Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.12.2 (MEK)
18,161 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The classical paradigm for G protein-coupled receptor (GPCR) signal transduction involves the agonist-dependent interaction of GPCRs with heterotrimeric G proteins at the plasma membrane and the subsequent generation, by membrane-localized effectors, of soluble second messengers or ion currents. Termination of GPCR signals follows G protein-coupled receptor kinase (GRK)- and beta-arrestin-mediated receptor uncoupling and internalization. Here we show that these paradigms are inadequate to account for GPCR-mediated, Ras-dependent activation of the mitogen-activated protein (MAP) kinases Erk1 and -2. In HEK293 cells expressing dominant suppressor mutants of beta-arrestin or dynamin, beta2-adrenergic receptor-mediated activation of MAP kinase is inhibited. The inhibitors of receptor internalization specifically blocked Raf-mediated activation of MEK. Plasma membrane-delimited steps in the GPCR-mediated activation of the MAP kinase pathway, such as tyrosine phosphorylation of Shc and Raf kinase activation by Ras, are unaffected by inhibitors of receptor internalization. Thus, GRKs and beta-arrestins, which uncouple GPCRs and target them for internalization, function as essential elements in the GPCR-mediated MAP kinase signaling cascade.
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PMID:Essential role for G protein-coupled receptor endocytosis in the activation of mitogen-activated protein kinase. 942 17

Using both confocal immunofluorescence microscopy and biochemical approaches, we have examined the role of beta-arrestins in the activation and targeting of extracellular signal-regulated kinase 2 (ERK2) following stimulation of angiotensin II type 1a receptors (AT1aR). In HEK-293 cells expressing hemagglutinin-tagged AT1aR, angiotensin stimulation triggered beta-arrestin-2 binding to the receptor and internalization of AT1aR-beta-arrestin complexes. Using red fluorescent protein-tagged ERK2 to track the subcellular distribution of ERK2, we found that angiotensin treatment caused the redistribution of activated ERK2 into endosomal vesicles that also contained AT1aR-beta-arrestin complexes. This targeting of ERK2 reflects the formation of multiprotein complexes containing AT1aR, beta-arrestin-2, and the component kinases of the ERK cascade, cRaf-1, MEK1, and ERK2. Myc-tagged cRaf-1, MEK1, and green fluorescent protein-tagged ERK2 coprecipitated with Flag-tagged beta-arrestin-2 from transfected COS-7 cells. Coprecipitation of cRaf-1 with beta-arrestin-2 was independent of MEK1 and ERK2, whereas the coprecipitation of MEK1 and ERK2 with beta-arrestin-2 was significantly enhanced in the presence of overexpressed cRaf-1, suggesting that binding of cRaf-1 to beta-arrestin facilitates the assembly of a cRaf-1, MEK1, ERK2 complex. The phosphorylation of ERK2 in beta-arrestin complexes was markedly enhanced by coexpression of cRaf-1, and this effect is blocked by expression of a catalytically inactive dominant inhibitory mutant of MEK1. Stimulation with angiotensin increased the binding of both cRaf-1 and ERK2 to beta-arrestin-2, and the association of beta-arrestin-2, cRaf-1, and ERK2 with AT1aR. These data suggest that beta-arrestins function both as scaffolds to enhance cRaf-1 and MEK-dependent activation of ERK2, and as targeting proteins that direct activated ERK to specific subcellular locations.
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PMID:Activation and targeting of extracellular signal-regulated kinases by beta-arrestin scaffolds. 1122 59

beta-Arrestin-1 mediates agonist-dependent desensitization and internalization of G protein-coupled receptors (GPCRs) and is also essential for GPCR mitogenic signaling. In addition, insulin-like growth factor I receptor (IGF-IR) endocytosis is facilitated by beta-arrestin-1, and internalization is necessary for IGF-I-stimulated mitogen-activated protein (MAP) kinase activation. Here, we report that treatment of cells for 12 h with insulin (100 ng/ml) induces an approximately 50% decrease in cellular beta-arrestin-1 content due to ubiquitination of beta-arrestin-1 and proteosome-mediated degradation. This insulin-induced decrease in beta-arrestin-1 content was blocked by inhibition of phosphatidylinositol-3 kinase (PI-3 kinase) and MEK with wortmannin and PD98059, respectively. We also found a marked decrease in the association of beta-arrestin-1 with the IGF-IR and a 55% inhibition of IGF-I-stimulated MAP kinase phosphorylation. In insulin-treated, beta-arrestin-1-downregulated cells, there was complete inhibition of lysophosphatidic acid (LPA) or isoproterenol (ISO)-stimulated MAP kinase phosphorylation. This was associated with a decrease in beta-arrestin-1 association with the beta2-AR as well as a decrease in beta-arrestin-1-Src and Src-beta2-AR association. Ectopic expression of wild-type beta-arrestin-1 in insulin-treated cells in which endogenous beta-arrestin-1 had been downregulated rescued IGF-I- and LPA-stimulated MAP kinase phosphorylation. In conclusion, we found the following. (i) Chronic insulin treatment leads to enhanced beta-arrestin-1 degradation. (ii) This downregulation of endogenous beta-arrestin-1 is associated with decreased IGF-I-, LPA-, and ISO-mediated MAP kinase signaling, which can be rescued by ectopic expression of wild-type beta-arrestin-1. (iii) Finally, these results describe a novel mechanism for heterologous desensitization, whereby insulin treatment can impair GPCR signaling, and highlight the importance of beta-arrestin-1 as a target molecule for this desensitization mechanism.
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PMID:Insulin induces heterologous desensitization of G-protein-coupled receptor and insulin-like growth factor I signaling by downregulating beta-arrestin-1. 1216 19

