UNIPROT:P04626 - FACTA Search

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Query: UNIPROT:P04626 (erbB-2)
5,251 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effect of herbimycin A on the biosynthesis of epidermal growth factor (EGF) receptor was examined in human epidermoid carcinoma A431 cells. Cells were pulse-labelled with [35S]methionine, and EGF receptor biosynthesis was quantified by immunoprecipitation using a monoclonal anti-(EGF receptor) antibody. In the presence of herbimycin A, an immature 160 kDa EGF receptor precursor accumulated in 1 h and disappeared completely in 4 h. Pulse-labelled 160 kDa receptor precursor in the absence of herbimycin A, however, was converted normally into a 170 kDa one by chase with herbimycin A. Herbimycin A affected neither the synthesis of the secreted form of EGF receptor devoid of cytoplasmic domain, nor that of the transferrin receptor in A431 cells. The herbimycin A-induced degradation of 160 kDa EGF receptor precursor was not inhibited by an inhibitor of lysosomal enzymes, NH4Cl. Endoglycosidase H digestion of the 160 kDa precursor converted it into the deglycosylated 130 kDa precursor peptide. These results suggested that herbimycin A selectively acted on the EGF receptor precursor during the synthesis of the 160 kDa form, probably on the cytoplasmic domain, to form an aberrant molecule which was subjected to rapid degradation in the endoplasmic reticulum.
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PMID:Accelerated degradation of 160 kDa epidermal growth factor (EGF) receptor precursor by the tyrosine kinase inhibitor herbimycin A in the endoplasmic reticulum of A431 human epidermoid carcinoma cells. 803 92

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

Treatment of SKBr3 human breast carcinoma cells with the benzoquinoid ansamycin, geldanamycin, rapidly depletes p185c-erbB-2 protein-tyrosine kinase. Loss of p185c-erbB-2 is initiated by disruption of a heteromeric complex between p185c-erbB-2 and the 94-kDa glucose-regulated protein, GRP94, to which geldanamycin binds avidly. Here we report that within minutes of exposure to geldanamycin, mature p185c-erbB-2 becomes polyubiquitinated. Treatment of cells with the specific proteasome proteolytic inhibitor, lactacystin, blocked geldanamycin-induced degradation of p185c-erbB-2 and enhanced the accumulation of polyubiquitinated p185c-erbB-2. Following geldanamycin and lactacystin treatment, a higher molecular weight form of p185c-erbB-2, which likely represents ubiquitin-p185c-erbB-2 conjugates, was detected by anti-p185c-erbB-2 immunoblotting. Nascent p185c-erbB-2 synthesized in the presence of geldanamycin is incompletely glycosylated and remains sequestered in the endoplasmic reticulum. While this immature form of the protein is not ubiquitinated in the presence of geldanamycin, its marked, drug-induced instability is nonetheless antagonized by lactacystin. Thus, the rapid depletion of mature p185c-erbB-2 caused by geldanamycin and the marked, drug-stimulated decrease in half-life of the newly synthesized protein are both mediated by the proteasome, although only the former phenomenon involves polyubiquitination.
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PMID:Polyubiquitination and proteasomal degradation of the p185c-erbB-2 receptor protein-tyrosine kinase induced by geldanamycin. 879 56

erbB-2 is known to be overexpressed in several human malignancies including lung cancer. Because of its role in neoplastic transformation as well as its association with poor prognosis, this oncogene has been targeted through various anti-cancer methodologies. In this regard, we have recently demonstrated that erbB-2-overexpressing ovarian tumor cell lines transfected with an endoplasmic reticulum form of an anti-erbB-2 single-chain antibody undergo a specific cytotoxicity through the induction of apoptosis. Since certain forms of lung cancer are also associated with overexpression of erbB-2, we evaluated the use of this novel therapeutic in this context. For these studies, several human lung adenocarcinoma cell lines were stably and transiently transfected with the anti-erbB-2 sFv gene. We demonstrate here that the anti-erbB-2 sFv can cause specific cytotoxicity in lung cancer cells. As a first step toward clinical translation of this strategy, we constructed a replication-deficient recombinant adenoviral vector expressing the anti-erbB-2 sFv construct. We further demonstrate that our anti-erbB-2 sFv-encoding adenoviral vector can accomplish high levels of cytotoxicity in lung cancer cells. Based on these results, it is proposed that this strategy of oncoprotein ablation may have use in the treatment of some forms of human lung cancer.
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PMID:erbB-2 knockout employing an intracellular single-chain antibody (sFv) accomplishes specific toxicity in erbB-2-expressing lung cancer cells. 881 Jun 38

