p38MAP kinase is a negative regulator for ERK1/2-mediated growth of AIDS-associated Kaposi's sarcoma cells.

AIDS-associated Kaposi's sarcoma (KS) is a cytokine-mediated tumor, at least in the early stages of this disease; however, there is at present no definitive consensus regarding the exact role of intracellular signaling pathways involved in growth of KS cells. We found that KS cell growth factors oncostatin M, sIL-6R/IL-6, TNFalpha, and IL-1beta all activate ERK1/2, and selective blockage of this kinase by PD98059 resulted in a profound inhibition of the cytokine-induced KS cell growth. Concurrently with activation of ERK1/2, these growth factors phosphorylated and activated p38MAPK. The selective inhibition of p38MAPK by SB203580 prominently enhanced the cytokine-induced proliferation of KS cells, thereby indicating that p38MAPK has a negative feedback on mitogenic signals. As these KS cell growth factors lead to simultaneous activation of ERK1/2 and p38MAPK signaling pathways, the concerted effects of these kinase activities may well determine the intensity of cellular proliferative responses to these growth factors.     

Prostacyclin receptor-mediated activation of extracellular signal-regulated kinases 1 and 2

The prostacyclin mimetic cicaprost increased phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in Chinese hamster ovary cells transiently expressing human (hIP-CHO) or mouse prostacyclin (mIP-CHO) receptors, but not in human neuroblastoma SK-N-SH cells or rat/mouse neuroblastoma-glioma NG108-15 cells which endogenously express IP receptors. Cicaprost stimulated ERK1/2 activity in hIP-CHO and mIP-CHO cells with EC50 values of 60 and 83 nM, respectively, and this response was significantly inhibited by protein kinase C inhibitors and agents which elevate cyclic AMP. A poor correlation was discovered between the level of ERK1/2 activity and the ability of agents to increase or decrease cyclic AMP production. The potent inhibitory effect of 3-isobutyl-1-methyl xanthine on cicaprost-stimulated phospho-ERK1/2 may be due to inhibition of phosphoinositide 3-kinase. Therefore, IP receptor-mediated activation of ERK1/2 in CHO cells occurs through a Gq/11/protein kinase C-dependent and a phosphoinoside 3-kinase-dependent process which is insensitive to IP receptor-generated cyclic AMP.     

Mitochondrial extracellular signal-regulated kinases 1/2 (ERK1/2) are modulated during brain development

Intracellular activation and trafficking of extracellular signal-regulated protein kinases (ERK) play a significant role in cell cycle progression, contributing to developmental brain activities. Additionally, mitochondria participate in cell signalling through energy-linked functions, redox metabolism and activation of pro- or anti-apoptotic proteins. The purpose of the present study was to analyze the presence of ERK1/2 in mitochondria during rat brain development. Immunoblotting, immune electron microscopy and activity assays demonstrated that ERK1/2 are present in fully active brain mitochondria at the outer membrane/intermembrane space fraction. Besides, it was observed that ERK1/2 translocation to brain mitochondria follows a developmental pattern which is maximal between E19-P2 stages and afterwards declines at P3, just before maximal translocation to nucleus, and up to adulthood. Most of mitochondrial ERK1/2 were active; upstream phospho-MAPK/ERK kinases (MEK1/2) were also detected in the brain organelles. Mitochondrial phospho-ERK1/2 increased at 1 microm hydrogen peroxide (H(2)O(2)) concentration, but it decreased at higher 50-100 microm H(2)O(2), almost disappearing after the organelles were maximally stimulated to produce H(2)O(2) with antimycin. Our results suggest that developmental mitochondrial activation of ERK1/2 cascade contributes to its nuclear translocation effects, providing information about mitochondrial energetic and redox status to the proliferating/differentiating nuclear pathways.     

