MEK inhibitors block AICAR-induced maturation in mouse oocytes by a MAPK-independent mechanism

The present study was carried out to assess the possible role of mitogen-activated protein kinase (MAPK) in the meiosis-inducing action of the AMP-activated protein kinase (AMPK) activator, 5-aminoimidazole-4-carboxamide 1-beta-ribofuranoside (AICAR). Cumulus cell-enclosed oocytes (CEO) or denuded oocytes (DO) from immature, eCG-primed mice were cultured 4 hr in Eagle's minimum essential medium containing dbcAMP plus increasing concentrations of AICAR or okadaic acid (OA). OA is a phosphatase inhibitor known to stimulate both meiotic maturation and MAPK activation and served as a positive control. Both OA and AICAR were potent inducers of meiotic resumption in mouse oocytes and brought about the phosphorylation (and thus, activation) of MAPK, but by different kinetics: MAPK phosphorylation preceded GVB in OA-treated oocytes, while that resulting from AICAR treatment appeared only after GVB. The MEK inhibitors, PD98059 and U0126, blocked the meiotic resumption induced by AICAR but not that induced by OA. Although the MEK inhibitors suppressed MAPK phosphorylation in both OA- and AICAR-treated oocytes, meiotic resumption was not causally linked to MAPK phosphorylation in either group. Furthermore, AICAR-induced meiotic resumption in Mos-null oocytes (which are unable to stimulate MAPK) was also abrogated by PD98059 treatment. A non-specific effect of the MEK inhibitors on AICAR accessibility to the oocyte was discounted by showing that they failed to suppress either nucleoside uptake or AICAR-stimulated phosphorylation of acetyl CoA carboxylase (ACC), a substrate of AMPK. The suppression of AICAR-induced maturation by MEK inhibitors must, therefore, be occurring by actions unrelated to MEK stimulation of MAPK; consequently, it would be prudent to consider this possible non-specific action of the inhibitors when they are used to block MAPK activation in mouse oocytes.     

Positive regulation of ERK activation and MKP-1 expression by peroxovanadium complex bpV (phen)

Lower micromolar concentrations of peroxovanadium compound potassium bisperoxo(1,10-phenanthroline)oxovanadate (V) [bpV (phen)] stimulate RINm5F cell metabolic activity. 1 and 3 mol/L bpV (phen) induces strong and sustained activation of extracellular signal-regulated kinase (ERK). However, it seems that bpV (phen) does not effect c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) phosphorylation. In addition, bpV (phen) induces mitogen-activated protein kinase phosphatase-1 (MKP-1) expression. We found that ERK activation could be completely abolished if RINm5F cells were incubated with both bpV (phen) and PD 98059, a specific inhibitor of upstream ERK kinase MEK1. On the other hand, this combined treatment up-regulated activation of stress kinases, JNK and p38 MAPK, significantly suppressed MKP-1 expression and induced cell death. Thus, our results suggest that the mechanism underlying bpV (phen) survival-enhancing effect could be associated with induced ERK activation and MKP-1 expression.     

Mos activates MAP kinase in mouse oocytes through two opposite pathways

Activation of mitogen-activated protein kinase (MAPK) in maturing mouse oocytes occurs after synthesis of Mos, a MAPKKK. To investigate whether Mos acts only through MEK1, we microinjected constitutively active forms of MEK1 (MEK1S218D/S222D referred herein as MEK*) and Raf (DeltaRaf) into mouse oocytes. In mos(-/-) oocytes, which do not activate MAPK during meiosis and do not arrest in metaphase II, MEK* and DeltaRaf did not rescue MAPK activation and metaphase II arrest, whereas Mos induced a complete rescue. MEK* and DeltaRaf induced cleavage arrest of two-cell blastomeres. They induced MAPK activation when protein phosphatases were inhibited by okadaic acid, suggesting that Mos may inhibit protein phosphatases. Finally, in mos(-/-) oocytes, MEK* induced the phosphorylation of Xp42(mapk)D324N, a mutant less sensitive to dephosphorylation, showing that a MAPK phosphatase activity is present in mouse oocytes. We demonstrate that active MAPKK or MAPKKK cannot substitute for Mos to activate MAPK in mouse oocytes. We also show that a phosphatase activity inactivates MAPK, and that Mos can overcome this inhibitory activity. Thus Mos activates MAPK through two opposite pathways: activation of MEK1 and inhibition of a phosphatase.     

