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:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ligand-induced activation of many receptors leads to dissociation of the alpha- and beta gamma-subunit complexes of heterotrimeric G proteins, both of which regulate a variety of effector molecules involved in cellular signaling processes. In one case, a cytosolic enzyme, the beta-adrenergic receptor kinase (beta ARK) binds to the dissociated, prenylated, membrane-anchored beta gamma-subunits of heterotrimeric G proteins (G beta gamma) and is thereby targeted to its membrane-bound receptor substrate. Quite recently, numerous proteins involved in cellular signal transduction have been shown to contain sequences homologous with a "domain" originally identified in the protein "pleckstrin" (pleckstrin homology domain; PH domain) and subsequently found in the G beta gamma interaction region of the beta ARK sequence. Here we demonstrate that glutathione S-transferase-fusion proteins, containing sequences encompassing the PH domain of nine proteins from this group, bind G beta gamma to varying extents. Binding of G beta gamma to these fusion proteins was documented either by a direct binding assay or by ability to block G beta gamma-mediated membrane translocation of beta ARK1. G beta gamma binding to these fusion proteins was inhibited by the alpha subunit of Go (Go alpha), indicating that the binding of G beta gamma to G alpha and the PH domain-containing fusion proteins is mutually exclusive. Studies with a series of truncated PH domains derived from the Ras-guanine-nucleotide-releasing factor indicate that the G beta gamma binding domain includes only the C-terminal portion of the PH domain and sequences just distal to this. Protein-protein interactions between G beta gamma and PH domain-containing proteins may play a significant role in cellular signaling analogous to that previously demonstrated for Src homology 2 and 3 domains.
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PMID:Binding of G protein beta gamma-subunits to pleckstrin homology domains. 814 1

beta-Adrenergic receptor kinase (beta ARK) is a regulatory enzyme involved in the modulation of beta-adrenergic and other G protein-coupled receptors. It has been described that beta ARK is a cytosolic protein that transiently translocates to the plasma membrane in order to specifically phosphorylate agonist-occupied receptors. In this report, we used beta ARK-specific antibodies to demonstrate that a significant amount of this kinase is present in rat liver microsomal membranes. beta ARK seems to be peripherally associated with the cytosolic side of microsomal membranes since it can be stripped from the membranes by mild salt treatment. Cell-free association experiments indicate that the interaction of beta ARK is reversible, saturable, and strongly inhibited by protease or heat treatment of the microsomes, thus suggesting that beta ARK interacts with a protein component of the microsomal membrane. Gradient fractionation studies indicate that the highest beta ARK-specific activity co-migrates with endoplasmic reticulum enzymatic markers. Furthermore, indirect immunofluorescence and immunogold electron microscopy experiments performed in cultured cells using affinity-purified anti-beta ARK antibodies are consistent with this subcellular localization pattern. Taken together, our data suggest that several beta ARK pools (i.e. microsome-bound, plasma membrane-bound, and cytosolic) may exist inside the cell. Such results are in line with recent reports showing that proteins involved in plasma membrane signal transduction, such as heterotrimeric G proteins, are also associated with membranes of different intracellular organelles.
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PMID:Association of the regulatory beta-adrenergic receptor kinase with rat liver microsomal membranes. 828

The purpose of this study was to investigate whether angiotensin-converting enzyme (ACE; EC 3.4.15.1) and neutral endopeptidase (NEP; EC 3.4.24.11), two membrane-bound metalloenzymes that are widely distributed in the peripheral microcirculation and degrade kinins very effectively, modulate bradykinin-induced arteriolar dilation in vivo. Using intravital microscopy, we measured diameter of second-order arterioles in the hamster cheek pouch during suffusion of bradykinin (0.1-10.0 microM) before and after topical application of captopril (10.0 microM) and phosphoramidon (10.0 nM). We found that each inhibitor significantly potentiated bradykinin-induced increase in arteriolar diameter (P < 0.05). Suffusion of other proteinase inhibitors (excluding ACE and NEP inhibitors) had no significant effect on bradykinin-induced responses. Captopril and phosphoramidon did not potentiate isoproterenol (0.1 microM)-induced arteriolar dilation in the cheek pouch. Collectively, these data indicate that ACE and NEP each plays an important role in regulating bradykinin-induced vasorelaxation in the peripheral microcirculation in vivo.
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PMID:Peptidases modulate bradykinin-induced arteriolar dilation in the hamster cheek pouch. 830 27

