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: UNIPROT:Q9UL75 (A431)
5,640 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Using as a starting material either a detergent extract or a protein fraction eluted from membranes with ethylene glycol bis (beta-aminoethyl ether)-N,N'-tetraacetic acid, we have isolated from human placental membranes a major substrate for the epidermal growth factor (urogastrone) receptor kinase (EGF kinase). The substrate was isolated both in an intact form, having a molecular mass of approximately 38-kDa (p38), and in a 35-kDa form (p35) representing a proteolytic cleavage product of p38. Both p38 and p35 cross-reacted with antibodies directed against bovine retinal transducin, but did not cross-react with antibodies directed against the 35-kDa beta subunit of human placental G-protein. Antisera directed against the placental EGF kinase substrate failed to react with either bovine or human placental src kinase substrate, p36. Conversely, antisera directed against p36 reacted only poorly with placental p38 or p35. Although p38 had a blocked amino terminus that precluded sequence analysis, p35 yielded an N-terminal sequence that was identical with residues 13-36 of human lipocortin. Our data clearly distinguish p38 from the previously described intestinal calcium binding protein calpactin I or p36 that is also a tyrosine kinase substrate, and our work points to a close relationship (if not identity) between p35 and a 35-kDa EGF receptor kinase substrate previously characterized in A431 cells. We conclude that p38 and p35, which very likely represent human placental lipocortin, may share only limited epitope homology with transducin alpha subunit; however, the possibility that p38, along with intestinal p36 and with a family of related calcium binding proteins, may, like transducin, play a role in receptor-mediated transmembrane signaling is discussed.
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PMID:Isolation of a major human placental substrate for the epidermal growth factor (urogastrone) receptor kinase: immunological cross-reactivity with transducin and sequence homology with lipocortin. 296 37

Addition of amiloride to A431 human epidermoid carcinoma cell membranes inhibited autophosphorylation of the epidermal growth factor (EGF) receptor. The tyrosine phosphorylation of histone H2B catalyzed by an affinity-purified preparation of EGF receptor was also inhibited by amiloride. The inhibition was noncompetitive with respect to histone but competitive with ATP, suggesting that amiloride may act as an ATP analogue which causes the formation of nonproductive enzyme-substrate complexes. The tyrosine phosphorylation of histone H2B catalyzed by the purified EGF receptor was inhibited by amiloride at concentrations identical to those previously reported to block EGF action on cell proliferation (Ki = 350 microM). Amiloride similarly inhibited the tyrosine phosphorylation of the human placental insulin receptor and the platelet-derived growth factor receptor of Swiss 3T3 cells. Immunoprecipitation of the EGF receptor from A431 cells labeled for 24 h with [32P]phosphate demonstrated that amiloride decreased the phosphorylation of the EGF receptor on serine and threonine residues and blocked the effect of EGF to cause phosphorylation of the receptor on tyrosine residues. Phosphoamino acid analysis of total cell proteins indicated that amiloride inhibited the increase in phosphotyrosine levels caused by EGF. We conclude that amiloride directly inhibits the tyrosine kinase activity of the receptors for EGF, insulin, and platelet-derived growth factor in in vitro and can mediate such actions in vivo. This effect of amiloride demonstrates that it is unsuitable as a drug to test the hypothesis that the stimulation of the Na+/H+ antiporter is essential for mitogenic signaling by growth factor receptors.
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PMID:Amiloride directly inhibits growth factor receptor tyrosine kinase activity. 298 24