Eicosanoid activation of extracellular signal-regulated kinase1/2 in human epidermoid carcinoma cells

12(S)-Hydroxyeicosatetraenoic acid (12(S)-HETE), a 12-lipoxygenase metabolite of arachidonic acid, has multiple effects on tumor and endothelial cells, including stimulation of invasion and angiogenesis. However, the signaling mechanisms controlling these physiological processes are poorly understood. In a human epidermoid carcinoma cell line (i.e. A431), 12(S)-HETE activates extracellular signal-regulated kinases 1/2 (ERK1/2), which is mediated by upstream kinases MEK and Raf. 12(S)-HETE stimulates phosphorylation of phospholipase Cgamma1 and activity of protein kinase Calpha (PKCalpha). In addition, independent of PKC 12(S)-HETE increases tyrosine phosphorylation of Shc, and Grb2, stimulates association between Shc and Src, and increases the activity of Ras, via Src family kinases. Furthermore, at low (10-100 nm) concentrations 12(S)-HETE counteracts epidermal growth factor-stimulated activation of ERK1/2 via stimulating protein tyrosine phosphatases. We also present evidence that 12(S)-HETE stimulates ERK1/2 via G proteins and that A431 cells have multiple binding sites for 12(S)-HETE. Finally, inhibition of 12-lipoxygenase induced apoptosis of A431 cells, which was reversed by addition of exogenous 12(S)-HETE. Collectively we demonstrate that the activation of ERK1/2 by 12(S)-HETE may be regulated by multiple receptors triggering PKC-dependent and PKC-independent pathways in A431 cells.     

Extracellular signal-regulated kinases in T cells: characterization of human ERK1 and ERK2 cDNAs.

Extracellular signal-regulated kinases 1 and 2 are growth factor-sensitive serine/threonine kinases. cDNAs for both human kinases were isolated and sequenced. The nucleic acid and deduced protein sequences of human extracellular signal-regulated kinase 1 were 88% and 96% identical, respectively, to the homologous rat sequences. The nucleic acid and deduced protein sequences of human extracellular signal-regulated kinase 2 were 90% and 98% identical, respectively, to the corresponding rat sequences. A human extracellular signal-regulated kinase 2 specific probe was used to demonstrate that the mRNA for this kinase was present in T cells and did not change with activation. The deduced protein sequences of both human kinases were greater than 95% identical to two Xenopus kinase sequences, indicating that these enzymes are highly conserved across species.     

Acquisition of chemoresistance in intrahepatic cholangiocarcinoma cells by activation of AKT and extracellular signal-regulated kinase (ERK)1/2

Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignant tumor and is refractory to conventional chemotherapy. The aim of this study is therefore to elucidate the mechanism of chemoresistance in ICC which is not fully understood. We generated cisplatin resistant ICC cells via long term exposure to cisplatin and found that these cells are also resistant to 5-fluorouracil (5-FU) and gemcitabine. The chemoresistant cells showed enhanced Bcl-2 expression and reduced Bax expression compared to parental ICC cells. In addition, the resistant cells showed enhanced activation of AKT and extracellular signal-regulated kinase (ERK) 1/2. Inhibition of AKT activation by phosphoinocitide 3-kinase (PI3K) inhibitor LY294002 resulted in reduced Bcl-2 expression and enhanced Bax expression and thus induced apoptosis in the resistant cells, whereas inhibition of ERK1/2 activation by mitogen-activated protein kinase (MEK) inhibitor U0126 did not induce apoptosis without affecting the expression of Bcl-2 and Bax but decreased cell growth. Moreover, the inhibition of AKT or ERK1/2 sensitized the resistant cells to cisplatin and therefore resulted in greatly enhanced cisplatin-induced apoptosis and growth inhibition in the cells. The results indicate that AKT and ERK1/2 signaling mediate chemoresistance in the cells and could be important therapeutic targets for overcoming chemoresistance in ICC.     

Epidermal growth factor activates extracellular signal-regulated protein kinases (ERK) in freshly isolated porcine granulosa cells.

