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)

Insulin receptor substrate-1 (IRS-1) is a major substrate of the insulin receptor and acts as a docking protein for Src homology 2 domain containing signaling molecules that mediate many of the pleiotropic actions of insulin. Insulin stimulation elicits serine/threonine phosphorylation of IRS-1, which produces a mobility shift on SDS-PAGE, followed by degradation of IRS-1 after prolonged stimulation. We investigated the molecular mechanisms and the functional consequences of these phenomena in 3T3-L1 adipocytes. PI 3-kinase inhibitors or rapamycin, but not the MEK inhibitor, blocked both the insulin-induced electrophoretic mobility shift and degradation of IRS-1. Adenovirus-mediated expression of a membrane-targeted form of the p110 subunit of phosphatidylinositol (PI) 3-kinase (p110CAAX) induced a mobility shift and degradation of IRS-1, both of which were inhibited by rapamycin. Lactacystin, a specific proteasome inhibitor, inhibited insulin-induced degradation of IRS-1 without any effect on its electrophoretic mobility. Inhibition of the mobility shift did not significantly affect tyrosine phosphorylation of IRS-1 or downstream insulin signaling. In contrast, blockade of IRS-1 degradation resulted in sustained activation of Akt, p70 S6 kinase, and mitogen-activated protein (MAP) kinase during prolonged insulin treatment. These results indicate that insulin-induced serine/threonine phosphorylation and degradation of IRS-1 are mediated by a rapamycin-sensitive pathway, which is downstream of PI 3-kinase and independent of ras/MAP kinase. The pathway leads to degradation of IRS-1 by the proteasome, which plays a major role in down-regulation of certain insulin actions during prolonged stimulation.
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PMID:A rapamycin-sensitive pathway down-regulates insulin signaling via phosphorylation and proteasomal degradation of insulin receptor substrate-1. 1084 81

Cotreatment with a minimally toxic concentration of the protein kinase C (PKC) activator (and down-regulator) bryostatin 1 (BRY) induced a marked increase in mitochondrial dysfunction and apoptosis in U937 monocytic leukemia cells exposed to the proteasome inhibitor lactacystin (LC). This effect was blocked by cycloheximide, but not by alpha-amanitin or actinomycin D. Qualitatively similar interactions were observed with other PKC activators (eg, phorbol 12-myristate 13-acetate and mezerein), but not phospholipase C, which does not down-regulate the enzyme. These events were examined in relationship to functional alterations in stress (eg, SAPK, JNK) and survival (eg, MAPK, ERK) signaling pathways. The observations that LC/BRY treatment failed to trigger JNK activation and that cell death was unaffected by a dominant-interfering form of c-JUN (TAM67) or by pretreatment with either curcumin or the p38/RK inhibitor, SB203580, suggested that the SAPK pathway was not involved in potentiation of apoptosis. In marked contrast, perturbations in the PKC/Raf/MAPK pathway played an integral role in LC/BRY-mediated cell death based on evidence that pretreatment of cells with bisindolylmaleimide I, a selective PKC inhibitor, or geldanamycin, a benzoquinone ansamycin, which destabilizes and depletes Raf-1, markedly suppressed apoptosis. Furthermore, ERK phosphorylation was substantially prolonged in LC/BRY-treated cells compared to those exposed to BRY alone, and pretreatment with the highly specific MEK inhibitors, PD98059, U0126, and SL327, opposed ERK activation while protecting cells from LC/BRY-induced lethality. Together, these findings suggest a role for activation and/or dysregulation of the PKC/MAPK cascade in modulation of leukemic cell apoptosis following exposure to the proteasome inhibitor LC. (Blood. 2001;97:2105-2114)
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PMID:Synergistic induction of apoptosis in human leukemia cells (U937) exposed to bryostatin 1 and the proteasome inhibitor lactacystin involves dysregulation of the PKC/MAPK cascade. 1126 78

