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)

We describe the molecular cloning of a receptor tyrosine kinase from a cell line (LK63) derived from a case of human pre-B-cell leukemia. We have previously shown that a monoclonal antibody (IIIA4) raised against LK63 recognized a glycosylated, cell-surface 135-kDa molecule (HEK), which displayed tyrosine kinase activity in vitro. The HEK protein was purified by using a IIIA4 antibody column and both N-terminal and internal amino acid sequences were obtained. A 51-mer degenerate oligonucleotide based on the internal amino acid sequence was used to screen an LK63-derived lambda gt10 cDNA library under low-stringency hybridization conditions. One clone of 2.5 kilobases (kb) was isolated and characterized and used to rescreen the library under more-stringent hybridization conditions. A 4.5-kb clone containing the entire HEK coding region was isolated and its complete DNA sequence was determined. The 4.5-kb insert was subcloned into the expression vector CDM8 and transfected into COS cells. COS cells transfected with the sense HEK/CDM8 construct stained specifically with the IIIA4 antibody, thereby confirming that the antigen recognized by the IIIA4 antibody and the expressed protein product of the HEK cDNA clone were identical. DNA sequence analysis revealed that HEK is a newly discovered member of the EPH/ELK family of receptor tyrosine kinases. Northern blot analysis of a number of cell lines demonstrated the expression of 5.5- to 6.0-kb HEK transcripts in LK63 and the T-cell lines JM and HSB-2. Southern blot analysis of DNA from LK63 suggested that the HEK gene was neither amplified nor rearranged in the LK63 tumor.
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PMID:Molecular cloning of HEK, the gene encoding a receptor tyrosine kinase expressed by human lymphoid tumor cell lines. 131 45

Neutral endopeptidase (NEP; EC 3.4.24.11) is an integral membrane protein found at the plasma membrane of many cell types. A secreted form of NEP (sec-NEP) was recently obtained by transfection of COS-1 cells with a recombinant expression vector consisting of the cDNA encoding the signal peptide of pro-opiomelanocortin fused in-frame to the cDNA sequence of the complete ectodomain of rabbit NEP [Lemay, Waksman, Roques, Crine & Boileau (1989) J. Biol. Chem. 264, 15620-15623]. In order to produce large quantities of this enzyme for structural studies we have expressed this recombinant soluble form of NEP at high yields using a baculovirus/insect-cell system. A recombinant Autographa californica nuclear polyhedrosis-virus genome containing the sec-NEP sequence was used to infect host Spodoptera frugiperda Sf9 cells. Infected cells secreted an N-glycosylated soluble form of neutral endopeptidase which was enzymically active. The yield was about 80 nmol of enzyme/litre of culture. The soluble form of the recombinant enzyme purified by immunoaffinity showed the same catalytic properties as the wild-type enzyme extracted from the kidney brush-border membranes. Treatment of the recombinant enzyme with endo-beta-N-acetylglucosaminidase H showed, however, that invertebrate cells did not glycosylate the enzyme to the same extent as did mammalian cells. Our findings demonstrate that insect cells can be used as hosts for the production of the soluble form of neutral endopeptidase. We also conclude that neither a full complement of carbohydrate side chains nor the membrane anchor appear to be essential for the production and targeting to the cell surface of a fully functional enzyme in this expression system.
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PMID:Secretion of a functional soluble form of neutral endopeptidase-24.11 from a baculovirus-infected insect cell line. 159 10

cDNA clones coding for novel protein kinase C delta (nPKC delta) were isolated from a mouse brain cDNA library. Mouse nPKC delta consists of 674 amino acid residues and has sequence identity of 95% with rat nPKC delta. Antiserum raised against a C-terminal peptide of rat nPKC delta identified a 79-kDa protein in COS cells transfected with a mouse nPKC delta cDNA expression plasmid. nPKC delta expressed in COS1 cells had phorbol-ester-binding activity and protein kinase activity in a phorbol-ester- or diacylglycerol-dependent manner, like conventional protein kinase C (cPKC) isozymes and nPKC epsilon. However, nPKC delta, like nPKC epsilon, is not activated by Ca2+, a known activator of cPKCs, and requires lower concentrations of Mg2+ for full activation than cPKCs. Moreover, apparent kinetic constants for synthetic oligopeptides (MBP4-14, EGFR peptide and epsilon-peptide) were quite different between nPKC delta and cPKC in two different conditions. Among various phospholipids tested, phosphatidylinositol is the most potent activator of nPKC delta, in clear contrast to cPKCs and nPKC epsilon. Limited proteolysis of nPKC delta generated a C-terminal active fragment with a cofactor-independent kinase activity. Northern blot analysis indicated that nPKC delta, like cPKC alpha, is widely distributed in almost all the tissues and cells examined and, in some cases such as fibroblast cells, exists as a major PKC type. These results suggest that nPKC delta is involved in fundamental cellular functions regulated by diacylglycerols and mimicked by phorbol esters.
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PMID:Structure and properties of a ubiquitously expressed protein kinase C, nPKC delta. 176 3

