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:3.4.25.1 (proteasome)
28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The cellular responses to activated Ras vary depending on cell type. Normal cells are often induced into pathways that lead to cell growth arrest, senescence, and/or apoptosis in response to activated Ras expression. These are important protective anti-tumorigenic responses that restrict the propagation of cells bearing activated oncogenes. Here we show that induction of Ha-Ras(Val-12) in Rat-1 fibroblasts resulted in G(1) growth arrest and apoptosis with loss of viable cells that is accompanied by a marked decrease in cyclin D1 levels via increased ubiquitin-proteasome-dependent cyclin D1 turnover. This is in contrast with a rat intestinal epithelial cell line in which induction of Ha-Ras(Val-12) results in transformation associated with sustained proliferation and increased levels of cyclin D1, that is not accompanied by anoikis or apoptosis. Expression of the cyclin D1 mutant (T286A) that contains an alanine for threonine 286 substitution and is resistant to ubiquitin-proteasome degradation in the Ha-Ras(Val-12) expressing Rat-1 cells resulted in a sustained transformed phenotype with no accumulation of cells in G(1). Inhibition of mitogen-activated protein kinase (MEK1/2) pathway partially reversed the Ras-mediated decrease in cyclin D1. Induction of Ha-Ras(Val-12) resulted in activation of Akt kinase and inactivation of glycogen-synthase-3beta kinase that are associated with reduction of cyclin D1 protein. These results suggest that Ras-mediated cyclin D1 degradation in Rat-1 cells appears to be partially dependent on activation of mitogen-activated protein kinase pathway and independent of glycogen-synthase-3beta kinase pathway.
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PMID:Oncogenic Ras-mediated cell growth arrest and apoptosis are associated with increased ubiquitin-dependent cyclin D1 degradation. 1078 97

Overexpression of a constitutively active mutant of the mitogen-activated protein kinase kinase MEK1 (caMEK1) in epithelial Madin-Darby canine kidney (MDCK)-C7 cells disrupts morphogenesis, induces an invasive phenotype, and is associated with a reduced rate of cell proliferation. The role of cell-cell adhesion molecules and cell cycle proteins in these processes, however, has not been investigated. We now report loss of E-cadherin expression as well as a marked reduction of beta- and alpha-catenin expression in transdifferentiated MDCK-C7 cells stably expressing caMEK1 (C7caMEK1) compared with epithelial mock-transfected MDCK-C7 (C7Mock1) cells. At least part of the remaining alpha-catenin was coimmunoprecipitated with beta-catenin, whereas no E-cadherin was detected in beta-catenin immunoprecipitates. In both cell types, the proteasome-specific protease inhibitors N-acetyl-Leu-Leu-norleucinal (ALLN) and lactacystin led to a time-dependent accumulation of beta-catenin, including the appearance of high-molecular-weight beta-catenin species. Quiescent as well as serum-stimulated C7caMEK1 cells showed a higher cyclin D expression than epithelial C7Mock1 cells. The MEK inhibitor U-0126 inhibited extracellular signal-regulated kinase phosphorylation and cyclin D expression in C7caMEK1 cells and almost abolished their already reduced cell proliferation rate. We conclude that the transdifferentiated and invasive phenotype of C7caMEK1 cells is associated with a diminished expression of proteins involved in cell-cell adhesion. Although beta-catenin expression is reduced, C7caMEK1 cells show a higher expression of U-0126-sensitive cyclin D protein.
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PMID:Differential expression of cell-cell adhesion proteins and cyclin D in MEK1-transdifferentiated MDCK cells. 1102 95

Activation of Jun N-kinase (JNK) and NF-kappaB transcription factor are the hallmarks of cellular response to stress. Phosphorylation of NF-kappaB inhibitor (IkappaB) by respective stress-inducible kinases (IKK) is a key event in NF-kappaB activation. beta-TrCP F-box protein mediates ubiquitination of phosphorylated IkappaB via recruitment of SCF(beta-TrCP)-Roc1 E3 ubiquitin ligase complex. Subsequent proteasome-dependent degradation of IkappaB results in activation of the NF-kappaB pathway. We found that a variety of cellular stress stimuli induce an increase in the steady state levels of beta-TrCP mRNA and protein levels in human cells. Activation of stress-activated protein kinases JNK (and, to a lesser extent, p38) by forced expression of constitutively active mutants of JNKK2 and MKK6 (but not MEK1 or IKKbeta) also leads to accumulation of beta-TrCP. Transcription of the beta-TrCP gene is not required for JNK-mediated induction of beta-TrCP. A synergistic effect of stimulation of IKK and JNK on the transcriptional activity of NF-kappaB was observed. The mechanisms of beta-TrCP induction via stress and its role in NF-kappaB activation are discussed.
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PMID:Induction of beta-transducin repeat-containing protein by JNK signaling and its role in the activation of NF-kappaB. 1137 88

