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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 have expressed in COS-1 cells mutants of neprilysin (neutral endopeptidase-24.11; NEP) in which the hydrophilic sequence S-Q-N-S was either substituted for V42-T-M-I or inserted after T38 in the signal peptide/membrane anchor (SA) domain. These mutations were introduced in full-length NEP (mutants NEP(H1) and NEP(H2), respectively) and a form of NEP lacking its cytosolic tail (mutants NEP delta cyto(H1) and NEP delta cyto(H2), respectively). Immunoblotting showed that NEP(H1) was membrane-bound while NEP delta cyto(H1), NEP(H2), and NEP delta cyto(H2) were secreted. Furthermore, carbonate treatment of isolated intracellular membranes suggested that cleavage of the SA domain was performed in the endoplasmic reticulum, presumably by signal peptidase. Sequencing of the secreted proteins indicated that cleavage of the SA domain mostly occurred at the carboxy side of Ala46 but also at the carboxy side of Ala41 in NEP(H2) and NEP delta cyto(H2). We conclude that the position of the S-Q-N-S sequence influences the accessibility of the cleavage site and, in the case of NEP(H1) and NEP(H2), the efficiency of cleavage of the SA domain.
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PMID:Insertion of hydrophilic amino acid residues in the signal peptide/membrane anchor domain of neprilysin (neutral endopeptidase-24.11) results in its cleavage: role of the position of insertion. 798 81

Stable clones of NIH 3T3 fibroblasts transfected with the cDNA of either the wild-type or deletion forms of the rat type I (or cerebellar) inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) were investigated. The delta form, missing the NH2-terminal sequence that includes the IP3-binding site, is expected to be still assembled with wild-type subunits to yield a tetrameric Ca2+ channel across the endoplasmic reticulum membrane; the s form, missing the membrane-spanning sequences, is expected to remain as a soluble monomer in the cytosol. With respect to control clones transfected with the vector only, the synthesis fo IP3Rs was markedly stimulated in the receptor-transfected clones. The mass accumulation, however, was increased only moderately (deletion forms = 15-30% of the endogenous IP3R), apparently because of a compensatory increase in receptor turnover. Coordinate changes in IP3 generation and Ca2+ release were revealed in the delta clones by experiments in both intact and permeabilized cells. In these clones, the IP3R was more sensitive to IP3, and IP3 generation at the ATP P2u surface receptor was decreased. This latter effect was due neither to a defect in G protein coupling nor to changes in phospholipase C expression, but to down-regulation of the P2u receptor. In the cells expressing the s- and delta-IP3R subunits, no differences with respect to the controls were observed in epidermal growth factor-induced DNA synthesis, whereas long-term growth stimulated by serum was reduced. Even more marked, especially in the delta clones (-90%), was the inhibition of cell transformation induced by autocrine stimulation with transforming growth factor alpha of the overexpressed epidermal growth factor receptors or by other growth factor receptors and oncogenes (platelet-derived growth factor/platelet-derived growth factor receptor beta, HER2/neu, and v-erbB). These effects appear not to be connected to the signaling processes mediated by tyrosine phosphorylation since the latter was unchanged in the delta clones. These results demonstrate for the first time (a) that the changes in cellular homeostasis directly induced by deleted IP3R subunits (increased receptor synthesis and increased IP3R sensitivity) are largely compensated by indirect coordinate changes apparently aimed to keep near normal the signaling properties of the cells; (b) that modulation of intracellular Ca2+ channels induces profound consequences that differentially affect growth and oncogenesis; and (c) that IP3Rs and the Ca2+ stores are important cross-roads of intracellular signaling pathways.
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PMID:Stable expression of truncated inositol 1,4,5-trisphosphate receptor subunits in 3T3 fibroblasts. Coordinate signaling changes and differential suppression of cell growth and transformation. 803 82