Neutral endopeptidase 24.11 (EC 3.4.24.11; NEP) is a membrane-bound Zn-metalloendopeptidase with a catalytic activity and a specificity very similar to that of thermolysin, a bacterial zinc-endoprotease. NEP can be inactivated by reaction with diethylpyrocarbonate, due to the modification of a histidine residue present in the active site of the enzyme. This histidine residue was proposed to be analogous to His231 in thermolysin, which is involved in the stabilization of the tetrahedral intermediate during the transition state. Using site-directed mutagenesis of the cDNA encoding rabbit NEP, we have created two mutants of NEP where His711 was replaced by either Gln or Phe (NEP-Gln711 and NEP-Phe711). Determination of kinetic parameters showed that both mutants had Km values very similar to that of the non-mutated enzyme but that their kcat values were 25-fold lower. The calculated difference in free energy needed to form the transition state complex was increased by 2.2 kcal/mol for both mutants. These observations strongly suggest that His711 is involved in the stabilization of the transition state by forming an hydrogen bond with the oxyanion of the tetrahedral intermediate.
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PMID:Kinetic evidence that His-711 of neutral endopeptidase 24.11 is involved in stabilization of the transition state. 844 Mar 86

Neutral endopeptidase (NEP; also known as neprilysin and enkephalinase; EC 3.4.24.11) is a cell-surface metallopeptidase that is present in many mammalian tissues. It is particularly abundant on the brush-border membranes of the kidney proximal tubule. In this paper, the presence of NEP in purified glomeruli from dog kidney was assessed by measuring phosphoramidon- and thiorphan-sensitive [D-Ala2,Leu5]enkephalin-degrading activity. Using this assay, the Km and kcat. of the glomerular enzyme were found to be identical to those of the tubular enzyme. By Western blotting the apparent M(r) of the glomerular enzyme was found to be 104,000, compared with 94,000 for the tubular enzyme. This might be due to a different glycosylation pattern, since endoglycosidase F treatment of NEP obtained from both tissues yielded deglycosylated enzymes with similar electrophoretic mobilities. The glomerular enzyme also appears to be membrane-bound, since it was retained in the detergent-rich phase after phase separation with Triton X-114. Autoradiography experiments performed with RB104, a new highly selective and potent NEP inhibitor, showed that NEP was expressed in both glomeruli and proximal tubules. The presence in glomeruli of NEP and some other brush-border peptidases (dipeptidyl-dipeptidase IV, aminopeptidase N and angiotensin I-converting enzyme) suggests that cell-surface peptidases might play an important role as regulators of plasma-derived peptides in this part of the nephron.
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PMID:Characterization of neutral endopeptidase 24.11 in dog glomeruli. 848 5

Neutral endopeptidase (NEP; also known as EC 3.4.24.11, CALLA) is a widely distributed membrane-bound enzyme that hydrolyzes many biologically important endogenous peptides. To evaluate the influence of glucocorticoids on airway epithelial cell NEP expression, we used the human airway epithelial cell line Calu-1. Cells, grown to confluency in Dulbecco's modified Eagle medium with 10% fetal bovine serum and penicillin-streptomycin, were incubated with different concentrations of dexamethasone or vehicle alone in the presence or absence of actinomycin D or cycloheximide for planned times. NEP activity was assayed at the end of treatment employing reverse-phase, high-pressure liquid chromatography. In some experiments, changes in NEP-specific mRNAs in the presence or absence of dexamethasone and/or the inhibitors were also evaluated by Northern blot analysis. We found that dexamethasone increased Calu-1 NEP activity in a dose- and time-dependent manner. Northern blot analysis indicated that NEP-specific mRNAs were also increased by dexamethasone. Furthermore, neither actinomycin D nor cycloheximide inhibited the increases in NEP activity and NEP-specific mRNAs caused by dexamethasone stimulation. We speculate, therefore, that dexamethasone increases NEP expression of these airway epithelial cells by enhancing transcription and new protein synthesis.
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PMID:Dexamethasone increases airway epithelial cell neutral endopeptidase by enhancing transcription and new protein synthesis. 850 56