The muscle protein catabolism present in rats with insulin-dependent diabetes and other catabolic conditions is generally associated with increased glucocorticoid production and mRNAs encoding components of the ubiquitin-proteasome system. The mechanisms that increase ubiquitin (UbC) expression have not been identified. We studied the regulation of UbC expression in L6 muscle cells because dexamethasone stimulates the transcription of this gene and others encoding components of the ubiquitin-proteasome pathway. Results of in vivo genomic DNA footprinting experiments indicate that a protein(s) binds to Sp1 sites approximately 50 bp upstream from the UbC transcription start site; dexamethasone changes the methylation pattern at these sites. Sp1 binds to DNA probes corresponding to the rat or human UbC promoter, and treating cells with dexamethasone increases this binding. Deletion and mutation analyses of the rat and human UbC promoters are consistent with an important role of Sp1 in UbC induction by glucocorticoids. Dexamethasone-induced ubiquitin expression is blocked by mithramycin, an inhibitor of Sp1 binding. UO126, a pharmacologic inhibitor of MEK1, also blocks UbC transcriptional activation by dexamethasone; L6 cells transfected to express constitutively active MEK1 exhibit increased UbC promoter activity. Thus, glucocorticoids increase UbC expression in muscle cells by a novel transcriptional mechanism involving Sp1 and MEK1.
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PMID:Ubiquitin (UbC) expression in muscle cells is increased by glucocorticoids through a mechanism involving Sp1 and MEK1. 1187 50

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

A key task for the multifunctional von Hippel-Lindau protein (pVHL) is regulation of the activity of hypoxia-inducible factor-1alpha (HIF-1alpha) by targeting it to the proteasome for degradation under normoxia. pVHL binding to HIF-1alpha is lost under low O2 tension, leading to transcription of several genes involved in the hypoxia response. However, regulation of pVHL by hypoxia remains to be investigated. We evaluated the effects of hypoxia on pVHL expression in carcinoma and endothelial cells. We showed that hypoxia stimulates pVHL levels (2.5-fold) in renal Caki-1 cells expressing wild-type VHL (VHL+/+). This upregulation was independent of VHL status, because hypoxia also increased pVHL expression in renal 786-O cells carrying mutated VHL (VHL-/-). Hypoxia did not affect pVHL expression in endothelial cells. Hypoxia-induced pVHL in Caki-1 cells was RhoA dependent, because inhibition by exotoxin C3 prevented pVHL stimulation. Furthermore, inhibition of Rho kinase by Y-27632 blocked pVHL induction by hypoxia. During normoxia, pVHL expression was also induced in cells transfected with dominant-active RhoA. Furthermore, disruption of actin organization by chemical agents or by hypoxia stimulated pVHL expression in kidney cells. On the other hand, inhibition of MAP kinases p38 and JNK, but not MAP kinase kinase (MEK1/2), reduced pVHL upregulation by 30 and 72%, respectively, during hypoxia, supporting a significant role for these signaling pathways. Expression and phosphorylation of c-Jun were stimulated in cells transfected with dominant-active RhoA. Together, these findings demonstrate that hypoxia induces pVHL expression in renal cancer cells, and this induction is mediated by RhoA-dependent pathways.
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PMID:Hypoxia upregulates von Hippel-Lindau tumor-suppressor protein through RhoA-dependent activity in renal cell carcinoma. 1458 36

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

Mitogen-activated protein (MAP) kinase phosphatases (MKPs) are dual-specificity phosphatases that dephosphorylate phosphothreonine and phosphotyrosine residues within MAP kinases. Here, we describe a novel posttranslational mechanism for regulating MKP-3/Pyst1/DUSP6, a member of the MKP family that is highly specific for extracellular signal-regulated kinase 1 and 2 (ERK1/2) inactivation. Using a fibroblast model in which the expression of either MKP-3 or a more stable MKP-3-green fluorescent protein (GFP) chimera was induced by tetracycline, we found that serum induces the phosphorylation of MKP-3 and its subsequent degradation by the proteasome in a MEK1 and MEK2 (MEK1/2)-ERK1/2-dependent manner. In vitro phosphorylation assays using glutathione S-transferase (GST)-MKP-3 fusion proteins indicated that ERK2 could phosphorylate MKP-3 on serines 159 and 197. Tetracycline-inducible cell clones expressing either single or double serine mutants of MKP-3 or MKP-3-GFP confirmed that these two sites are targeted by the MEK1/2-ERK1/2 module in vivo. Double serine mutants of MKP-3 or MKP-3-GFP were more efficiently protected from degradation than single mutants or wild-type MKP-3, indicating that phosphorylation of either serine by ERK1/2 enhances proteasomal degradation of MKP-3. Hence, double mutation caused a threefold increase in the half-life of MKP-3. Finally, we show that the phosphorylation of MKP-3 has no effect on its catalytic activity. Thus, ERK1/2 exert a positive feedback loop on their own activity by promoting the degradation of MKP-3, one of their major inactivators in the cytosol, a situation opposite to that described for the nuclear phosphatase MKP-1.
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PMID:Extracellular signal-regulated kinases phosphorylate mitogen-activated protein kinase phosphatase 3/DUSP6 at serines 159 and 197, two sites critical for its proteasomal degradation. 1563 84

