Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.11.18 (MAP)
 7,412 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mammalian Cdc25 phosphatase is responsible for the dephosphorylation of Cdc2 and other cyclin-dependent kinases at Thr14 and Tyr15, thus activating the kinase and allowing cell cycle progression. The catalytic domain of this dual-specificity phosphatase has recently been mapped to the 180 most C-terminal amino acids. Apart from a CX3R motif, which is present at the active site of all known tyrosine phosphatases, Cdc25 does not share any obvious sequence similarity with any of those enzymes. Until very recently, the Cdc25 family was the only subfamily of tyrosine phosphates for which no three-dimensional structural data were available. Using the generalized profile technique, a sensitive method for sequence database searches, we found an extended and highly significant sequence similarity between the Cdc25 catalytic domain and similarly sized regions in other proteins: the non-catalytic domain of two distinct families of MAP-kinase phosphates, the non-catalytic domain of several ubiquitin protein hydrolases, the N and C-terminal domain of rhodanese, and a large and heterogeneous groups of stress-response proteins from all phyla. The relationship of Cdc25 to the structurally well-characterized rhodanese spans the entire catalytic domain and served as template for a structural model for human Cdc25a, which is fundamentally different from previously suggested models for Cdc25 catalytic domain organization. The surface positioning of subfamily-specific conserved residues allows us to predict the sites of interaction with Cdk2, a physiological target of Cdc25a. Based on the results of this analysis, we also predict that the budding yeast arsenate resistance protein Acr2 and the ORF Ygr203w encode protein phosphatases with catalytic properties similar to that of the Cdc25 family. Recent determination of the crystal structure of the Cdc25a catalytic domain supports the validity of the model and demonstrates the power of the generalized sequence profile technique in homology-based modeling of the three-dimensional structure of a protein having a weak but significant sequence similarity with a structurally characterized protein.
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PMID:A model of Cdc25 phosphatase catalytic domain and Cdk-interaction surface based on the presence of a rhodanese homology domain. 973 50

The first amino acid of "authentic" poliovirus RNA-dependent RNA polymerase, 3D(pol), is a glycine. As a result, production of 3D(pol) in Escherichia coli requires addition of an initiation codon; thus, a formylmethionine is added to the amino terminus. The formylmethionine should be removed by the combined action of a cellular deformylase and methionine aminopeptidase. However, high-level expression of 3D(pol) in E. coli yields enzyme with a heterogeneous amino terminus. To preclude this problem, we developed a new expression system for 3D(pol). This system exploits the observation that proteins fused to the carboxyl terminus of ubiquitin can be processed in E. coli to produce proteins with any amino acid as the first residue when expressed in the presence of a ubiquitin-specific, carboxy-terminal protease. By using this system, authentic 3D(pol) can be obtained in yields of 30-60 mg per liter of culture. While addition of a single glycine, alanine, serine, or valine to the amino terminus of 3D(pol) produced derivatives with a specific activity reduced by at least 25-fold relative to wild-type enzyme, addition of a methionine to the amino terminus resulted in some processing to yield enzyme with a glycine amino terminus. Addition of a hexahistidine tag to the carboxyl terminus of 3D(pol) had no deleterious effect on the activity of the enzyme. The utility of this expression system for production of other viral polymerases and accessory proteins is discussed.
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PMID:Production of "authentic" poliovirus RNA-dependent RNA polymerase (3D(pol)) by ubiquitin-protease-mediated cleavage in Escherichia coli. 1049 78

The intracellular levels of many proteins are regulated by ubiquitin-dependent proteolysis. One of the best-characterized enzymes that catalyzes the attachment of ubiquitin to proteins is a ubiquitin ligase complex, Skp1-Cullin-F box complex containing Hrt1 (SCF). We sought to artificially target a protein to the SCF complex for ubiquitination and degradation. To this end, we tested methionine aminopeptidase-2 (MetAP-2), which covalently binds the angiogenesis inhibitor ovalicin. A chimeric compound, protein-targeting chimeric molecule 1 (Protac-1), was synthesized to recruit MetAP-2 to SCF. One domain of Protac-1 contains the I kappa B alpha phosphopeptide that is recognized by the F-box protein beta-TRCP, whereas the other domain is composed of ovalicin. We show that MetAP-2 can be tethered to SCF(beta-TRCP), ubiquitinated, and degraded in a Protac-1-dependent manner. In the future, this approach may be useful for conditional inactivation of proteins, and for targeting disease-causing proteins for destruction.
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PMID:Protacs: chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation. 1143 90