The beta-adrenergic receptor kinase (beta ARK) specifically phosphorylates the agonist-occupied form of the beta-adrenergic and related G protein-coupled receptors. Structural features of this enzyme have been elucidated recently by the isolation of a cDNA that encodes bovine beta ARK. Utilizing a catalytic domain fragment of the beta ARK cDNA to screen a bovine brain cDNA library we have isolated a clone encoding a beta ARK-related enzyme which we have termed beta ARK2. Overall, this enzyme has 85% amino acid identity with beta ARK, with the protein kinase catalytic domain having 95% identity. The ability of beta ARK2 to phosphorylate various substrates was studied after expression in COS 7 cells. Although beta ARK2 is essentially equiactive with beta ARK in phosphorylating an acid-rich synthetic model peptide it was only approximately 50% as active when the substrate was the agonist-occupied beta 2-adrenergic receptor and only approximately 20% as active toward light-bleached rhodopsin. As with beta ARK, phosphorylation of the receptor substrates by beta ARK2 was completely stimulus dependent. RNA blot analysis with selected bovine tissues reveals an mRNA of 8 kilobases with a distribution similar to that of beta ARK. More detailed RNA analysis using a ribonuclease protection assay in various rat tissues suggests that the beta ARK2 message is present at much lower levels (typically 10-20%) than the beta ARK message. In the rat the beta ARK2 mRNA is localized predominantly in neuronal tissues although low levels are also observed in various peripheral tissues. The beta ARK2 gene has been localized to a region of mouse chromosome 5 whereas the beta ARK gene is localized on mouse chromosome 19. These data suggest the existence of a "family" of receptor kinases which may serve broadly to regulate receptor function.
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PMID:Cloning, expression, and chromosomal localization of beta-adrenergic receptor kinase 2. A new member of the receptor kinase family. 186 33

Epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), and amphiregulin are structurally and functionally related growth regulatory proteins. These secreted polypeptides all bind to the 170-kDa cell-surface EGF receptor, activating its intrinsic kinase activity. However, amphiregulin exhibits different activities than EGF and TGF-alpha in a number of biological assays. Amphiregulin only partially competes with EGF for binding EGF receptor, and amphiregulin does not induce anchorage-independent growth of normal rat kidney cells (NRK) in the presence of TGF-beta. Amphiregulin also appears to abrogate the stimulatory effect of TGF-alpha on the growth of several aggressive epithelial carcinomas that overexpress EGF receptor. These findings suggest that amphiregulin may interact with a separate receptor in certain cell types. Here we report the cloning of another member of the human EGF receptor (HER) family of receptor tyrosine kinases, which we have named "HER3/ERRB3." The cDNA was isolated from a human carcinoma cell line, and its 6-kilobase transcript was identified in various human tissues. We have generated peptide-specific antisera that recognizes the 160-kDa HER3 protein when transiently expressed in COS cells. These reagents will allow us to determine whether HER3 binds amphiregulin or other growth regulatory proteins and what role HER3 protein plays in the regulation of cell growth.
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PMID:Molecular cloning and expression of an additional epidermal growth factor receptor-related gene. 216 10

We have isolated, from a human tumor cDNA library, a gene encoding a putative receptor-like protein-tyrosine kinase that we call TK14. The amino acid sequence of the TK14 protein is closely related to the available partial sequence of the mouse protein bek, and more distantly related to the sequences of a chicken basic fibroblast growth factor receptor (73% sequence homology) and the apparent human equivalent of this receptor, the FLG protein (encoded by the fms-like tyrosine kinase gene). Overexpression of the TK14 protein by transfection of COS-1 cells with the corresponding cDNA in a simian virus 40-based expression vector leads to the appearance of new cell-surface binding sites for both acidic and basic fibroblast growth factors. This has been demonstrated by specific binding assays and chemical cross-linking experiments using 125I-labeled growth factors. It appears, therefore, that the human genome contains at least two distinct genes, for TK14 and FLG, that code for related fibroblast growth factor receptors.
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PMID:Related fibroblast growth factor receptor genes exist in the human genome. 217 78