ATMPK4, an Arabidopsis homolog of mitogen-activated protein kinase, is activated in vitro by AtMEK1 through threonine phosphorylation

The modulation of mitogen-activated protein kinase (MAPK) activity regulates many intracellular signaling processes. In animal and yeast cells, MAP kinases are activated via phosphorylation by the dual-specificity kinase MEK (MAP kinase kinase). Several plant homologs of MEK and MAPK have been identified, but the biochemical events underlying the activation of plant MAPKs remain unknown. We describe the in vitro activation of an Arabidopsis homolog of MAP kinase, ATMPK4. ATMPK4 was phosphorylated in vitro by an Arabidopsis MEK homolog, AtMEK1. This phosphorylation occurred principally on threonine (Thr) residues and resulted in elevated ATMPK4 kinase activity. A second Arabidopsis MEK isoform, ATMAP2Kalpha, failed to phosphorylate ATMPK4 in vitro. Tyr dephosphorylation by the Arabidopsis Tyr-specific phosphatase AtPTP1 resulted in an almost complete loss of ATMPK4 activity. Immunoprecipitates of Arabidopsis extracts with anti-ATMPK4 antibodies displayed myelin basic protein kinase activity that was sensitive to treatment with AtPTP1. These results demonstrate that a plant MEK can phosphorylate and activate MAPK, and that Tyr phosphorylation is critical for the catalytic activity of MAPK in plants. Surprisingly, in contrast to the animal enzymes, AtMEK1 may not be a dual-specificity kinase but, rather, the required Tyr phosphorylation on ATMPK4 may result from autophosphorylation.     

Involvement of Ca2+-dependent proteasome in the degradation of both cyclin B1 and Mos during spontaneous activation of matured rat oocytes

In matured rat oocytes, spontaneous activation from the metaphase-II (MII) stage occurred after collection from the oviducts. It is well known that the mitogen-activated protein kinase (MAPK) pathway and p34(cdc2) kinase play an important role in the arrest at MII in other species. However, there is no information about the difference in these factors among strains of rats. In the present study, in spontaneously activated oocytes from the Wistar rat, the Mos protein level and the activity of MAPK kinase (MEK)/MAPK were decreased at 120 min (13.8, 25.7, and 19.3, respectively, P<0.05), whereas Sprague-Dawley (SD) oocytes, which were not spontaneously activated, had a high level of Mos protein and MEK/MAPK activity (75.9, 76.2, and 87.9, respectively, P<0.05). Phosphorylation of MAPK in the SD oocytes was significantly suppressed by MEK inhibitor, U0126 at 60 min; this treatment decreased p34(cdc2) kinase activity via cyclin B1 degradation in a time-dependent manner. The treatment with proteasome inhibitor, MG132 or Ca2+-chelator, BAPTA-AM, overcame the spontaneous degradation of both Mos and cyclin B1 in a dose-dependent manner in Wistar oocytes. More than 90% of Wistar oocytes treated with BAPTA-AM were arrested at MII until 120 min. In conclusion, SD oocytes carrying Mos/MEK/MAPK, maintained a high activity of p34(cdc2) kinase by stabilizing cyclin B1, thus involved in their meiotic arrest. In contrast, Wistar oocytes had a relatively low cytostatic factor activity; rapid decrease of Mos/MEK/MAPK failed to stabilize both cyclin B1 and Mos, and these oocytes were likely to spontaneously activate.     