The role of ERK, Jun N-terminal kinase (JNK), p38, and c-Src in GnRH-stimulated FSHbeta-subunit promoter activity was examined in the LbetaT-2 gonadotroph cell line. Incubation of the cells with a GnRH agonist resulted in activation of ERK, JNK, p38, and c-Src. The peak of ERK activation was observed at 5 min, whereas that of JNK, p38, and c-Src at 30 min, declining thereafter. ERK activation by GnRH is dependent on protein kinase C (PKC), as evident by activation, inhibition, and depletion of 12-O-tetradecanoylphorbol-13-acetate-sensitive PKC subspecies. Ca(2+) influx, but not Ca(2+) mobilization, is required for ERK activation. GnRH signaling to ERK is partially mediated by dynamin and a protein tyrosine kinase, apparently c-Src. ERK activation by GnRH in LbetaT-2 cells does not involve transactivation of epidermal growth factor receptor or mediation via Gbetagamma or beta-arrestin. Once activated by GnRH, ERK translocates to the nucleus. We examined the role of ERK, JNK, p38, and c-Src in GnRH-stimulated ovine FSHbeta promoter, linked to a luciferase reporter gene (-4741oFSHbeta-LUC). The PKC activator 12-O-tetradecanoylphorbol-13-acetate, but not the Ca(2+) ionophore ionomycin, stimulated FSHbeta-luciferase (LUC) activity. Furthermore, down-regulation of PKC, but not removal of Ca(2+), inhibited the GnRH response. Cotransfection of FSHbeta-LUC and the constitutively active forms of Raf-1 and MEK stimulated FSHbeta-LUC activity, whereas the dominant negatives of Ras, Raf-1, and MEK and the selective MEK inhibitor PD98059, abolished GnRH-induced FSHbeta-LUC activity. The dominant negatives of CDC42 and JNK reduced the GnRH response by 36 and 49%, respectively. Incubation of the cells with the p38 or the c-Src inhibitors SB203580 and PP1 also reduced the GnRH response. Surprisingly, two proximal activator protein-1 sites contribute very little to the GnRH response. Thus, PKC, ERK, JNK, p38, and c-Src, but not Ca(2+), are involved in GnRH induction of the ovine FSHbeta gene.
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PMID:Extracellular signal-regulated kinase, Jun N-terminal kinase, p38, and c-Src are involved in gonadotropin-releasing hormone-stimulated activity of the glycoprotein hormone follicle-stimulating hormone beta-subunit promoter. 1473 35

Protease-activated receptor-2 (PAR-2) is activated by trypsin-like serine proteases and can promote cell migration through an ERK1/2-dependent pathway, involving formation of a scaffolding complex at the leading edge of the cell. Previous studies also showed that expression of a dominant negative fragment of beta-arrestin-1 reduces PAR-2-stimulated internalization, ERK1/2 activation, and cell migration; however, this reagent may block association of many proteins, including beta-arrestin-2 with clathrin-coated pits. Here we investigate the role of PAR-2 in the constitutive migration of a metastatic breast cancer cell line, MDA MB-231, and use small interfering RNA to determine the contribution of each beta-arrestin to this process. We demonstrate that a trypsin-like protease secreted from MDA MB-231 cells can promote cell migration through autocrine activation of PAR-2 and this correlates with constitutive localization of PAR-2, beta-arrestin-2, and activated ERK1/2 to pseudopodia. Addition of MEK-1 inhibitors, trypsin inhibitors, a scrambled PAR-2 peptide, and silencing of beta-arrestins with small interfering RNA also reduce base-line migration of MDA MB-231 cells. In contrast, a less metastatic PAR-2 expressing breast cancer cell line does not exhibit constitutive migration, pseudopodia formation, or trypsin secretion; in these cells PAR-2 is more uniformly distributed around the cell periphery. These data demonstrate a requirement for both beta-arrestins in PAR-2-mediated motility and suggest that autocrine activation of PAR-2 by secreted proteases may contribute to the migration of metastatic tumor cells through beta-arrestin-dependent ERK1/2 activation.
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PMID:Constitutive protease-activated receptor-2-mediated migration of MDA MB-231 breast cancer cells requires both beta-arrestin-1 and -2. 1548 20