The identification of substrates of protein tyrosine phosphatases (PTPs) is an essential step toward a complete understanding of the physiological function of members of this enzyme family. PTPs are defined by a conserved catalytic domain harboring 27 invariant residues. From a mutagenesis study of these invariant residues that was guided by our knowledge of the crystal structure of PTP1B, we have discovered a mutation of the invariant catalytic acid (Asp-181 in PTP1B) that converts an extremely active enzyme into a "substrate trap." Expression of this D181A mutant of PTP1B in COS and 293 cells results in an enzyme that competes with endogenous PTP1B for substrates and promotes the accumulation of phosphotyrosine primarily on the epidermal growth factor (EGF) receptor as well as on proteins of 120, 80, and 70 kDa. The association between the D181A mutant of PTP1B and these substrates was sufficiently stable to allow isolation of the complex by immunoprecipitation. As predicted for an interaction between the substrate-binding site of PTP1B and its substrates, the complex is disrupted by vanadate and, for the EGF receptor, the interaction absolutely requires receptor autophosphorylation. Furthermore, from immunofluorescence studies, the D181A mutant of PTP1B appeared to retain the endogenous EGF receptor in an intracellular complex. These results suggest that the EGF receptor is a bona fide substrate for PTP1B in vivo and that one important function of PTP1B is to prevent the inappropriate, ligand-independent, activation of newly synthesized EGF receptor in the endoplasmic reticulum. This essential catalytic aspartate residue is present in all PTPs and has structurally equivalent counterparts in the dual-specificity phosphatases and the low molecular weight PTPs. Therefore we anticipate that this method may be widely applicable to facilitate the identification of substrates of other members of this enzyme family.
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PMID:Development of "substrate-trapping" mutants to identify physiological substrates of protein tyrosine phosphatases. 905 Aug 38

We previously demonstrated that delivery of a gene encoding an anti-erbB-2 intracellular single-chain antibody (sFv) resulted in down-regulation of cell surface erbB-2 levels and induction of apoptosis in erbB-2 overexpressing ovarian cancer cells. Based upon these findings, we hypothesized that human breast carcinomas overexpressing erbB-2 would be similarly affected by this genetic intervention. We evaluated the phenotypic effects resulting from intracellular expression of the anti-erbB-2 sFv on the human breast cancer cell lines MDA-MB-361, SK-BR-3, BT-474, MCF-7 and MDA-MB-231. Recombinant adenoviruses encoding either a reporter gene (AdCMVLacZ) or the endoplasmic reticulum (ER) directed anti-erbB-2 sFv (Ad21) were delivered to various breast cancer cell lines. Cell viability was determined by a proliferation assay and fluorescent microscopy allowed visualization of apoptotic cells. An erbB-2 ELISA quantified the endogenous erbB-2 levels of each cell line. The anti-erbB-2 sFv-encoding-adenovirus, Ad21, but not the beta-galactosidase encoding adenovirus, AdCMVLacZ, was cytotoxic to > 95% of the tumor cells in the MDA-MB-361 and SK-BR-3 lines, and > 60% of the tumor cells in the BT-474 line. In marked contrast, the MCF-7 and MDA-MB-231 cell lines showed no change in the rate of cell proliferation following this treatment. The cytotoxic effects generated in the first three lines were a consequence of the induction of apoptosis by the anti-erbB-2 sFv. An ELISA specific for erbB-2 showed that the breast cancer cell lines most susceptible to the anti-erbB-2 sFv, MDA-MB-361, SK-BR-3 and BT-474, overexpressed the erbB-2 protein while the cell lines demonstrating no response to the anti-erbB-2 sFv, MCF-7 and MDA-MB-231, expressed the lowest levels of erbB-2. These results demonstrate that targeted killing of erbB-2 overexpressing cells via intracellular knockout can be accomplished in the context of breast carcinoma. Furthermore, erbB-2 levels in breast tumor cells may be predictive of their sensitivity to sFv-mediated killing. The ability to accomplish selective cytotoxicity of breast cancer cell lines overexpressing the erbB-2 tumor marker should allow for derivation of clinical gene therapy strategies for breast cancer utilizing this approach.
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PMID:An intracellular anti-erbB-2 single-chain antibody is specifically cytotoxic to human breast carcinoma cells overexpressing erbB-2. 917 17