Prolonged eosinophil survival is an essential step in the late and chronic phases of allergic inflammation and is regulated by the eosinophil survival cytokines. Our work has demonstrated that tumour necrosis factor (TNF)-alpha enhances survival (Trypan blue exclusion test) of human peripheral blood eosinophils from mildly allergic patients in a dose-dependent manner. The survival activity of TNF-alpha was inhibited by anti-TNF-RI, anti-TNF-RII antagonist antibodies and anti-granulocyte-monocyte colony-stimulating factor (GM-CSF) neutralizing antibodies but not by anti-interleukin (IL)-3 or anti-IL-5 antibodies. Furthermore, TNF-alpha-induced GM-CSF release from eosinophils. Anti-TNF-alpha antibodies also inhibited GM-CSF release from eosinophils induced by rat mast cell sonicate, which enhances eosinophil survival. To define the signal transduction pathway involved in GM-CSF production, eosinophils were incubated either with various mitogen-activated protein kinases (MAPK) inhibitors (MEK, JNK, P38), or Cyclosporin A (calcineurin inhibitor), or MG-132 (proteasome inhibitor). Only the proteasome inhibitor significantly decreased both TNF-alpha-enhanced eosinophil survival (from 38.1+/-4.1% to 13.3+/-1.4%) and GM-CSF release (from 6.2+/-0.7 pg/ml to 0.3+/-0.1 pg/ml). TNF-alpha also induced nuclear factor-kappaB (NF-kappaB) translocation to the nucleus, an essential step in GM-CSF mRNA production. All these findings provide evidence that NF-kappaB is involved in TNF-alpha-enhanced eosinophil survival through the regulation of GM-CSF production by eosinophils.
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PMID:Mechanism of tumour necrosis factor alpha mediated eosinophil survival. 1150 5

Enterohemorrhagic Escherichia coli (EHEC) infections are associated with hemorrhagic colitis and the hemolytic-uremic syndrome (HUS). In vivo, elevated plasma levels of the proinflammatory cytokine interleukin-8 (IL-8) in EHEC-infected children are correlated with a high risk of developing HUS. As IL-8 gene transcription is regulated by the transcription factors NF-kappaB and AP-1, we analyzed the role of these factors in the regulation of IL-8 production after infection of the epithelial intestinal T84 cell line by EHEC. By 6 h of infection, EHEC had induced significant secretion of IL-8 (35.84 +/- 6.76 ng/ml versus 0.44 +/- 0.04 ng/ml in control cells). EHEC induced AP-1 and NF-kappaB activation by 3 h of infection. Moreover, the three mitogen-activated protein kinases (MAPK) (ERK1/2, p38, and JNK) were phosphorylated in EHEC-infected T84 cells concomitant with induction of AP-1 DNA binding activity, and IkappaB-alpha was phosphorylated and then degraded concomitant with induction of NF-kappaB DNA binding activity. Pretreatment of cells with the highly specific MEK1/2 inhibitor U0126, the p38 inhibitor SB203580, and/or the proteasome inhibitor ALLN led to inhibition of the IL-8 secretion induced in EHEC-infected T84 cells. These findings demonstrate that (i) EHEC can induce in vitro a potent proinflammatory response by secretion of IL-8 and (ii) the secretion of IL-8 is due to the involvement of MAPK, AP-1, and NF-kappaB signaling pathways.
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PMID:Enterohemorrhagic Escherichia coli infection induces interleukin-8 production via activation of mitogen-activated protein kinases and the transcription factors NF-kappaB and AP-1 in T84 cells. 1195 64

Helicobacter pylori has been reported to induce interleukin-6 (IL-6) production in monocytes/macrophages and in chronically inflamed gastric tissues. The mechanism by which H. pylori induces IL-6 production in macrophages, however, has not been investigated. To identify the H. pylori factor responsible for this activity, we fractionated soluble proteins from H. pylori strain 26695 by ion exchange and size exclusion chromatography and screened the fractions for IL-6-inducing activity on RAW 264.7 macrophages. A single protein was purified and identified by mass spectrometry as H. pylori heat shock protein 60 (HSP60). Consistent with the observed IL-6-inducing activity of H. pylori HSP60, soluble protein extracts of H. pylori 26695 and SS1 strains that were depleted of this protein by affinity chromatography had dramatically reduced IL-6-inducing activities. The immunopurified HSP60 stimulated IL-6 production in macrophages. When stimulated with H. pylori HSP60 or intact bacteria, peritoneal macrophages from mice deficient in Toll-like receptor (TLR)-2, TLR-4, TLR-2/TLR-4, and myeloid differentiation factor 88 produced the same amount of IL-6 than macrophages from wild-type mice, demonstrating the independence of H. pylori HSP60 responses from these signaling molecules. H. pylori HSP60-induced IL-6 mRNA expression, and NF-kappaB activation in RAW 264.7 cells was abrogated in the presence of MG-132, a proteasome inhibitor. In contrast, inhibitors of protein kinase A or C, mitogen-activated protein kinase kinase, and phosphoinositide 3-kinase had no effect on IL-6 mRNA levels. This study demonstrates the induction of innate immune responses by H. pylori HSP60, thereby implicating this highly conserved protein in the pathophysiology of chronic gastritis.
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PMID:Helicobacter pylori heat shock protein 60 mediates interleukin-6 production by macrophages via a toll-like receptor (TLR)-2-, TLR-4-, and myeloid differentiation factor 88-independent mechanism. 1457 21