We investigated the ability of EGF to stimulate the phosphorylation (i.e. activation) of extracellular signal-regulated kinases (ERKs) in freshly isolated porcine granulosa cells (pGC) held in suspension. pGCs were isolated from the ovaries of prepubertal pigs at slaughter, and equilibrated for 24 h at 37 degrees C in 12 x 75 mm culture tubes. The cells were then treated with 0-10 ng/ml EGF for 1-240 min. Treatments were terminated, and the total cell lysates were subjected to SDS-PAGE and Western analysis. The Westerns were blotted with anti-panERK and with anti-phosphoERK, antibodies that recognize all forms of ERKs and the phosphorylated (i.e. activated) forms of ERKs, respectively. Western blot analysis with the panERK antibody revealed a gel shift of ERKs, suggesting hyperphosphorylation after treatment with as little as 0.1 ng/ml of EGF. Phosphorylation of the ERKs was confirmed by using the phosphoERK antibody, which indicated increased phosphorylation of ERKs above control with 0.1 ng/ml EGF and maximal phosphorylation of ERK with 5-10 ng/ml EGF. Activation of ERK by EGF, as measured by both gel shift analysis and active ERK blotting, in the freshly isolated pGC was rapid, increasing above controls after 1 min of treatment, maintaining high levels through 40 min, and declining from 60 to 240 min. These data indicate that EGF stimulates active ERK in a time- and concentration-dependent manner in freshly isolated pGCs and that this experimental approach represents an effective manner with which to evaluate the role of EGF and the ERK signal transduction pathway in freshly harvested pGC.     

PKC alpha regulates the hypertrophic growth of cardiomyocytes through extracellular signal-regulated kinase1/2 (ERK1/2)

Members of the protein kinase C (PKC) isozyme family are important signal transducers in virtually every mammalian cell type. Within the heart, PKC isozymes are thought to participate in a signaling network that programs developmental and pathological cardiomyocyte hypertrophic growth. To investigate the function of PKC signaling in regulating cardiomyocyte growth, adenoviral-mediated gene transfer of wild-type and dominant negative mutants of PKC alpha, beta II, delta, and epsilon (only wild-type zeta) was performed in cultured neonatal rat cardiomyocytes. Overexpression of wild-type PKC alpha, beta II, delta, and epsilon revealed distinct subcellular localizations upon activation suggesting unique functions of each isozyme in cardiomyocytes. Indeed, overexpression of wild-type PKC alpha, but not betaI I, delta, epsilon, or zeta induced hypertrophic growth of cardiomyocytes characterized by increased cell surface area, increased [(3)H]-leucine incorporation, and increased expression of the hypertrophic marker gene atrial natriuretic factor. In contrast, expression of dominant negative PKC alpha, beta II, delta, and epsilon revealed a necessary role for PKC alpha as a mediator of agonist-induced cardiomyocyte hypertrophy, whereas dominant negative PKC epsilon reduced cellular viability. A mechanism whereby PKC alpha might regulate hypertrophy was suggested by the observations that wild-type PKC alpha induced extracellular signal-regulated kinase1/2 (ERK1/2), that dominant negative PKC alpha inhibited PMA-induced ERK1/2 activation, and that dominant negative MEK1 (up-stream of ERK1/2) inhibited wild-type PKC alpha-induced hypertrophic growth. These results implicate PKC alpha as a necessary mediator of cardiomyocyte hypertrophic growth, in part, through a ERK1/2-dependent signaling pathway.     