A soluble form of the kidney membrane metalloendopeptidase, meprin, is present in urine. Urinary meprin is expressed in BALB/C mice with the Mep-1 alpha/alpha genotype (high meprin, expressing meprin-alpha and meprin-beta ) but not in BALB.K mice of the Mep-1b/b genotype (that only express meprin-beta ). Western blotting with antisera specific to the meprin-alpha and the meprin-beta subunits established that the only form of meprin present in urine samples was derived from meprin-alpha. This form of meprin is partially active, and comprises at least three variants by non-reducing SDS/PAGE and by zymography and two protein bands on reducing SDS/PAGE. Sequencing of these two bands established that the N-terminus of the larger protein band begins with the pro-peptide sequence of the alpha-subunit (VSIKH..), whereas the smaller band possessed the mature meprin N-terminal sequence (NAMRDP..). Trypsin is able to remove the pro-peptide, with a concomitant activation in proteolytic activity. After deglycosylation, the size of the pro- and mature forms of urinary meprin are consistent with cleavage in the region of the X-I boundary. There is a pronounced sexual dimorphism in urinary meprin expression. Females secrete a slightly larger form, and its proteolytic activity is about 50% of that released by males. The urinary meprin is therefore a naturally occurring secreted form of this membrane-bound metalloendopeptidase and is more likely to be generated by alternative processing pathways than by specific release mechanisms.
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PMID:Characterization of the soluble, secreted form of urinary meprin. 861 15

Insulin stimulates the Ras/Raf/MEK/ERK pathway leading to feedback phosphorylation of the Ras guanylnucleotide exchange protein SOS and dissociation of Grb2 from SOS. Even though epidermal growth factor (EGF) also stimulates ERK activity and phosphorylation of SOS similar to insulin, EGF induces a dissociation of the Grb2-SOS complex from Shc. To determine the molecular basis for this difference, we examined the signaling properties of a mutant EGF receptor lacking the five major autophosphorylation sites. Although EGF stimulation of the mutant EGF receptor activates ERK and phosphorylation of both Shc and SOS, it fails to directly associate with either Shc or Grb2. However, under these conditions EGF induces a dissociation of the Grb2-SOS complex suggesting a role for receptor and/or plasma membrane targeting in the stabilization of Grb2-SOS interaction. Consistent with this hypothesis, expression of an SH2 domain Grb2 mutant which is unable to mediate plasma membrane targeting of the Grb2-SOS complex results in both insulin- and EGF-stimulated uncoupling of Grb2 from SOS. Furthermore, a plasma membrane-bound Grb2 fusion protein remains constitutively associated with SOS. Together, these data demonstrate that EGF stimulation prevents the feedback uncoupling of Grb2 from SOS by inducing a persistent plasma membrane receptor targeting of the Grb2-SOS complex.
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PMID:Epidermal growth factor receptor targeting prevents uncoupling of the Grb2-SOS complex. 862 25

HTK is a receptor tyrosine kinase that belongs to the Eph subfamily. An extensive screening using BIAcore system revealed that a colon cancer cell line, C-1, expressed the ligand for HTK. From the conditioned medium of C-1 cells, a soluble form of ligand was purified by receptor affinity chromatography, and the isolation of full-length cDNA revealed that this ligand is identical to the human HTK ligand (HTKL) previously reported. HTK receptor tyrosine phosphorylation was induced by membrane-bound or clustered soluble HTKL but not by unclustered soluble HTKL, indicating that HTKL requires cell-to-cell interaction for receptor activation. Binding analysis demonstrated that HTKL binds to HTK with a much higher affinity (Kd: 1.23 nM) than the other transmembrane-type ligand for Eph family, LERK-2/ELKL (Kd: 135 nM). The expression of HTK in cord blood cells was upregulated after the culture in the presence of stem cell factor. Clustered soluble HTKL stimulated the proliferation of sorted HTK+ cord blood cells and a hematopoietic cell line, UT-7/EPO from which HTK was isolated. These findings suggest the involvement of HTK-HTKL system in the proliferation of HTK+ hematopoietic progenitor cells in the hematopoietic environment.
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PMID:Characterization of a ligand for receptor protein-tyrosine kinase HTK expressed in immature hematopoietic cells. 876 3

A search of the nucleic acid database of expressed sequence tags (ESTs) revealed several partial cDNA sequences that could encode proteins homologous to the ligands for Eph-related kinases (LERKs). Oligonucleotides designed from the ESTs were used to probe a human brain cDNA library and obtain overlapping clones that encoded two different novel LERKS (NLERK-1 and NLERK-2). NLERK-1 and NLERK-2 are most closely related to human LERK-2/Elk-ligand and they form a subclass of LERKs that contain a transmembrane domain and a conserved cytoplasmic domain. Full-length NLERK-1 was expressed as a glycosylated membrane protein in COS cells and was not secreted into the medium. Full-length NLERK-2 was similarly expressed in COS cells but both membrane-bound and a truncated, proteolytically-released form were detected. Engineered forms of both NLERK-1 and NLERK-2 lacking transmembrane and cytoplasmic domains were also expressed in COS cells and each was detected in the extracellular medium.
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PMID:Molecular cloning of two novel transmembrane ligands for Eph-related kinases (LERKS) that are related to LERK-2. 880 96


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