Effects of MEK inhibitor U0126 on meiotic progression in mouse oocytes: microtuble organization, asymmetric division and metaphase II arrest

In this study we used U0126, a potent and specific inhibitor of MEK, to study the roles of MEK/ERK/p90rsk signaling pathway in the meiotic cell cycle of mouse oocytes. The phosphorylation of MAP kinase and p90rsk in the oocytes treated with 1.5 microM U0126 was the same as that in oocytes cultured in drug-free medium. With 1.5 microM U0126 treatment, the spindles appeared normal as they formed in oocytes, but failed to maintain its structure. Instead, the spindle lost one pole or elongated extraordinarily. After further culture, some oocytes extruded gigantic polar bodies (>30 microm) that later divided into two small ones. Some oocytes underwent symmetric division and produced two equal-size daughter cells in which normal spindles formed. In oocytes with different division patterns, MAP kinase was normally phosphorylated. When the concentration of U0126 was increased to 15 mM, the phosphorylation of both MAPK and p90rsk were inhibited, while symmetric division was decreased. When incubating in medium containing 15 microM U0126 for 14 h, oocytes were activated, but part of them failed to emit polar bodies. MII oocytes were also activated by 15 microM U0126, at the same time the dephosphorylation of MAP kinase and p90rsk was observed. Our results indicate that 1) MEK plays important but not indispensable roles in microtubule organization; 2) MEK keeps normal meiotic spindle morphology, targets peripheral spindle positioning and regulates asymmetric division by activating some unknown substrates other than MAP kinase /p90rsk; and 3) activation of MEK/ERK/p90rsk cascade maintains MII arrest in mouse oocytes.     

Xp42(Mpk1) activation is not required for germinal vesicle breakdown but for Raf complete phosphorylation in insulin-stimulated Xenopus oocytes

Fully grown G2-arrested Xenopus oocytes resume meiosis in vitro upon exposure to hormonal stimulation. Progesterone triggers oocyte meiosis resumption through a Ras-independent pathway that involves a p39Mos-dependent activation of the mitogen-activated protein (MAP) kinases. Insulin also triggers meiosis resumption through a tyrosine kinase receptor that activates a Ras-dependent pathway leading to the MAP kinases activation. Antisense phosphorothioate oligonucleotides were used to prevent p39Mos accumulation and Erk-like Xp42(Mpk1) activation during insulin-induced Xenopus oocytes maturation. In contrast to previous works, prevention of p39Mos-induced activation of Xp42(Mpk1) in insulin-treated oocytes did not inhibit but delayed meiotic resumption, like in progesterone-stimulated oocytes. Activations of Xp42(Mpk1), the unique Erk of the oocyte, and of its downstream target p90Rsk, were impaired and phosphorylation of the MAPKK kinase Raf was partially inhibited. Similarly, oocytes treated with the MEK inhibitor U0126, stimulated by insulin exhibited delayed germinal vesicle breakdown, absence of Xp42(Mpk1) activation, and partial phosphorylation of Raf. To summarize, whereas p39Mos-induced activation of MEK/MAPK pathway is dispensable for insulin-induced germinal vesicle breakdown, Xp42(Mpk1) activation induced by insulin is dependent upon p39Mos synthesis. Raf complete phosphorylation appears to require the MEK/MAPK pathway activation both in progesterone and insulin-stimulated oocytes.     

Characterization of ribosomal S6 protein kinase p90rsk during meiotic maturation and fertilization in pig oocytes: mitogen-activated protein kinase-associated activation and localization