BAY 43-9006 is a kinase inhibitor that induces apoptosis in a variety of tumor cells. Here we report that treatment with BAY 43-9006 results in marked cytochrome c and AIF release into the cytosol, caspase-9, -8, -7, and -3 activation, and apoptosis in human leukemia cells (U937, Jurkat, and K562). Pronounced apoptosis was also observed in blasts from patients with acute myeloid leukemia. These events were accompanied by ERK1/2 inactivation and caspase-independent down-regulation of Mcl-1. Inducible expression of a constitutively active MEK1 construct did not prevent Mcl-1 down-regulation, suggesting that this event is not related to MEK/ERK pathway inactivation. Furthermore, BAY 43-9006 did not induce major changes in Mcl-1 mRNA levels monitored by real-time PCR or Mcl-1 promoter activity demonstrated by luciferase reporter assays, but it did enhance Mcl-1 down-regulation in actinomycin D-treated cells. Inhibition of protein synthesis by cycloheximide or proteasome function with MG132 and pulse-chase studies with [35S]methionine demonstrated that BAY 43-9006 did not diminish Mcl-1 protein stability, nor did it enhance Mcl-1 ubiquitination, but instead markedly attenuated Mcl-1 translation in association with the rapid and potent dephosphorylation of the eIF4E translation initiation factor. Finally, ectopic expression of Mcl-1 in leukemic cells markedly inhibited BAY 43-9006-mediated cytochrome c cytosolic release, caspase-9, -7, and -3 activation, as well as cell death, indicating that Mcl-1 operates upstream of cytochrome c release and caspase activation. Together, these findings demonstrate that BAY 43-9006 mediates cell death in human leukemia cells, at least in part, through down-regulation of Mcl-1 via inhibition of translation.
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PMID:Apoptosis induced by the kinase inhibitor BAY 43-9006 in human leukemia cells involves down-regulation of Mcl-1 through inhibition of translation. 1610 13

Transcription factor nuclear factor-kappaB (NF-kappaB) is held in the cytoplasm in an inactive state by IkappaB inhibitors. Oncogenic activation of NF-kappaB is achieved by stimulus-induced ubiquitination and subsequent proteasome-mediated degradation of IkappaBalpha. Once released from the inhibitor, NF-kappaB/p65 enters the nucleus. A pre-requisite for cytokine-induced IkappaBalpha ubiquitination and degradation is the phosphorylation of IkappaBalpha at S32/S36. Phosphorylation of IkappaBalpha alone, however, is not sufficient to trigger its degradation, suggesting other events must be required for regulating IkappaBalpha degradation. In this study, we tested the hypothesis that phosphorylation of p65 at 536 is required for TNF-alpha induced IkappaBalpha proteolysis that in turn controls p65 nuclear translocation. We observed that, without affecting IkappaBalpha phosphorylation, MEK1 inhibitor U0126 treatment inhibited not only p65-S536 phosphorylation but also TNF-alpha-induced polyubiquitination of IkappaBalpha thereby inhibiting IkappaBalpha degradation. With p65 S536 phosphorylation mutants and mimics, we further observed that the structural mutation of p65 serine 536 to alanine inhibited the recruitment of ubiquitin to the p65-containing complex. As a consequence of suppressing polyubiquitination of the p65-containing complex, degradation of p65 phosphorylation mutant-bound IkappaBalpha was also inhibited. Accordingly, the nuclear translocation of phosphorylation-impaired p65 was significantly reduced. These findings suggest that p65 phosphorylation plays a key role in stimulus-induced IkappaBalpha ubiquitination.
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PMID:Suppression of p65 phosphorylation coincides with inhibition of IkappaBalpha polyubiquitination and degradation. 1616 8


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