Cloned sequences encoding a truncated form of the HER2 receptor were obtained from cDNA libraries derived from two HER2-overexpressing human breast cancer cell lines, BT-474 and SK-BR-3. The 5' 2.1 kb of the encoded transcript is identical to that of full-length 4.6-kb HER2 transcript and would be expected to produce a secreted form of HER2 receptor containing only the extracellular ligand binding domain (ECD). The 3' end of the truncated transcript diverges 61 nucleotides before the receptor's transmembrane region, reads through a consensus splice donor site containing an in-frame stop codon, and contains a poly(A) addition site, suggesting that the truncated transcript arises by alternative RNA processing. S1 nuclease protection assays show a 40-fold variation in the abundance of the truncated 2.3-kb transcript relative to full-length 4.6-kb transcript in a panel of eight HER2-expressing tumor cell lines of gastric, ovarian, and breast cancer origin. Expression of this truncated transcript in COS-1 cells produces both secreted and intracellular forms of HER2 ECD; however, immunofluorescent labeling of HER2 ECD protein in MKN7 tumor cells that natively overexpress the 2.3-kb transcript suggests that transcriptionally generated HER2 ECD is concentrated within the perinuclear cytoplasm. Metabolic labeling and endoglycosidase studies suggest that this HER2 ECD (100 kDa) undergoes differential trafficking between the endoplasmic reticulum and Golgi compartments compared with full-length (185-kDa) HER2 receptor. Transfection studies indicate that excess production of HER2 ECD in human tumor cells overexpressing full-length HER2 receptor can result in resistance to the growth-inhibiting effects of anti-HER2 monoclonal antibodies such as muMAb4D5. These findings demonstrate alternative processing of the HER2 transcript and implicate a potentially important growth regulatory role for intracellularly sequestered HER2 ECD in HER2-amplified human tumors.
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PMID:A truncated intracellular HER2/neu receptor produced by alternative RNA processing affects growth of human carcinoma cells. 809 58

In vivo infection of human T cell lymphocytes by HIV-1 is mediated by the specific binding of the HIV-1 envelope glycoprotein gp120 to the T cell CD4 receptor. One of the post-infection events observed in vivo is the progressive loss of CD4+ T cells. One possible mechanism is the production of infected T cells which are lacking in surface expression of the CD4 receptor protein. We have analysed this possibility utilizing the two HIV-1 chronically-infected CD4- cell lines, 8E5 and ACH-2, both of which are derived from a CD4+ parental strain (A3.01) after HIV-1 infection. In each cell (8E5 and ACH-2) the loss of CD4 surface expression was found to occur by different mechanisms. In ACH-2 cells, neither CD4 protein nor the 3 kb CD4 RNA transcript could be detected. However, treatment of ACH-2 cells with cycloheximide elicited production of the 3 kb transcript suggesting the possibility for a repressor protein(s) to act at the level of transcription and/or stability of the 3 kb mRNA. In contrast, in 8E5 cells the level of the 3 kb CD4 RNA was comparable with that found in the CD4+ A3.01 parental strain. Analysis of the 8E5 strain revealed the presence of a CD4- gp160 bimolecular protein complex sequestered internally in the rough endoplasmic reticulum (RER). Finally, the protein tyrosine kinase p56lck, normally associated with the cellular membrane, appeared to be linked to the RER and bound to the CD4- gp160 proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Transcriptional and post-transcriptional mechanisms are involved in the absence of CD4 surface expression in two HIV-1 chronically infected T cell lines. 810 73

The subtilisin-like enzyme PC1 (also known as PC3) cleaves the neuropeptide precursor proopiomelanocortin at paired basic residues in transfection experiments, thus providing evidence for a critical role in precursor processing. While mRNA for this enzyme is highly enriched in neuroendocrine tissues, little is known about the tissue and subcellular distribution of the PC1 protein. This study used immunocytochemical techniques to investigate the anatomical distribution of PC1, both alone and compared to met-enkephalin (MET-enk), in AtT-20 pituicytes transfected with proenkephalin cDNA. A high density of PC1 immunostaining was observed in a small region adjacent to the nucleus and in the tips of the processes of these cells. Dual-staining immunocytochemistry of whole cells illustrated that both PC1 and MET-enk immunoreactivity were present in the tips, but PC1 was concentrated in a region adjacent to the nucleus while MET-enk punctate staining was dispersed throughout the soma. This codistribution was confirmed in semithin sections of dual-stained cells cut at 1-1.5 microns through the thickness of the cells. PC1 staining resembled that of TGN38, a marker for the trans-Golgi network. When PC1 immunocytochemistry was performed in cells that were pretreated with brefeldin A, a drug that redistributes the proximal Golgi compartments to the endoplasmic reticulum, there was a complete disruption of the defined locus of PC1 immunoreactivity. Taken together, our data indicate that (1) PC1 is concentrated in a region of the cell body resembling the trans-Golgi network and (2) both the enzyme and the processed peptide are transported to the tips of the processes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Immunocytochemical localization of the neuropeptide-synthesizing enzyme PC1 in AtT-20 cells. 811 23