Incubation of murine C2C12 myotubes with tumour necrosis factor-alpha (TNF-alpha) leads to significant changes in protein content and turnover, suggesting that the cytokine exerts direct effects in skeletal muscle. The effects of the cytokine on protein content show a clear bimodal behaviour. At low concentrations (1 U/ml or less), TNF-alpha decreases both total and myofibrillar protein content, while at relatively high concentrations (100 U/ml or more), the effects are opposite and TNF-alpha increases the total and myofibrillar protein content in C2C12 myotubes. The mechanisms responsible for this latter, unexpected anabolic effect of the cytokine on muscle cells are related to a 40% increase in the rate of protein synthesis and to a significant decrease (14%) in the rate of protein degradation. At high concentrations, TNF-alpha decreased the expression of the mRNA of components of both the ATP- (ubiquitin, E2, C8) and Ca2+-dependent (m-calpain) proteolytic systems. The effects of TNF-alpha (10 U/ml or higher) on protein content of cultured murine myotubes (differentiated myogenic cells) were similar to those induced by insulin (1 or 5 microg/ml), but the effects of TNF-alpha and those of insulin were not additive. Experiments using inhibitors of the signalling pathways mediated by PI3K and MAP kinases (MAPKs) ERK1/2 and p38 suggest that insulin and TNF-alpha may share some intracellular signalling pathways involving MAPKs in the enhanced protein accretion observed in the muscle cell cultures.
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PMID:Direct effects of tumor necrosis factor alpha (TNF-alpha) on murine skeletal muscle cell lines. Bimodal effects on protein metabolism. 1156 20

ERK1/2 MAP kinases are important regulators in cellular signaling, whose activity is normally reversibly regulated by threonine-tyrosine phosphorylation. In contrast, we have found that stress-induced ERK1/2 activity is downregulated by ubiquitin/proteasome-mediated degradation of ERK1/2. The PHD domain of MEKK1, a RING finger-like structure, exhibited E3 ubiquitin ligase activity toward ERK2 in vitro and in vivo. Moreover, both MEKK1 kinase activity and the docking motif on ERK1/2 were involved in ERK1/2 ubiquitination. Significantly, cells expressing ERK2 with the docking motif mutation were resistant to sorbitol-induced apoptosis. Therefore, MEKK1 functions not only as an upstream activator of the ERK and JNK through its kinase domain, but also as an E3 ligase through its PHD domain, providing a negative regulatory mechanism for decreasing ERK1/2 activity.
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PMID:The PHD domain of MEKK1 acts as an E3 ubiquitin ligase and mediates ubiquitination and degradation of ERK1/2. 1204 32

Interleukin-6 (IL-6) is a multifunctional cytokine that plays an important role in inflammatory reactions. We have addressed the possible regulation of IL-6 expression by the ubiquitin-protease system in human umbilical vein endothelial cells. Cultured endothelial cells were treated with MG-132, a protease inhibitor, and the levels of IL-6 mRNA and protein were measured by reverse transcription-PCR and ELISA. MG-132 increased the expression of IL-6 mRNA and protein;and this effect was abolished by the pretreatment of the cells with U0126, an inhibitor of MAP or ERK kinases (MEK 1/2). MG-132 treatment was also found to enhance the level of phosphorylated MEK 1/2. Treatment of the cells with actinomycin D inhibited IL-6 expression in response to MG-132, suggesting the transcriptional upregulation of IL-6 under proteasomal inhibition. We conclude that a protease inhibitor MG-132 upregulates IL-6 expression in vascular endothelial cells, at least in part, through the activation of MEK 1/2.
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PMID:Proteasome inhibitor MG-132 enhances the expression of interleukin-6 in human umbilical vein endothelial cells: Involvement of MAP/ERK kinase. 1206 9

Autophagy is a process for the bulk degradation of cytosolic compartments by lysosomes/vacuoles. The formation of autophagosomes involves a dynamic rearrangement of the membrane for which two ubiquitin-like modifications (the conjugation of Apg12p and the modification of a soluble form of MAP-LC3 to a membrane-bound form) are essential. In yeast, Apg10p is an E2-like enzyme essential for Apg12p conjugation. The isolated mouse APG10 gene product interacts with mammalian Apg12p dependent on mammalian Apg7p (E1-like enzyme), and facilitates Apg12p conjugation. The interaction of Apg10p with Apg12p is dependent on the carboxyl-terminal glycine of Apg12p. Mutational analysis of the predicted active site cysteine (Cys161) within mouse Apg10p shows that mutant Apg10pC161S, which can form a stable intermediate with Apg12p, inhibits Apg12p conjugation even in the presence of Apg7p, while overexpression of Apg7p facilitates formation of an Apg12p-Apg5p conjugate. Furthermore, the coexpression of Apg10p with Apg7p facilitates the modification of a soluble form of MAP-LC3 to a membrane-bound form, a second modification essential for autophagy. Mouse Apg10p interacts with MAP-LC3 in HEK293 cells, while no mutant Apg10pC161S forms any intermediate with MAP-LC3. Direct interaction between Apg10p and MAP-LC3 is also demonstrated by yeast two-hybrid analysis. The inability of mutant Apg10pC161S to form any intermediate with MAP-LC3 has ruled out the possibility that MAP-LC3 interacts with Apg10p as a substrate.
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PMID:The mouse APG10 homologue, an E2-like enzyme for Apg12p conjugation, facilitates MAP-LC3 modification. 1289 Jun 87