Mitogen-activated protein kinase (MAPK) becomes activated during the meiotic maturation of pig oocytes, but its physiological substrate is unknown. The 90-kDa ribosome S6 protein kinase (p90rsk) is the best known MAPK substrate in Xenopus and mouse oocytes. The present study was designed to investigate the expression, phosphorylation, subcellular localization, and possible roles of p90rsk in porcine oocytes during meiotic maturation, fertilization, and parthenogenetic activation. This kinase was partially phosphorylated in oocytes at germinal vesicle (GV) stage through a MAPK-independent mechanism, but its full phosphorylation is dependent on MAPK activity. After fertilization or electrical activation, p90rsk was dephosphorylated shortly before pronucleus formation, which coincided with the inactivation of MAPK. A protein phosphatase inhibitor, okadaic acid, accelerated the phosphorylation of p90rsk during meiotic maturation and induced its rephosphorylation in activated eggs. MAPK kinase (MAPKK or MEK) inhibitor U0126 inhibited the activation of MAPK and p90rsk in both cumulus-enclosed and denuded pig oocytes, but prevented GV breakdown (GVBD) only in cumulus-enclosed oocytes. Active MAPK and p90rsk were detected in pig cumulus cells, and U0126 induced their dephosphorylation. In meiosis II arrested eggs, U0126 led to the inactivation of MAPK and p90rsk, as well as the interphase transition of the eggs. P90rsk was distributed evenly in GV oocytes, but it accumulated in the nucleus before GVBD. It was localized to the meiotic spindle after GVBD and concentrated in the spindle mid zone during emission of the polar bodies. All these results suggest that p90rsk is downstream of MAPK and plays functional roles in the regulation of nuclear status and microtubule organization. Although MAPK and p90rsk activity are not essential for the spontaneous meiotic resumption in denuded oocytes, activation of this cascade in cumulus cells is indispensable for the gonadotropin-induced meiotic resumption of pig oocytes.     

B-Raf is critical for MAPK activation during mitosis and is regulated in an M phase-dependent manner in Xenopus egg extracts

Activation of the MAPK cascade during mitosis is critical for spindle assembly and normal mitotic progression. The underlying regulatory mechanisms that control activation of the MEK/MAPK cascade during mitosis are poorly understood. Here we purified and characterized the MEK kinase activity present in Xenopus M phase-arrested egg extracts. Our results show that B-Raf was the critical MEK kinase required for M phase activation of the MAPK pathway. Consistent with this, B-Raf was activated and underwent hyperphosphorylation in an M phase-dependent manner. Interestingly B-Raf hyperphosphorylation at mitosis occurred, at least in part, as a consequence of a feedback loop involving MAPK-mediated phosphorylation within a conserved C-terminal SPKTP motif. The kinase activity of a B-Raf mutant defective at both phosphorylation sites was substantially greater than its wild type counterpart when incubated in Xenopus M phase egg extracts. Furthermore suppression of MAPK feedback at mitosis enhanced B-Raf activity, whereas constitutive activation of MAPK at mitosis strongly suppressed B-Raf activity. These results suggest that feedback phosphorylation by MAPK negatively regulates B-Raf activity at mitosis. Collectively our data demonstrate for the first time a role for B-Raf at mitosis and provide new insight into understanding the regulation and function of B-Raf during cell proliferation.     

The effect of PD98059 on MAPK regulation in cumulus-enclosed and cumulus-free mouse oocytes

The effect of the p42/44 mitogen-activated kinase (MAPK) inhibitor, PD98059, on MAPK activation and meiosis resumption in mouse oocytes was studied. When germinal vesicle (GV)-stage denuded oocytes (DOs) were cultured continuously in 50 microM PD98059, germinal vesicle breakdown (GVBD) was postponed for 2-3 h. MAPK phosphorylation and activation was delayed as well. However, PD98059 did not impair histone H1 kinase activation. After 14 h of culture there was no significant difference in the rate of DOs reaching metaphase II (MII) arrest in either control or experimental conditions. The effect of PD98059 on MAPK inhibition was further tested in epidermal growth factor (EGF)-treated oocytecumulus complexes (OCCs). Exposure of GV-stage OCCs for 5 min to EGF (10 ng/ml) induced a considerable increase in MAPK phosphorylation. After OCCs were further cultured in 50 microM PD98059 a rapid dephosphorylation of MAPK was induced. Already after 1 min of treatment the non-phosphorylated form of MAPK dominated, indicating the high effectivity of PD98059. This result indicates that short EGF/PD98059 treatment of OCCs induced MAPK phosphorylation/dephosphorylation in cumulus cells only. As only a transient delay in MAPK phosphorylation and activation was observed in PD98059-treated DOs we conclude that there is also another PD98059-nonsensitive pathway(s) leading to MAPK activation in mouse oocytes. The data obtained suggest that meiosis resumption in mouse oocytes is somehow influenced by the MEK/MAPK activation pathway.     