Metastatic melanoma still has a very poor prognosis since it withstands conventional therapies like surgery or chemotherapy. A paraneoplastic autoimmune manifestation of this disease is melanoma-associated retinopathy (MAR). MAR has been associated with prolonged survival and may be an early marker of tumor progression. By screening a retina and a melanoma cDNA phage library by SEREX using sera of patients suffering from melanoma and, in some cases, clinical symptoms of MAR, we identified 20 new antigens (HD-MM-28-47), of which 14 clones had high homology to well-known genes. Six of these genes had previously been associated with retina: rhodopsin, visual arrestin, MEK1, SRPX, BBS1 and galectin-3. Individual clones were recognized by up to 43% of patients' sera, while sera of healthy volunteers were negative except in 2 cases. The expression profile of the antigens identified on the basis of homologous EST database entries in healthy tissues was ubiquitous to differential. Using RT-PCR, we found frequent expression of preselected antigens in melanoma cell lines. For rhodopsin, this could be quantified by quantitative PCR. Retinal proteins were recognized by serum antibodies of melanoma patients but not healthy controls. The role of these antigens in MAR awaits further investigation. (Supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html.)
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PMID:SEREX identification of new tumor antigens linked to melanoma-associated retinopathy. 1552 88

Activation of seven-transmembrane region receptors typically causes their phosphorylation with consequent arrestin binding and desensitization. Arrestins also act as scaffolds, mediating signaling to Raf and ERK and, for some receptors, inhibiting nuclear translocation of ERK. GnRH receptors (GnRHRs) act via Gq/11 to stimulate the phospholipase C/Ca2+/protein kinase C (PKC) cascade and the Raf/MEK/ERK cassette. Uniquely, type I mammalian GnRHRs lack the C-tails that are found in other seven-transmembrane region receptors (including nonmammalian GnRHRs) and are implicated in arrestin binding. Here we have compared ERK signaling by human GnRHRs (hGnRHRs) and Xenopus GnRHRs (XGnRHRs). In HeLa cells, XGnRHRs underwent rapid and arrestin-dependent internalization and caused arrestin/green fluorescent protein (GFP) translocation to the membrane and endosomes, whereas hGnRHRs did not. Internalized XGnRHRs were co-localized with arrestin-GFP, whereas hGnRHRs were not. Both receptors mediated transient ERK phosphorylation and nuclear translocation (revealed by immunohistochemistry or by imaging of co-transfected ERK2-GFP), and for both, ERK phosphorylation was reduced by PKC inhibition but not by inhibiting epidermal growth factor receptor autophosphorylation. In the presence of PKC inhibitor, Deltaarrestin-(319-418) blocked XGnRHR-mediated, but not hGnRHR-mediated, ERK phosphorylation. When receptor number was varied, hGnRHRs activated phospholipase C and ERK more efficiently than XGnRHRs but were less efficient at causing ERK2-GFP translocation. At high receptor number, XGnRHRs and hGnRHRs both caused ERK2-GFP translocation to the nucleus, but at low receptor number, XGnRHRs caused ERK2-GFP translocation, whereas hGnRHRs did not. Thus, experiments with XGnRHRs have revealed the first direct evidence of arrestin-mediated (probably G protein-independent) GnRHR signaling, whereas those with hGnRHRs imply that scaffolds other than arrestins can determine GnRHR effects on ERK compartmentalization.
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PMID:Arrestin-mediated ERK activation by gonadotropin-releasing hormone receptors: receptor-specific activation mechanisms and compartmentalization. 1631 13

