor PD98059 and by tyrosine kinase inhibition but only modestly attenuated by PKC downregulation or inhibition. Pretreatment of cannulated mesenteric arteries (50 mm Hg internal pressure) with PD98059 significantly reduced the contractile responsiveness of the vessels to NA (1.56 +/- 0.14 microM, EC(50) control; 3.32 +/- 0.49 microM, EC(50) + PD98059, p < 0.01). Thus, NA induces time-dependent increases in protein-tyrosine phosphorylation and ERK activation in rat mesenteric arteries that could suggest a role for Ca(2+)-dependent non-receptor tyrosine kinases and ERKs in the response of small arteries to NA. In addition, the modulation of NA-induced mesenteric artery contraction by inhibition of the MEK/ERK pathway further implicates ERK in the regulation of, though perhaps not the mediation of NA-induced small artery contraction.     

Extracellular signal-regulated protein kinase, but not c-Jun N-terminal kinase, is activated by type II gonadotropin-releasing hormone involved in the inhibition of ovarian cancer cell proliferation

Although a novel second form of GnRH (GnRH-II) has been reported to have an antiproliferative effect on gynecologic cancer cells, its biological mechanism remains to be elucidated. We have previously demonstrated that GnRH-II activates p38 MAPK. There is accumulating evidence that activation of MAPKs by GnRH-I and -II is important for cell proliferation, differentiation, and apoptosis. In the present study, we further investigated the involvement of GnRH-II in the inhibition of cell proliferation and activation of ERK1/2 and c-Jun N-terminal protein kinase/stress-activated protein kinase (JNK/SAPK) in ovarian cancer cells, OVCAR-3. The [(3)H]thymidine incorporation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays revealed that treatment with GnRH-II suppresses cell proliferation of ovarian cancer cells. Western blot analysis demonstrated that ERK1/2 was activated by GnRH-II (100 nm). Moreover, PD98059 (10 mum), an inhibitor of a MAPK/ERK kinase, reversed the activation of ERK1/2 induced by GnRH-II. The activation of ERK1/2 by GnRH-II subsequently phosphorylated Elk-1 as a downstream pathway, which was blocked by PD98059. On the other hand, it is not likely that GnRH-II activates the JNK/SAPK pathway. Taken together, these results indicate that the ERK1/2 pathway is involved in the effect of GnRH-II on antiproliferation and may be an important target for ovarian cancer therapy.     

Mixed-lineage kinase 3 regulates B-Raf through maintenance of the B-Raf/Raf-1 complex and inhibition by the NF2 tumor suppressor protein

The Ras --> Raf --> MEK1/2 --> extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway couples mitogenic signals to cell proliferation. B-Raf and Raf-1 function within an oligomer wherein they are regulated in part by mutual transactivation. The MAPK kinase kinase (MAP3K) mixed-lineage kinase 3 (MLK3) is required for mitogen activation of B-Raf and cell proliferation. Here we show that the kinase activity of MLK3 is not required for support of B-Raf activation. Instead, MLK3 is a component of the B-Raf/Raf-1 complex and is required for maintenance of the integrity of this complex. We show that the activation of ERK and the proliferation of human schwannoma cells bearing a loss-of-function mutation in the neurofibromatosis 2 (NF2) gene require MLK3. We find that merlin, the product of NF2, blunts the activation of both ERK and c-Jun N-terminal kinase (JNK). Finally, we demonstrate that merlin and MLK3 can interact in situ and that merlin can disrupt the interactions between B-Raf and Raf-1 or those between MLK3 and either B-Raf or Raf-1. Thus, MLK3 is part of a multiprotein complex and is required for ERK activation. The levels of this complex may be negatively regulated by merlin.