MAP and cdc2 Kinase Activities at Germinal Vesicle Breakdown inChaetopterus

In frog oocytes, activation of mitogen-activated protein kinase (MAPK, ERK) leads to activation of cdc2 and germinal vesicle breakdown (GVBD). By contrast, in starfish, MAPK is activated after GVBD. Here we have examined the relative involvements of MAPK and cdc2 in GVBD ofChaetopterusoocytes. MAPK was rapidly tyrosine-phosphorylated and activated (within 1–2 min) in response to exposure of the oocytes either to natural seawater (the normal trigger of GVBD in this organism) or to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), which can also elicit GVBD. This response preceded the tyrosine dephosphorylation and activation of cdc2 by several minutes. MAPK phosphorylation and activation were transient, lasting only until GVBD occurred and the spindle migrated to the cortex. The enzyme was not phosphorylated again as a result of egg activation. These results are consistent with the hypothesis that the activation of MAPK has a role in GVBD. However, PD 98059, a potent and selective inhibitor of MEK, the protein kinase that phosphorylates and activates MAPK, blocked the phosphorylation of MAPK but did not block GVBD, the dephosphorylation and activation of cdc2, or spindle formation and migration. Oocytes that underwent GVBD in PD 98059 could be fertilized and cleaved normally. Ionophore A23187, although it caused germinal vesicles to disappear and caused transient phosphorylation of MAPK, did not cause dephosphorylation of cdc2, and therefore this disappearance is artifactual. These results suggest that MAPK activation is neither obligatory nor sufficient for either GVBD or meiotic metaphase arrest inChaetopterusand that activation of MAPK and cdc2 occur on independent, parallel pathways.     

Phosphorylation pattern of the p90rsk and mitogen-activated protein kinase (MAPK) molecule: comparison of in vitro and in vivo matured porcine oocytes

The overall objective was to elucidate the phosphorylation pattern and activity of the kinase p90rsk, a substrate of mitogen-activated protein kinase (MAPK), during in vitro and in vivo maturation of pig oocytes. Cumulus-oocyte complexes were collected from slaughtered pigs and matured in vitro (0, 22, 26, 30, 34, 46 h) with and without the MEK inhibitor U0126. For in vivo maturation, gilts were stimulated with equine chorionic gonadotrophin (eCG) (600-800 IU). Maturation was induced 72 h later with hCG (500 IU). Oocytes were obtained surgically (0, 22, 30 h). The samples were submitted to electrophoresis and protein blotting analysis. Enhanced chemiluminescence was used for visualization. In vitro matured oocytes were further submitted to a commercially available radioactive kinase assay to determine kinase activity. It was shown that oocytes, as well as cumulus cells, already possess a partially phosphorylated p90rsk at the time of removal from follicles, with a further phosphorylation of the molecule occurring between 22-24 h after the initiation of culture, and in vivo maturation. The phosphorylation of p90rsk coincides with the phosphorylation of MAPK and can be prevented by U0126, indicating a MAPK-dependent phosphorylation of p90rsk. Phosphorylation of the in vivo matured oocytes occurred shown as a band of less than 200 kDa. This is presumably a molecule complex, with MAPK not being a component. Therefore, the p90rsk molecule in vivo exists as a dimer. Determination of kinase activity demonstrated decreasing enzyme activities. This led to the conclusion that the assay is not specific for p90rsk, instead measuring p70S6 kinase activities.     