In Alzheimer's disease (AD), neuronal thread protein (NTP) accumulates in cortical neurons and colocalizes with phospho- tau-immunoreactive cytoskeletal lesions that correlate with dementia. To generate additional information about the potential role of NTP in AD, we characterized its expression and regulation in human SH-Sy5y neuronal cells. Quantitative real-time reverse transcription-polymerase chain reactin and Western blot analysis demonstrated prominent insulin, moderate insulin-like growth factor, type 1 (IGF-1) and minimal nerve growth factor stimulation of NTP expression. In addition, NTP protein was more stable and it progressively accumulated in cells that were stimulated with insulin for 24 or 48 h. Metabolic labeling and phospho-amino acid analysis demonstrated phosphorylation of NTP on Serine residues, 30-60 min after insulin or IGF-1 stimulation, when glycogen synthase kinase 3beta (GSK-3beta) activity would no longer have been suppressed. Kinase inhibitor and in vitro phosphorylation studies demonstrated a role for GSK-3beta in the positive regulation of NTP expression and phosphorylation. Coimmunoprecipitation studies demonstrated physical interactions between NTP and tau or microtubule-associated protein 1b (MAP-1b), and ubiquitin immunoreactivity in NTP immunoprecipitates. In summary, these studies showed that (i) NTP expression is regulated at the level of transcription by insulin and IGF-1 stimulation; (ii) NTP is phosphorylated by GSK-3beta; (iii) NTP can physically interact with tau and MAP-1b and (iv) NTP-MAP complexes are ubiquitinated. The results suggest a functional role for NTP in relation to the turnover or processing of neuronal cytoskeletal proteins, attributes that may be modulated by insulin/IGF-1-mediated signaling.
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PMID:Neuronal thread protein regulation and interaction with microtubule-associated proteins in SH-Sy5y neuronal cells. 1468 91

ERK7 is a unique member of the extracellular signal-regulated kinase (ERK) subfamily of MAP kinases. Although ERK7 shares a TEY motif in the activation loop of the kinase, it displays constitutive activation, nuclear localization, and growth inhibitory properties that are regulated by its C-terminal domain. Because ERK7 is expressed at low levels compared with ERK2 and its activity is dependent upon its expression level, we investigated the mechanism by which ERK7 expression is regulated. We now show that ERK7 expression is regulated by ubiquitination and rapid proteosomal turnover. Furthermore, both the kinase domain and the C-terminal tail are independently degraded at a rate comparable with that of the intact protein. Analysis of a series of chimeras between ERK2 and ERK7 reveal that the N-terminal 20 amino acids of the kinase domain are a primary determinant of ERK7 degradation. Fusion of the N-terminal 20 amino acids is both necessary and sufficient to cause proteolytic degradation of both ERK2 and green fluorescent protein. Finally, ERK7 is stabilized by an N-terminal mutant of Cullin-1 suggesting that ERK7 is ubiquitinated by the Skip1-Cullin-F box complex. These results indicate that ERK7 is a highly regulated enzyme whose cellular expression and kinase activation level is tightly controlled by the ubiquitin-proteosome pathway.
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PMID:ERK7 expression and kinase activity is regulated by the ubiquitin-proteosome pathway. 1503 83

The ubiquitin-proteasome pathway plays a critical role in the degradation of cellular proteins related to signal transduction. Cytokine and growth factor-dependent aberrant proliferation has been implicated in renal cell carcinoma (RCC). We hypothesized that inhibiting the proteasome function might activate a proapoptotic signal transduction by modulating the cytokine and growth factor related signal transduction pathway. We therefore investigated the effectiveness of a proteasome inhibitor in the treatment of RCC regarding the involvement of Mitogen-activated protein kinases (MAP kinases), because MAP kinases are major signal transduction molecules that are known to play a pivotal role in cancer cell proliferation or apoptosis triggered by extra-cellular cytokines and growth factors. A proteasome inhibitor, MG132 inhibited the proliferation of RCC cell lines, 786-O and KU20-01 in a time and dose-dependent manner. 786-O cells have truncated von-Hippel Lindau (VHL) tumor suppressor gene protein due to a one base pair deletion at exon 1, whereas KU20-01 cells have a wild-type VHL protein. MG132 induced apoptosis in both cell lines. The inhibition of the ubiquitin-proteasome pathways was confirmed by the accumulation of ubiquitin-tagged proteins. MG132 induced the phosphorylation of ERK at 4 h and thereafter persisted for 8 to 16 h. In contrast, JNK and p38 activation persisted for longer periods and remained enhanced until 24 h. The concomitant activation of effector caspases, caspase-3 and caspase-7 was observed in 786-O cells. The inhibition of the proteasome function can induce apoptosis in RCC irrespective of the VHL protein status. The persistence of JNK and p38 activation may therefore be a unique mechanism underlying MG132 induced apoptosis.
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PMID:Inhibition of the ubiquitin-proteasome pathway activates stress kinases and induces apoptosis in renal cancer cells. 1528 72


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