Stromal cell-derived factor-1alpha induces astrocyte proliferation through the activation of extracellular signal-regulated kinases 1/2 pathway

Stromal cell-derived factor-1 (SDF-1), the ligand of the CXCR4 receptor, is a chemokine involved in chemotaxis and brain development that also acts as co-receptor for HIV-1 infection. We previously demonstrated that CXCR4 and SDF-1alpha are expressed in cultured type-I cortical rat astrocytes, cortical neurones and cerebellar granule cells. Here, we investigated the possible functions of CXCR4 expressed in rat type-I cortical astrocytes and demonstrated that SDF-1alpha stimulated the proliferation of these cells in vitro. The proliferative activity induced by SDF-1alpha in astrocytes was reduced by PD98059, indicating the involvement of extracellular signal-regulated kinases (ERK1/2) in the astrocyte proliferation induced by CXCR4 stimulation. This observation was further confirmed showing that SDF-1alpha treatment selectively activated ERK1/2, but not p38 or stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). Moreover, both astrocyte proliferation and ERK1/2 phosphorylation, induced by SDF-1alpha, were inhibited by pertussis toxin (PTX) and wortmannin treatment indicating the involvement of a PTX sensitive G-protein and of phosphatidyl inositol-3 kinase in the signalling of SDF-1alpha. In addition, Pyk2 activation represent an upstream components for the CXCR4 signalling to ERK1/2 in astrocytes. To our knowledge, this is the first report demonstrating a proliferative effect for SDF-1alpha in primary cultures of rat type-I astrocytes, and showing that the activation of ERK1/2 is responsible for this effect. These data suggest that CXCR4/SDF-1 should play an important role in physiological and pathological glial proliferation, such as brain development, reactive gliosis and brain tumour formation.     

Monophosphothreonyl extracellular signal-regulated kinases 1 and 2 (ERK1/2) are formed endogenously in intact cardiac myocytes and are enzymically active

ERK1 and ERK2 (ERK1/2) are central to the regulation of cell division, growth and survival. They are activated by phosphorylation of the Thr- and the Tyr- residues in their Thr-Glu-Tyr activation loops. The dogma is that dually-phosphorylated ERK1/2 constitute the principal activities in intact cells. We previously showed that, in neonatal rat cardiac myocytes, endothelin-1 and phorbol 12-myristate 13-acetate (PMA) powerfully and rapidly (maximal at ~ 5 min) activate ERK1/2. Here, we show that dually-phosphorylated ERK1/2 rapidly (< 2 min) appear in the nucleus following stimulation with endothelin-1. We characterized the active ERK1/2 species in myocytes exposed to endothelin-1 or PMA using MonoQ FPLC. Unexpectedly, two peaks of ERK1 and two peaks of ERK2 activity were resolved using in vitro kinase assays. One of each of these represented the dually-phosphorylated species. The other two represented activities for ERK1 or ERK2 which were phosphorylated solely on the Thr- residue. Monophosphothreonyl ERK1/2 represented maximally ~ 30% of total ERK1/2 activity after stimulation with endothelin-1 or PMA, and their k_cat values were estimated to be minimally ~ 30% of the dually-phosphorylated species. Appearance of monophosphothreonyl ERK1/2 was rapid but delayed in comparison with dually-phosphorylated ERK1/2. Of 10 agonists studied, endothelin-1 and PMA were most effective in terms of ERK1/2 activation and in stimulating the appearance of monophosphothreonyl and dually-phosphorylated ERK1/2. Thus, enzymically active monophosphothreonyl ERK1/2 are formed endogenously following activation of the ERK1/2 cascade and we suggest that monophosphothreonyl ERK1/2 arise by protein tyrosine phosphatase-mediated dephosphorylation of dually-phosphorylated ERK1/2.     