OxLDL induces mitogen-activated protein kinase activation mediated via PI3-kinase/Akt in vascular smooth muscle cells

Oxidized low-density lipoprotein (OxLDL) is a risk factor in atherosclerosis and stimulates multiple signaling pathways, including activation of phosphatidylinositol 3-kinase (PI3-K)/Akt and p42/p44 mitogen-activated protein kinase (MAPK), which are involved in mitogenesis of vascular smooth muscle cells (VSMCs). We therefore investigated the relationship between PI3-K/Akt and p42/p44 MAPK activation and cell proliferation induced by OxLDL. OxLDL stimulated Akt phosphorylation in a time- and concentration-dependent manner, as determined by Western blot analysis. Phosphorylation of Akt stimulated by OxLDL and epidermal growth factor (EGF) was attenuated by inhibitors of PI3-K (wortmannin and LY294002) and intracellular Ca2+ chelator (BAPTA/AM) plus EDTA. Pretreatment of VSMCs with pertussis toxin, cholera toxin, and forskolin for 24 h also attenuated the OxLDL-stimulated Akt phosphorylation. In addition, pretreatment of VSMCs with wortmannin or LY294002 inhibited OxLDL-stimulated p42/p44 MAPK phosphorylation and [3H]thymidine incorporation. Furthermore, treatment with U0126, an inhibitor of MAPK kinase (MEK)1/2, attenuated the p42/p44 MAPK phosphorylation, but had no effect on Akt activation in response to OxLDL and EGF. Overexpression of p85-DN or Akt-DN mutants attenuated MEK1/2 and p42/p44 MAPK phosphorylation stimulated by OxLDL and EGF. These results suggest that the mitogenic effect of OxLDL is, at least in part, mediated through activation of PI3-K/Akt/MEK/MAPK pathway in VSMCs.     

Inhibition of the MEK/ERK signaling pathway by the novel antimetastatic agent NAMI-A down regulates c-myc gene expression and endothelial cell proliferation.

Imidazolium trans-imidazoledimethyl sulfoxide-tetrachlororuthenate (NAMI-A) is a novel ruthenium-containing experimental antimetastatic agent. Compelling evidence ascribes a pivotal role to endothelial cells in the orchestration of tumor angiogenesis and metastatic growth, suggesting antiangiogenic therapy as an attractive approach for anticancer treatment. In this context, activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathway has been found fundamental in transducing extracellular stimuli that modulate a number of cellular process including cell proliferation, migration and invasion. Here we show that exposure of the transformed endothelial cell line ECV304 to NAMI-A significantly inhibited DNA synthesis, as well as the expression of the proliferating cell nuclear antigene (PCNA). These responses were associated with a marked down-regulation of ERK phosphorylation in serum-cultured cells. In addition, NAMI-A markedly reduced serum stimulated- and completely suppressed phorbol 12-myristate 13-acetate (PMA)-triggered MAPK/ERK kinase activity. NAMI-A was also able to inhibit the phosphorylation of MEK, the upstream activator of ERK, and, similar to both the protein kinase C (PKC) inhibitor GF109203X and the MAPK/ERK (MEK) inhibitor PD98059, it completely counteracted PMA-induced ERK phosphorylation. Finally, NAMI-A and PD98059 down regulated c-myc gene expression to the same extent in serum-cultured cells and dose-dependently counteracted, and ultimately abolished, the increase in c-myc gene expression elicited by PMA in serum-free cells. These results suggest that inhibition of MEK/ERK signaling by NAMI-A may have an important role in modulating c-myc gene expression and ECV304 proliferation.     