Membrane fatty acid desaturases are responsible for inserting double bonds into specific positions in fatty acids. We have cloned a new member of the human membrane fatty acid (lipid) desaturase gene family, MLD. The derived amino acid sequence of MLD contains three consensus motifs, HX3H, HX2HH, and HX2HHXFP, that are characteristic of a group of membrane fatty acid desaturases. MLD is predicted to be a multiple membrane-spanning protein and is found to be extractable from particulate fractions with detergent but not salt or urea. MLD is widely expressed in human tissues and is localized to the endoplasmic reticulum. Cotransfection of MLD with the epidermal growth factor (EGF) receptor resulted in decreased expression of the receptor but did not affect platelet-derived growth factor receptor expression. MLD overexpression inhibited biosynthesis of the EGF receptor, suggesting a possible role of a fatty acid desaturase in regulating biosynthetic processing of the EGF receptor.
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PMID:The product of the MLD gene is a member of the membrane fatty acid desaturase family: overexpression of MLD inhibits EGF receptor biosynthesis. 918 92

The protooncogene protein, Bcl-2, protects cells from apoptosis and ensures their survival in vitro by inhibiting the action of the apoptosis-inducer, Bax. Its expression in proliferative and long-lived cells in vivo also indicates that it protects against cell death. The chondrocytes of the epiphyseal plate cartilage undergo a series of maturation steps and deposit mineral in the cartilage matrix before dying. The possibility that Bcl-2 helps protect chondrocytes until mineral deposition is completed was investigated by determining the distribution of Bcl-2 immunoreactivity in the epiphyseal plate cartilage of growing rats and its subcellular localization, using a specific antibody. The involvement of Bax in the triggering of chondrocyte death was checked by immunocytochemistry. Bcl-2 expression in the osteoblasts and the final result of their evolution, the osteocytes, was also examined in trabecular bone. Bcl-2 immunoreactivity was non-uniformly distributed throughout the epiphyseal cartilage. It was maximal in proliferative chondrocytes, decreased in mature chondrocytes, and low in hypertrophic chondrocytes, whereas there was Bax immunoreactivity in all chondrocytes examined. Immunolabeling was intense in osteoblasts but considerably lower in fully differentiated osteocytes. Bcl-2 immunoreactivity was mainly in the cytoplasm of chondrocytes, osteoblasts, and early osteocytes; the nuclei appeared clear. The subcellular distribution of Bcl-2 immunolabeling in chondrocytes, revealed by gold particles in the electron microscope, showed that gold particles were frequently concentrated in the mitochondria in all the cartilage zones and lay mainly within the organelles, not at their periphery. The endoplasmic reticulum contained moderate immunoreactivity and there were few gold particles in the cytoplasm and nuclei. The number of gold particles decreased in all the subcellular compartments from proliferative to hypertrophic chondrocytes. In contrast, Bax immunoreactivity changed little during chondrocyte terminal evolution, and its subcellular distribution mirrored that of Bcl-2. These immunocytochemical data indicate that Bcl-2 helps maintain chondrocytes and osteoblasts until their terminal maturation.
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PMID:Expression of Bcl-2 protein in the epiphyseal plate cartilage and trabecular bone of growing rats. 937 24

We have established and characterized 3 new breast-cancer cell lines from pleural effusions of patients with advanced breast cancer. All 3 cell lines, designated IBEP-1, IBEP-2 and IBEP-3, showed typical ultrastructural characteristics of epithelial mammary tumor cells. Electron microscopy showed, among other characteristics, the presence of numerous microvilli, desmosomal junctions, intracytoplasmic duct-like vacuoles, well-developed endoplasmic reticulum and large nuclei. Immunohistochemical and biochemical studies revealed that the 3 cell lines expressed cytokeratin, epithelial membrane antigen, CEA and CA 15-3, but all showed negative immunoreaction for vimentin. On the other hand, other antigens (LEU-M1, GCDFP 15, c-erbB-2) were expressed by some of the cell lines, but in a variable manner. Ploidy studies confirmed the neoplastic origin of the cell lines. The doubling times were 68 hr for IBEP-1, 29 hr for IBEP-2 and 39 hr for IBEP-3. Only IBEP-2 cells expressed estrogen receptors (ER+), which were down-regulated after preincubation with E2, but they did not express progesterone receptors (PgR-). IBEP-1 and IBEP-3 cells were ER- but expressed PgR (PgR+). In these 2 cell lines, PgR were down-regulated after pre-incubation of the cells with progesterone (10(-8) M) for 24 hr. Estradiol (E2) increased the proliferation rate of IBEP-2 cells and progesterone increased the proliferation of IBEP-I and -3 cell lines. S.C. injection of the 3 IBEP cell lines into nude mice resulted in the growth of solid tumors between 11 and 16 weeks after inoculation. These cell lines could thus be new models for studying various aspects of the biology and the tumorigenicity of breast-cancer cells. A major interest of these new cell lines is that 2 of them were ER- and PgR+, which is an exceptional phenotypic feature. These 2 cell lines could be interesting models for studying the regulation of PgR and the effects of progestins and antiprogestins independently of the presence of ER.
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PMID:Establishment and characterization of three new breast-cancer cell lines. 961 Jul 25

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