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

We have derived highly enriched populations of ensheathing cells (ECs) from the olfactory nerve layer of neonatal rat olfactory bulbs. Contaminating cells, such as fibroblasts, were eliminated from EC cultures by cytosine arabinoside and immunoadsorption with antiserum to Thy-1.1. At the same time, ECs were stimulated to divide by the addition of bovine pituitary extract into the culture media. Confluent cultures containing 96-99% ECs, were comprised of either spindly bipolar cells or cells bearing multiple processes oriented on opposite poles. The ECs immunostained positively for GFAP, S-100 protein, N-CAMs and Neu 5, and were negative for the presence of neurofilaments. Scanning and transmission electron microscopy showed that the ultrastructure of the ECs resembled that in vivo. The nucleus was irregular in shape, intermediate filaments were usually scattered throughout the cytoplasm instead of being grouped into bundles, and rough endoplasmic reticulum existed as isolated expanded profiles.
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PMID:Cultures of ensheathing cells from neonatal rat olfactory bulbs. 843 68

Overexpression of the erbB-2 gene contributes to aggressive behavior of various human adenocarcinomas, including breast cancer, through an unknown molecular mechanism. The erbB-2-encoded protein is a member of the ErbB family of growth factor receptors, but no direct ligand of ErbB-2 has been reported. We show that in various cells ErbB-2 can form heterodimers with both EGF receptor (ErbB-1) and NDF receptors (ErbB-3 and ErbB-4), suggesting that it may affect the action of heterologous ligands without the involvement of a direct ErbB-2 ligand. This possibility was addressed in breast cancer cells through either overexpression of ErbB-2 or by blocking its delivery to the cell surface by means of an endoplasmic reticulum-trapped antibody. We report that ErbB-2 overexpression enhanced binding affinities to both EGF and NDF, through deceleration of ligand dissociation rates. Likewise, removal of ErbB-2 from the cell surface almost completely abolished ligand binding by accelerating dissociation of both growth factors. The kinetic effects resulted in enhancement and prolongation of the stimulation of two major cytoplasmic signaling pathways, namely: MAP kinase (ERK) and c-Jun kinase (SAPK), by either ligand. Our results imply that ErbB-2 is a pan-ErbB subunit of the high affinity heterodimeric receptors for NDF and EGF. Therefore, the oncogenic action of ErbB-2 in human cancers may be due to its ability to potentiate in trans growth factor signaling.
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PMID:ErbB-2 is a common auxiliary subunit of NDF and EGF receptors: implications for breast cancer. 861 1

The intracellular localization of Shc proteins was analyzed by immunofluorescence and immunoelectron microscopy in normal cells and cells expressing the epidermal growth factor receptor or the EGFR/erbB2 chimera. In unstimulated cells, the immunolabeling was localized in the central perinuclear area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane and endocytic structures, such as coated pits and endosomes, and with the peripheral cytosol. Receptor activation in cells expressing phosphorylation-defective mutants of Shc and erbB-2 kinase showed that receptor autophosphorylation, but not Shc phosphorylation, is required for redistribution of Shc proteins. The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein.
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PMID:Sch proteins are localized on endoplasmic reticulum membranes and are redistributed after tyrosine kinase receptor activation. 862 61

We investigated unusual structures produced in BHK-21 cells infected with rabies virus (HEP-Flury strain). Sellers' staining of the cells revealed, in addition to Negri body-like structures (inclusion bodies), production of a fuchsin-stained cytoplasmic structure (FCPS) which encircled the nucleus. The frequency of the FCPS-forming cells increased as replication progressed. The FCPS was different from the inclusion body because the former contained the viral glycoprotein (G) and matrix protein (M2) antigens, while the latter contained nucleocapsid antigens. In the early phase of infection, we observed accumulation of viral envelope antigens in a cytoplasmic structure that was considered to be expanded rough endoplasmic reticulum (rER) because of its concomitant increase in BiP content. Time-course studies suggested that the envelope antigen-containing structure, which was not stained with basic fuchsin, translocated to the perinuclear region to form the FCPS. FCPS formation was dependent on incubation temperature and was decreased at 30 degrees C, while the development of virus-induced cytopathic effect (CPE) was delayed. When the incubation temperature was shifted up to 37 degrees C, FCPS formation was induced again and progression of CPE was accelerated in approximate proportion to the increasing number of FCPS-positive cells. From these studies, we conclude that viral G proteins gradually accumulate in the rER with M2 protein and the expanded rER converts eventually into the FCPS, which may be closely related to accelerated host cell death.
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PMID:Studies on unusual cytoplasmic structures which contain rabies virus envelope proteins. 881 Oct 13


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