Zinc stimulates the activity of the insulin- and nutrient-regulated protein kinase mTOR

Recent studies indicate that zinc activates p70 S6 kinase (p70(S6k)) by a mechanism involving phosphatidylinositol 3-kinase (PI 3-kinase) and Akt (protein kinase B). Here it is shown that phenanthroline, a zinc and heavy metal chelator, inhibited both amino acid- and insulin-stimulated phosphorylation of p70(S6k). Both amino acid and insulin activations of p70(S6k) involve a rapamycin-sensitive step that involves the mammalian target of rapamycin (mTOR, also known as FRAP and RAFT). However, in contrast to insulin, amino acids activate p70(S6k) by an unknown PI 3-kinase- and Akt-independent mechanism. Thus the effects of chelator on amino acid activation of p70(S6k) were surprising. For this reason, we tested the hypothesis that zinc directly regulates mTOR activity, independently of PI 3-kinase activation. In support of this, basal and amino acid stimulation of p70(S6k) phosphorylation was increased by zinc addition to the incubation media. Furthermore, the protein kinase activities of mTOR immunoprecipitated from rat brain lysates were stimulated two- to fivefold by 10-300 microM Zn2+ in the presence of an excess of either Mn2+ or Mg2+, whereas incubation with 1,10-phenanthroline had no effect. These findings indicate that Zn2+ regulates, but is not absolutely required for, mTOR protein kinase activity. Zinc also stimulated a recombinant human form of mTOR. The stimulatory effects of Zn2+ were maximal at approximately 100 microM but decreased and became inhibitory at higher physiologically irrelevant concentrations. Micromolar concentrations of other divalent cations, Ca2+, Fe2+, and Mn2+, had no effect on the protein kinase activity of mTOR in the presence of excess Mg2+. Our results and the results of others suggest that zinc acts at multiple steps in amino acid- and insulin cell-signaling pathways, including mTOR, and that the additive effects of Zn2+ on these steps may thereby promote insulin and nutritional signaling.     

Mitogen-activated protein kinase signalling pathways triggered by the hepatitis C virus envelope protein E2: implications for the prevention of infection

OBJECTIVE: Hepatitis C virus (HCV) is a major pathogenic factor of liver diseases. During HCV infection, interaction of the envelope protein E2 of the virion, with target cells, is a crucial process for viral penetration into the cell and its propagation. We speculate that such interaction may trigger early signalling events required for HCV infection. MATERIALS AND METHODS: Human liver cell line L-02 was treated with HCV E2. The kinase phosphorylation levels of mitogen-activated protein kinase (MAPK) signalling pathways in the treated cells were analyzed by Western blotting. The proliferation of the E2-treated cells was evaluated by MTT assay. RESULTS: HCV E2 was shown to be an efficient activator for MAPK pathways. Levels of phosphorylation of upstream kinases Raf-1 and MEK1/2 were seen to be elevated following E2 treatment and similarly, phosphorylation levels of downstream kinases MAPK/ERK and p38 MAPK also increased in response to E2 treatment, and specificity of kinase activation by E2 was confirmed. E2-induced MAPK/ERK activation was inhibited by the MEK1/2 inhibitor U0126 in a concentration-dependent manner. Blockage of relevant cellular receptors reduced activation of Raf-1, MEK1/2, MAPK/ERK and p38 MAPK by E2, indicating efflux of the E2 signal from extracellular to the intracellular spaces. Thus, kinase cascades of MAPK pathways were continuously affected by E2 presence. Moreover, enhancement of cell proliferation by E2 appeared to be associated with the dynamic phosphorylation of MAPK/ERK and p38 MAPK. CONCLUSION: These results suggest that MAPK signalling pathways triggered by E2 may be a potential target for prevention of HCV infection.     

Bradykinin B<Subscript>2</Subscript> receptor-mediated proliferation via activation of the Ras/Raf/MEK/MAPK pathway in rat vascular smooth muscle cells