The beta-arrestins, a small family of G protein-coupled receptor (GPCR)-binding proteins involved in receptor desensitization, have been shown to bind extracellular signal-regulated kinases 1 and 2 (ERK1/2) and function as scaffolds for GPCR-stimulated ERK1/2 activation. To better understand the mechanism of beta-arrestin-mediated ERK1/2 activation, we compared ERK1/2 activation by the wild-type neurokinin 1 (NK1) receptor with a chimeric NK1 receptor having beta-arrestin1 fused to the receptor C terminus (NK1-betaArr1). The NK1 receptor couples to both G(s) and G(q/11), resides on the plasma membrane, and mediates rapid ERK1/2 activation and nuclear translocation in response to neurokinin A. In contrast, NK1-betaArr1 is a G protein-uncoupled "constitutively desensitized" receptor that resides almost entirely in an intracellular endosomal compartment. Despite its inability to respond to neurokinin A, we found that NK1-betaArr1 expression caused robust constitutive activation of cytosolic ERK1/2 and that endogenous Raf, MEK1/2, and ERK1/2 coprecipitated in a complex with NK1-betaArr1. While agonist-dependent ERK1/2 activation by the NK1 receptor was independent of protein kinase A (PKA) or PKC activity, NK1-betaArr1-mediated ERK1/2 activation was completely inhibited when basal PKA and PKC activity were blocked. In addition, the rate of ERK1/2 dephosphorylation was slowed in NK1-betaArr1-expressing cells, suggesting that beta-arrestin-bound ERK1/2 is protected from mitogen-activated protein kinase phosphatase activity. These data suggest that beta-arrestin binding to GPCRs nucleates the formation of a stable "signalsome" that functions as a passive scaffold for the ERK1/2 cascade while confining ERK1/2 activity to an extranuclear compartment.
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PMID:Constitutive ERK1/2 activation by a chimeric neurokinin 1 receptor-beta-arrestin1 fusion protein. Probing the composition and function of the G protein-coupled receptor "signalsome". 1667 94

Our previous studies have demonstrated that neither receptor endocytosis nor arrestin is required for ERK activation by the alpha2-adrenergic receptor (Wang, Q., Zhao, J., Brady, A. E., Feng, J., Allen, P. B., Lefkowitz, R. J., Greengard, P., and Limbird, L. E. (2004) Science 304, 1940-1944). The present studies address whether arrestin plays a role in determining the route of alpha2AR-evoked ERK signaling activation, taking advantage of endogenous expression of the alpha(2A)AR subtype in mouse embryonic fibroblasts (MEFs) and the availability of MEFs without arrestin expression (derived from Arr2,3-/- mice). Our data demonstrate that the endogenous alpha(2A)AR evokes ERK phosphorylation through both a Src-dependent and a Src-independent pathway, both of which are G protein dependent and converge on the Ras-Raf-MEK pathway. Arrestin is essential to recruit Src to this process, as alpha(2A)AR-mediated ERK signaling in Arr2,3-/- MEFs does not involve Src. Stimulation of alpha(2A)AR enhances arrestin-Src interaction and promotes activation of Src. alpha2 agonists have similar potencies in stimulating Src-dependent and Src-independent ERK phosphorylation in wild-type and Arr2,3-/- cells, respectively. However, Src-independent alpha(2A)AR-mediated ERK stimulation has both a longer duration of activation and a more rapid translocation of pERK into the nucleus when compared with Src-dependent activation. These data not only affirm the role of arrestin as an escort for signaling molecules such as Src family kinases but also demonstrate the impact of arrestin-dependent modulation on both the temporal and spatial properties of ERK activation.
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PMID:Arrestin serves as a molecular switch, linking endogenous alpha2-adrenergic receptor to SRC-dependent, but not SRC-independent, ERK activation. 1680 38

Toll-like receptors (TLRs) are a recently described receptor class involved in the regulation of innate and adaptive immunity. Here, we demonstrate that arrestin-2 and GRK5 (G protein-coupled receptor kinase 5), proteins that regulate G protein-coupled receptor signaling, play a negative role in TLR4 signaling in Raw264.7 macrophages. We find that lipopolysaccharide (LPS)-induced ERK1/2 phosphorylation is significantly enhanced in arrestin-2 and GRK5 knockdown cells. To elucidate the mechanisms involved, we tested the effect of arrestin-2 and GRK5 knockdown on LPS-stimulated signaling components that are upstream of ERK phosphorylation. Upon LPS stimulation, IkappaB kinase promotes phosphorylation and degradation of NFkappaB1 p105 (p105), which releases TPL2 (a MAP3K), which phosphorylates MEK1/2, which in turn phosphorylates ERK1/2. We demonstrate that knockdown of arrestin-2 leads to enhanced LPS-induced phosphorylation and degradation of p105, enhanced TPL2 release, and enhanced MEK1/2 phosphorylation. GRK5 knockdown also results in enhanced IkappaB kinase-mediated p105 phosphorylation and degradation, whereas GRK2 and GRK6 knockdown have no effect on this pathway. In vitro analysis demonstrates that arrestin-2 directly binds to the COOH-terminal domain of p105, whereas GRK5 binds to and phosphorylates p105. Taken together, these results suggest that p105 phosphorylation by GRK5 and binding of arrestin-2 negatively regulates LPS-stimulated ERK activation. These results reveal that arrestin-2 and GRK5 are important negative regulatory components in TLR4 signaling.
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PMID:Arrestin-2 and G protein-coupled receptor kinase 5 interact with NFkappaB1 p105 and negatively regulate lipopolysaccharide-stimulated ERK1/2 activation in macrophages. 1698 Mar 1


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