Activation of extracellular signal-regulated kinases (ERK 44/42) by chlorpyrifos oxon in Chinese hamster ovary cells

Acetylcholinesterase inhibition explains most but not all of the toxicological manifestations of exposure to the major organophosphorus insecticide chlorpyrifos (CP) and its metabolically activated form chlorpyrifos oxon (CPO); CPO is also reported to interact with muscarinic acetylcholine receptors and alter secondary messenger status. We find that CP and CPO activate extracellular signal-regulated kinases (ERK 44/42) in both wild-type (CHOK1) and human muscarinic receptor-expressing Chinese hamster ovary cells (CHO-M2). The degree of ERK 44/42 activation on treatment with 50 microM CPO for 40 minutes is 2- to 3-fold compared with control cells and is both concentration- and time-dependent. CP is at least 2-fold less potent than CPO as an activator of ERK 44/42 and the hydrolysis products 3,5,6-trichloropyridinol and diethyl phosphate are not activators. ERK 44/42 activation by CPO is insensitive to the protein kinase A inhibitor H-89, but is completely abolished by the phosphatidylinositol 3-kinase (P13-K) inhibitor wortmannin, the protein kinase C (PKC) inhibitor GF-109203X, and the mitogen-activated extracellular signal-regulated protein kinase kinase (MEK) inhibitor PD 098059. Therefore, CPO activates the ERK 44/42 signaling cascade in CHOK1 cells via a pathway dependent on P13-K, PKC, and MEK but not requiring PKA or the human M2 muscarinic receptor. In summary we find that CPO activates a mammalian signal transduction cascade involved in cell growth and differentiation. This occurs through a pathway common to growth factors and mitogens, consistent with a receptor-mediated event. However, CPO may also inhibit an enzyme involved in signal transduction. The specific target of CPO leading to the activation of ERK 44/42 and the potential effects of this activation on cell function remain to be determined.     

The role of muscarinic acetylcholine receptor-mediated activation of extracellular signal-regulated kinase 1/2 in pilocarpine-induced seizures

Pilocarpine-induced seizures are mediated by the M(1) subtype of muscarinic acetylcholine receptor (mAChR), but little is known about the signaling mechanisms linking the receptor to seizures. The extracellular signal-regulated kinase (ERK) signaling cascade is activated by M(1) mAChR and is elevated during status epilepticus. Yet, the role of ERK activation prior to seizure has not been evaluated. Here, we examine the role of pilocarpine-induced ERK activation in the induction of seizures in mice by pharmacological and behavioral approaches. We show that pilocarpine induces ERK activation prior to the induction of seizures by both western blot and immunocytochemistry with an antibody to phosphorylated ERK. In addition, we show that the ERK pathway inhibitor SL327 effectively blocks the pilocarpine-induced ERK activation. However, SL327 pretreatment has no effect on the initiation of seizures. In fact, animals treated with SL327 had higher seizure-related mortality than vehicle-treated animals, suggesting activated ERK may serve a protective role during seizures. In addition, ERK inhibition had no effect on the development of the long-term sequelae of status epilepticus (SE), including mossy fiber sprouting, neuronal death and spontaneous recurrent seizures.     

Prevention of TNF-alpha-induced apoptosis in polyamine-depleted IEC-6 cells is mediated through the activation of ERK1/2

It has been documented that polyamines play a critical role in the regulation of apoptosis in intestinal epithelial cells. We have recently reported that protection from TNF-alpha/cycloheximide (CHX)-induced apoptosis in epithelial cells depleted of polyamines is mediated through the inactivation of a proapoptotic mediator, JNK. In this study, we addressed the involvement of the MAPK pathway in the regulation of apoptosis after polyamine depletion of IEC-6 cells. Polyamine depletion by alpha-difluromethylornithine (DFMO) resulted in the sustained activation of ERK in response to TNF-alpha/CHX treatment. Pretreatment of polyamine-depleted IEC-6 cells with a cell membrane-permeable MEK1/2 inhibitor, U-0126, significantly inhibited TNF-alpha/CHX-induced ERK phosphorylation and significantly increased DNA fragmentation, JNK activity, and caspase-3 activity in response to TNF-alpha/CHX. Moreover, the dose dependency of U-0126-mediated inhibition of TNF-alpha/ CHX-induced ERK phosphorylation correlated with the reversal of the antiapoptotic effect of DFMO. IEC-6 cells expressing constitutively active MEK1 had decreased TNF-alpha/CHX-induced JNK phosphorylation and were significantly protected from apoptosis. Conversely, a dominant-negative MEK1 resulted in high basal activation of JNK, cytochrome c release, and spontaneous apoptosis. Polyamine depletion of the dominant-negative MEK1 cells did not prevent JNK activation or cytochrome c release and failed to confer protection from both TNF-alpha/CHX and camptothecin-induced apoptosis. Finally, expression of a dominant-negative mutant of JNK significantly protected IEC-6 cells from TNF-alpha/CHX-induced apoptosis. These data indicate that polyamine depletion results in the activation of ERK, which inhibits JNK activation and protects cells from apoptosis.     