It has been suggested that bradykinin (BK) plays an important role in regulating neointimal formation after vascular injury. However, implication of BK in the growth of rat vascular smooth muscle cells (VSMCs) is controversial. Therefore, we examined the mitogenic effect of BK on VSMCs associated with activation of mitogen-activated protein kinase (MAPK). Both [(3)H]thymidine incorporation and p42/p44 MAPK phosphorylation were activated by BK in time- and concentration-dependent manners. Pretreatment of these cells with neither pertussis toxin nor cholera toxin attenuated the BK-induced responses. Pretreatment of VSMCs with Hoe 140 (a selective B(2) receptor antagonist), U73122 (an inhibitor of phospholipase C), and BAPTA/AM (an intracellular Ca(2+) chelator) inhibited both [(3)H]thymidine incorporation and p42/p44 MAPK phosphorylation in response to BK. BK-induced [(3)H]thymidine incorporation and p42/p44 MAPK phosphorylation were inhibited by pretreatment of VSMCs with tyrosine kinase inhibitors (genistein and herbimycin A), protein kinase C (PKC) inhibitors (staurosporine, Go-6976, and Ro-318220), an MAPK kinase inhibitor (PD98059), and a p38 MAPK inhibitor (SB203580). Overexpression of the dominant negative mutants, H-Ras-15A and Raf-N4, suppressed p42/p44 MAPK activation induced by BK and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. From these results, we concluded that the mitogenic effect of BK is mediated through activation of the Ras/Raf/MEK/MAPK pathway similar to that of PDGF-BB. BK-mediated MAPK activation was modulated by Ca(2+), PKC, and tyrosine kinase all of which are associated with cell proliferation in rat cultured VSMCs.     

Mitogen-activated protein kinase phosphorylation patterns in pig oocytes and cumulus cells during gonadotrophin-induced resumption of meiosis in vitro

The present study investigated the phosphorylation pattern of mitogen-activated protein kinase (MAPK) in cumulus-oocyte complexes (COCs) during spontaneous and FSH/LH-induced in vitro maturation (IVM). Both isoforms of MAPK were unphosphorylated in oocytes recovered immediately after liberation from follicles and became phosphorylated following 25 h incubation, corresponding to the time of germinal vesicle breakdown (GVBD). In contrast, MAPK was already phosphorylated in minimal amounts in cumulus cells at the time of liberation from follicles and phosphorylation of MAPK increased after 0.5 h incubation. Supplementation of medium with gonadotrophins intensified phosphorylation at 0.5 h incubation, demonstrating the early and rapid action of FSH/LH on MAPK phosphorylation. Phosphorylation of MAPK in cumulus cells peaked after 21 h of incubation, whereas MAPK was almost completely dephosphorylated at the end of incubation (45 h). During subsequent incubation in the absence of added gonadotrophins, between 5 and 10 h exposure to FSH/LH-supplemented medium was required to induce resumption of meiosis in COCs. Phosphorylation of MAPK in oocytes was prevented by the MEK inhibitor U0126, but the inhibitor reduced phosphorylation of MAPK in cumulus cells only during the first 2 h of IVM. The data support the hypothesis that two different MAPK phosphorylation events occurred following gonadotrophin stimulation, one in cumulus cells and the other in oocytes. In cumulus cells, FSH/LH induced early and rapid U0126-insensitive phosphorylation of MAPK, whereas U0126-susceptible MAPK phosphorylation took place in the oocyte itself around the time of GVBD.     

An enzyme-linked immunosorbent assay for the Raf/MEK1/MAPK signaling cascade.

The Ras-MAPK signaling cascade transmits mitogenic stimuli from growth factor receptors and activated Ras to the cell nucleus. Inappropriate Ras activation is associated with approximately 30% of all human cancers. The kinase components of the Ras-MAPK signaling cascade are attractive targets for pharmaceutical intervention. Therefore, we have developed a high-throughput, nonradioactive ELISA method to monitor Raf and MEK1 kinase activity. In this assay system activated Raf phosphorylates and activates MEK1, which in turn phosphorylates MAPK. Antibodies that specifically detect phosphorylated MAPK (vs. nonphosphorylated MAPK) made enzyme-linked immunosorbent assay (ELISA) development possible. This assay detects inhibitors of Raf and/or MEK1 and has been used to screen large numbers of random compounds. The specific target of inhibition in the Raf/MEK1/MAPK ELISA can be subsequently identified by secondary assays which directly measure Raf phosphorylation of MEK1 or MEK1 phosphorylation of MAPK.