Growth related oncogene protein-alpha (GRO-alpha) is a member of C-X-C chemokine and plays an important role in inflammatory responses. Expression of GRO gene family is regulated by a number of factors at both transcriptional and posttranscriptional levels. In the present study, we have addressed the possible regulation of GRO-alpha expression by ubiquitin-proteasome system. Cultures of human umbilical vein endothelial cells were treated with a proteasome inhibitor, MG132, and the levels of GRO-alpha mRNA were analyzed by reverse transcription-polymerase chain reaction or northern blotting. Levels of GRO-alpha protein in the cell-conditioned medium were determined by enzyme-linked immunosorbent assay. MG132 alone increased the levels of GRO-alpha mRNA and protein; however, it did not affect the GRO-alpha mRNA induced by lipopolysaccharide (LPS) and inhibited the LPS-induced decrease in IkappaB levels. Other proteasome inhibitors, MG115 and lactacystin, also induced the expression of GRO-alpha mRNA. MG132 induced the phosphorylation of p38 MAPK, MEK and JNK. Pretreatment of the cells with SB203580, an inhibitor of p38 MAPK, suppressed the MG132-induced GRO-alpha expression, but pretreatment of the cells with U0126, PD98059 or SP600125, inhibitors of MEK1/2 or JNK, did not influence the effect of MG132. We conclude that MG132 upregulates GRO-alpha expression in vascular endothelial cells, at least in part, through the activation of p38 MAPK.
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PMID:Effect of MG132, a proteasome inhibitor, on the expression of growth related oncogene protein-alpha in human umbilical vein endothelial cells. 1458 Oct

In cultured rat vascular smooth muscle cells, sustained activation of ERK is required for interleukin-1beta to persistently activate NF-kappaB. Without ERK activation, interleukin-1beta induces only acute and transient NF-kappaB activation. The present study examined whether the temporal control of NF-kappaB activation by ERK could differentially regulate the expression of NF-kappaB-dependent genes, including inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), vascular cell adhesion molecule-1 (VCAM-1), and manganese-containing superoxide dismutase (Mn-SOD). Treatment of vascular smooth muscle cells with interleukin-1beta induced the expression of iNOS, COX-2, VCAM-1, and Mn-SOD in a time-dependent manner, but with different patterns. Either PD98059 or U0126, selective inhibitors of MEK, or overexpression of a dominant negative MEK-1 inhibited interleukin-1beta- induced ERK activation and the expression of iNOS and COX-2 but had essentially no effect on the expression of VCAM-1 and Mn-SOD. The expression of these genes was inhibited when NF-kappaB activation was down-regulated by MG132, a proteasome inhibitor, or by overexpression of an I-kappaBalpha mutant that prevented both the transient and the persistent activation of NF-kappaB. Inhibition of ERK did not affect interleukin-1beta-induced I-kappaBalpha phosphorylation and degradation but attenuated I-kappaBbeta degradation. Thus, although NF-kappaB activation was essential for interleukin-1beta induction of each of the proteins studied, gene expression was differentially regulated by ERK and by the duration of NF-kappaB activation. These results reveal a novel functional role for ERK as an important temporal regulator of NF-kappaB activation and NF-kappaB-dependent gene expression.
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PMID:Temporal control of NF-kappaB activation by ERK differentially regulates interleukin-1beta-induced gene expression. 1458 82

Connexins are membrane-spanning proteins that form gap junction channels between adjacent cells. Connexin43 (Cx43), the most widely expressed member of the connexin family in tissues and cell lines, has a rapid turnover rate and its degradation involves both the lysosomal and ubiquitin-proteasome pathway. It was previously shown that the proteasome is involved in regulating the number of functional gap junctions at the plasma membrane. However, little is known about how proteasome-dependent turnover of Cx43 is controlled. Epidermal growth factor (EGF) induces hyperphosphorylation of Cx43 and a rapid, transient decrease in gap junctional intercellular communication. In this study, we show that, along with inhibition of gap junctional intercellular communication, EGF induces disorganization, internalization and degradation of Cx43 gap junction plaques in IAR20 rat liver epithelial cells. These EGF-induced modifications of Cx43 were counteracted by the MEK1 inhibitor PD98059, indicating that the effects were mediated by the mitogen-activated protein kinase pathway. The EGF-induced destruction of Cx43 was proteasome-dependent, because the loss of Cx43 protein was counteracted by the proteasome inhibitor MG132 but not the lysosomal inhibitor leupeptin. Furthermore, EGF induced ubiquitination of Cx43, which was associated with the Cx43 hyperphosphorylation. The EGF-induced Cx43 ubiquitination was counteracted by PD98059. The EGF-induced internalization of Cx43 was blocked by hypertonic sucrose treatment, indicating that EGF mediates internalization of Cx43 via a clathrin-dependent mechanism. Our results indicate that ubiquitination of Cx43 occurs at the plasma membrane before Cx43 internalization. Taken together, these data provide the first evidence that EGF-induced phosphorylation of Cx43 induces binding of ubiquitin and targets Cx43 for internalization and degradation in a proteasome-dependent manner.
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PMID:Epidermal growth factor regulates ubiquitination, internalization and proteasome-dependent degradation of connexin43. 1497 Feb 63