Valproic acid induces extracellular signal-regulated kinase 1/2 activation and inhibits apoptosis in endothelial cells

The histone deacetylase (HDAC) inhibitor valproic acid (VPA) was recently shown to inhibit angiogenesis, but displays no toxicity in endothelial cells. Here, we demonstrate that VPA increases extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation in human umbilical vein endothelial cells (HUVEC). The investigation of structurally modified VPA derivatives revealed that the induction of ERK 1/2 phosphorylation is not correlated to HDAC inhibition. PD98059, a pharmacological inhibitor of the mitogen-activated protein kinase kinase 1/2, prevented the VPA-induced ERK 1/2 phosphorylation. In endothelial cells, ERK 1/2 phosphorylation is known to promote cell survival and angiogenesis. Our results showed that VPA-induced ERK 1/2 phosphorylation in turn causes phosphorylation of the antiapoptotic protein Bcl-2 and inhibits serum starvation-induced HUVEC apoptosis and cytochrome c release from the mitochondria. Moreover, the combination of VPA with PD98059 synergistically inhibited angiogenesis in vitro and in vivo.     

Pigment epithelium-derived factor inhibits oxidative stress-induced cell death by activation of extracellular signal-regulated kinases in cultured retinal pigment epithelial cells

Oxidative stress-induced retinal pigment epithelial (RPE) cell death is involved in the pathogenesis of age-related macular degeneration (AMD). Pigment epithelium-derived factor (PEDF) is an anti-angiogenic/neurotropic dual functional factor, and recently it was also shown to mediate anti-oxidative action. In the present study, the influence of PEDF in hydrogen peroxide (H(2)O(2))-induced RPE cell death was investigated using nontransformed human RPE cell line (ARPE-19). The recombinant PEDF was purified from E. coli. The MTT cell viability assay showed that PEDF rescued ARPE-19 from H(2)O(2)-induced cell death in a dose- and time-dependent manner. Western blot analysis revealed that PEDF stimulated the extracellular signal-regulated kinases (ERK1/2) phosphorylation. The PEDF cytoprotective effect was significantly attenuated by the ERK1/2 inhibitor PD98059. In this study, we demonstrate that PEDF induces ERK1/2 phosphorylation and we further suggest that the ERK signal cascade contributes to RPE cell's cytoprotection against oxidative stress.     

Visfatin promotes angiogenesis by activation of extracellular signal-regulated kinase 1/2

Adipose tissue is highly vascularized and requires the angiogenic properties for its mass growth. Visfatin has been recently characterized as a novel adipokine, which is preferentially produced by adipose tissue. In this study, we report that visfatin potently stimulates in vivo neovascularization in chick chorioallantoic membrane and mouse Matrigel plug. We also demonstrate that visfatin activates migration, invasion, and tube formation in human umbilical vein endothelial cells (HUVECs). Moreover, visfatin evokes activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) in endothelial cells, which is closely linked to angiogenesis. Inhibition of ERK activation markedly decreases visfatin-induced tube formation of HUVECs and visfatin-stimulated endothelial cell sprouting from rat aortic rings. Taken together, these results demonstrate that visfatin promotes angiogenesis via activation of mitogen-activated protein kinase ERK-dependent pathway and suggest that visfatin may play important roles in various pathophysiological angiogenesis including adipose tissue angiogenesis.     