Direct comparison of the primary structure of neutral endopeptidase (NEP, EC 3.4.24.11) with that of thermolysin, a bacterial metalloendopeptidase with a similar specificity, has revealed very few similarities between the two sequences, except for two conserved short segments. In thermolysin, these segments contain several of the residues involved in catalysis, including two zinc coordinating histidines (His-142 and His-146) and a third histidine (His-231) involved in stabilizing the transition state through hydrogen bonding. The role of the corresponding histidines in NEP (His-583, His-587 and His-637) was explored by site-directed mutagenesis of NEP cDNA and expression of the mutated cDNA in COS-1 cells. Substitution of either His-583 or His-587 of NEP for Phe completely abolished the activity and Zn-directed inhibitor recognition of the recombinant enzyme, suggesting that these residues play a role similar to His-142 and His-146 of thermolysin as zinc ligands. In contrast, substitution of His-637 for a phenylalanine residue was without effect on enzyme activity.
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PMID:Exploration of the catalytic site of endopeptidase 24.11 by site-directed mutagenesis. Histidine residues 583 and 587 are essential for catalysis. 316 86

In a previous study the BamHI-K fragment of Epstein-Barr virus DNA was shown to induce a nuclear antigen, Epstein-Barr virus nuclear antigen (EBNA), when cotransfected with the herpes simplex virus thymidine kinase gene into mouse LTK- cells. We have now inserted the BamHI-K fragment and a BamHI/HindIII subfragment, I1f , into shuttle vectors containing the origin of replication of simian virus 40. These plasmids have been introduced into COS-1, which are monkey kidney cells transformed by an origin-defective simian virus 40 genome. This expression system permitted rapid characterization of antigens, mRNAs, and proteins related to EBNA. The same-sized EBNA protein (approximately 78,000) was made after transfection with BamHI-K (5.2 kilobase pairs [kbp]) or the I1f subfragment (2.9 kbp). A deletion of about 600 bp occurred when the I1f fragment was propagated on the pSV2 plasmid in Escherichia coli. The deleted fragment gave rise to a smaller protein (approximately 52,000). These data provide evidence that EBNA is encoded by the 2.9-kbp I1f and is not an induced cellular protein. Nuclear antigen and polypeptide expression occurred equally well when the Epstein-Barr virus DNA was cloned on PSV2 -gpt or pSVOd . The latter plasmid lacks sequences allowing for efficient early gene transcription as well as splicing and polyadenylation signals which are present in pSV2 . Preliminary mapping of the EBNA gene transcripts demonstrated that two mRNAs (2.9 and 2.4 kilobases [kb]) are homologous to the I1f fragment. Taken together, the data suggest that the 2.9-kbp I1f fragment contains the structural gene for EBNA synthesis. COS-1 cells will thus provide a valuable system in which to analyze functional domains of the EBNA gene.
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PMID:Expression in COS-1 cells of Epstein-Barr virus nuclear antigen from a complete gene and a deleted gene. 632 12

Signaling through the B cell antigen receptor (BCR) results in rapid increases in tyrosine phosphorylation on a number of proteins. The BCR associates with two classes of tyrosine kinase: Src-family kinase (Src-protein-tyrosine kinase [PTK]; Lyn, Fyn, Blk, or Lck) and Syk kinase. We have investigated the interaction between the Src-PTK and the Syk kinase in the BCR signaling. In contrast to wild-type B cells, BCR-mediated tyrosine phosphorylation of Syk and activation of its in vitro kinase activity were profoundly reduced in lyn-negative cells. The requirement of the Src-PTK to induce tyrosine phosphorylation and activation of Syk was also demonstrated by cotransfection of syk and src-PTK cDNAs into COS cells. These results suggest that the Src-PTK associated with BCR phosphorylates the tyrosine residue(s) of Syk upon receptor stimulation, enhancing the activity of Syk.
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PMID:Syk activation by the Src-family tyrosine kinase in the B cell receptor signaling. 751 17

The FLT3 gene encodes an hematopoietic receptor related to the receptors for colony-stimulating factor 1, FMS, and for Steel factor, KIT. The extracellular part of these molecules is exclusively composed of five immunoglobulin (Ig)-like domains, designated 1 to 5, from the amino terminus to the carboxyl terminus of the extracellular region. We have isolated a unique murine FLT3 cDNA that codes for a variant isoform of FLT3, devoid of the fifth Ig-like domain, by comparison with the prototypic form. The corresponding mRNA is the result of a splicing event that leads to the elimination of two coding exons. mRNA coding for this variant was detected in almost all the tissues expressing the mRNA coding for the prototypic molecule, although at a lower level. Ligand-induced tyrosine phosphorylation of the two isoforms was equivalent in COS-1 transfected cells, indicating that the fifth Ig-like domain is not strictly necessary for either ligand-binding or kinase activation.
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PMID:Identification and characterization of a functional murine FLT3 isoform produced by exon skipping. 753


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