We have previously shown that nuclear factor-kappaB inhibition by adenovirus expressing mutated IkappaB-alpha or by proteasome inhibitor increases human articular chondrocytes sensibility to apoptosis. Moreover, the nuclear factor-kappaB inhibitor BAY11-7085, a potent anti-inflammatory drug in rat adjuvant arthritis, is itself a proapoptotic agent for chondrocytes. In this work, we show that BAY 11-7085 but not the proteasome inhibitor MG-132 induced a rapid and sustained phosphorylation of extracellular signal-regulated kinases (ERK1/2) in human articular chondrocytes. The level of ERK1/2 phosphorylation correlated with BAY 11-7085 concentration and chondrocyte apoptosis. 15-Deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2) and its precursor prostaglandin (PG) D2 but not PGE2 and PGF2alpha rescued chondrocytes from BAY 11-7085-induced apoptosis. 15d-PGJ2 markedly inhibited BAY 11-7085-induced phosphorylation of ERK1/2. BAY 11-7085 also induced ERK1/2 phosphorylation and apoptosis in human synovial fibroblasts, and these reactions were down-regulated by 15d-PGJ2. Further analysis in synovial fibroblasts showed that only molecules that suppressed BAY 11-7085-induced phosphorylation of ERK1/2 (i.e. 15d-PGJ2, PGD2, and to a lesser extent, MEK1/2 inhibitor UO126, but not prostaglandins E2 and F2alpha or peroxisome proliferator-activated receptor-gamma agonist ciglitazone) were able protect cells from apoptosis. These results suggested that the antiapoptotic effect of 15d-PGJ2 on chondrocytes and synovial fibroblasts might involve inhibition of ERK1/2 phosphorylation.
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PMID:15-deoxy-delta12,14-prostaglandin J2 inhibits Bay 11-7085-induced sustained extracellular signal-regulated kinase phosphorylation and apoptosis in human articular chondrocytes and synovial fibroblasts. 1500 16

Interactions between the cyclin-dependent kinase (CDK) inhibitor flavopiridol and the proteasome inhibitor bortezomib were examined in Bcr/Abl(+) human leukemia cells. Coexposure of K562 or LAMA84 cells to subtoxic concentration of flavopiridol (150-200 nM) and bortezomib (5-8 nM) resulted in a synergistic increase in mitochondrial dysfunction and apoptosis. These events were associated with a marked diminution in nuclear factor kappaB (NF-kappaB)/DNA binding activity; enhanced phosphorylation of SEK1/MKK4 (stress-activated protein kinase/extracellular signal-related kinase 1/mitogen-activated protein kinase kinase 4), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK); down-regulation of Bcr/Abl; and a marked reduction in signal transducer and activator of transcription 3 (STAT3) and STAT5 activity. In imatinib mesylate-resistant K562 cells displaying increased Bcr/Abl expression, bortezomib/flavopiridol treatment markedly increased apoptosis in association with down-regulation of Bcr/Abl and BclxL, and diminished phosphorylation of Lyn, Hck, CrkL, and Akt. Parallel studies were performed in imatinib mesylate-resistant LAMA84 cells exhibiting reduced expression of Bcr/Abl but a marked increase in expression/activation of Lyn and Hck. Flavopiridol/bortezomib effectively induced apoptosis in these cells in association with Lyn and Hck inactivation. The capacity of flavopiridol to promote bortezomib-mediated Bcr/Abl down-regulation and apoptosis was mimicked by the positive transcription elongation factor-b (P-TEFb) inhibitor DRB (5,6-dichloro 1-beta-d-ribofuranosylbenzinida-sole). Finally, the bortezomib/flavopiridol regimen also potently induced apoptosis in Bcr/Abl(-) human leukemia cells. Collectively, these findings suggest that a strategy combining flavopiridol and bortezomib warrants further examination in chronic myelogenous leukemia and related hematologic malignancies.
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PMID:Bortezomib and flavopiridol interact synergistically to induce apoptosis in chronic myeloid leukemia cells resistant to imatinib mesylate through both Bcr/Abl-dependent and -independent mechanisms. 1503 84


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