Potential role of phospholipase D2 in increasing interleukin-2 production by T-lymphocytes through activation of mitogen-activated protein kinases ERK1/ERK2

Hydrolysis of phosphatidylcholine by phospholipase D (PLD) leads to the generation of phosphatidic acid (PA), which is itself a source of diacylglycerol (DAG). These two versatile lipid second messengers are at the centre of a phospholipid signalling network and as such are involved in several cellular functions. However, their role in T-cell activation and functions are still enigmatic. In order to elucidate this role, we generated a human and a murine T-cell line that stably overexpressed the PLD2 isoform. Analysis of the Ras-MAPK pathway upon phorbol myristate acetate (PMA) and ionomycin stimulation revealed that PLD2 promoted an early and sustained increase in ERK1/2 phosphorylation in both cell lines. This response was inhibited by 1-butanol, a well known distracter of PLD activity, or upon overexpression of a dominant negative PLD2, and it was concomitant with a boost of PA/DAG production. As a functional consequence of this PLD2-dependent MAPK activation, interleukin-2 production evoked by PMA/ionomycin stimulation or CD3/CD28 engagement was enhanced in the two T-cell lines overexpressing PLD2. Thus, PLD2 emerged as an early player upstream of the Ras-MAPK-IL-2 pathway in T-cells via PA and DAG production, raising new possibilities of pharmacological manipulation in immune disorders.     

Abl interactor 1 binds to sos and inhibits epidermal growth factor- and v-Abl-induced activation of extracellular signal-regulated kinases

Recent studies have suggested that members of the Abl interactor (Abi) protein family negatively regulate cell growth and transformation. To date, however, no specific role in these cellular processes has been identified for the Abi family. Here we describe the inhibition by overexpressed Abi-1 of a mitogenic pathway activated by both growth factors and v-Abl. We have identified the guanine nucleotide exchange factors Sos1 and Sos2 as novel binding partners of Abi-1. A domain that is required for interaction with Sos in vivo has been mapped to the amino terminus of Abi-1. Overexpression of Abi-1 inhibits epidermal growth factor (EGF)-induced activation of extracellular signal-regulated kinases (Erks) but does not affect EGF-induced activation of c-Jun N-terminal kinase or Akt. In addition, overexpression of Abi-1 blocks Erk activation induced by v-Abl. In both cases, the maximal inhibitory effect requires an intact amino-terminal Sos-binding domain in Abi-1. Finally, we demonstrate that tyrosine phosphorylation of endogenous Abi-1 in fibroblasts is induced by both v-Abl and serum stimulation, further suggesting a role for Abi-1 in signal transduction initiated by v-Abl and growth factors. Taken together, these findings suggest that overexpressed Abi proteins negatively regulate cell growth and transformation by specifically targeting the Erk pathway.     

Evidence for the involvement of Gi2 in activation of extracellular signal-regulated kinases in hepatocytes

Background

Activation of the extracellular signal-regulated kinases ERK1 and ERK2 in hepatocytes by prostaglandin (PG)F_2α was recently found to be inhibited by pertussis toxin (PTX) suggesting a role for G_i proteins.

Results

Targeting the Gi_2α expression by a specific ribozyme inhibited the PGF_2α -induced ERK1/2 activation in hepatocytes. On the other hand a non-cleaving form of the Gi_2α ribozyme did not significantly decrease the ERK1/2 activation. In ribozyme-treated cells the Gi_2α protein level was reduced, while the G_qα level was not affected thus confirming the specificity of the ribozyme.

Conclusion

The present data suggest an important role of G_i2 in PGF_2α -induced ERK1/2 signaling in hepatocytes.