The effect of tumor-promoting phorbol diesters to potentiate the action of epidermal growth factor (EGF) on cell proliferation is associated with phosphorylation of EGF receptors, acute depression of EGF binding, and inhibition of EGF receptor tyrosine kinase activity. In the present studies, normal human fibroblasts and A431 carcinoma cells were labeled with [32P]phosphate and treated with and without 10 nM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). The EGF receptors then were isolated by immunoprecipitation and digested with trypsin. Analysis of the labeled receptor phosphopeptides by reversed-phase HPLC revealed that PMA induces the phosphorylation of a unique phosphopeptide containing [32P]phosphothreonine. Comparison of several chemical and physical properties of the 32P-labeled phosphopeptide with the primary structure of the EGF receptor suggested the identify Lys-Arg-Thr(P)-Leu-Arg. This was confirmed by direct demonstration that a synthetic peptide of this structure comigrates during HPLC and electrophoresis with the 32P-labeled phosphopeptide isolated from the EGF receptors of normal human fibroblasts. The phosphorylated site on the peptide corresponds to threonine-654 of the EGF receptor, which is located on the cytoplasmic side of the plasma membrane nine residues distant from the transmembrane domain. These data indicate that phosphorylation of the EGF receptor in human fibroblasts and A431 cells at threonine-654 may regulate the EGF receptor tyrosine kinase activity and the binding of EGF.
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PMID:Tumor-promoting phorbol diesters cause the phosphorylation of epidermal growth factor receptors in normal human fibroblasts at threonine-654. 298 76

The neu oncogene, identified in ethylnitrosourea-induced rat neuroglioblastomas, had strong homology with the erbB gene that encodes the epidermal growth factor receptor. This homology was limited to the region of erbB encoding the tyrosine kinase domain. It was concluded that the neu gene is a distinct novel gene, as it is not coamplified with sequences encoding the EGF receptor in the genome of the A431 tumor line and it maps to human chromosome 17.
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PMID:The neu gene: an erbB-homologous gene distinct from and unlinked to the gene encoding the EGF receptor. 299 90

The EGF receptor has been purified from human epidermoid carcinoma A431 cells by affinity chromatography on wheat germ agglutinin-agarose and tyrosine-Sepharose. The purified EGF receptor was shown to be homogeneous by SDS-polyacrylamide gel electrophoresis and possessed EGF-sensitive tyrosine kinase activity. Kinetic analysis of the autophosphorylation indicated that approximately 1.4 mol of phosphate was incorporated per mol of the EGF receptor. When a synthetic tyrosine-containing peptide was used as a phosphorylatable substrate, the specific activity of the EGF-stimulated kinase was 66 nmol/min/mg.
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PMID:Purification of the epidermal growth factor receptor by tyrosine-Sepharose affinity chromatography. 299 62

The receptor for epidermal growth factor (EGF) is a glycosylated transmembrane phosphoprotein that exhibits EGF-stimulable protein tyrosine kinase activity. On EGF stimulation, the receptor undergoes a self-phosphorylation reaction at tyrosine residues located primarily in the extreme carboxyl-terminal region of the protein. Using enzymatically active EGF receptor purified by immunoaffinity chromatography from A431 human epidermoid carcinoma cells, the self-phosphorylation reaction has been characterized as a rapid, intramolecular process which is maximal at 30-37 degrees C and exhibits a very low Km for ATP (0.2 microM). When phosphorylation of exogenous peptide substrates was measured as a function of receptor self-phosphorylation, tyrosine kinase activity was found to be enhanced two to threefold at 1-2 mol of phosphate per mol of receptor. Analysis of the dependence of the tyrosine kinase activity on ATP concentration yielded hyperbolic kinetics when plotted in double-reciprocal fashion, indicating that ATP can serve as an activator of the enzyme. Higher concentrations of peptide substrates were found to inhibit both the self- and peptide phosphorylation, but this inhibition could be overcome by first self-phosphorylating the enzyme. These results suggest that self-phosphorylation can remove a competitive/inhibitory constraint so that certain exogenous substrates can have greater access to the enzyme active site. In addition to self-phosphorylation, the EGF receptor can be phosphorylated on threonine residues by the calcium- and phospholipid-dependent protein kinase C. The sites on the EGF receptor phosphorylated in vitro by protein kinase C are identical to the sites phosphorylated on the receptor isolated from A431 cells exposed to the tumor promoters 12-O-tetradecanoylphorbol 13-acetate or teleocidin. This phosphorylation of the EGF receptor results in a suppression of its tyrosine kinase and EGF binding activities both in vivo and in vitro. The EGF receptor can thus be variably regulated by phosphorylation: self-phosphorylation can enhance tyrosine kinase activity whereas protein kinase C-catalyzed phosphorylation can depress enzyme activity. Because these two phosphorylations account for only a fraction of the phosphate present in the EGF receptor in vivo, other protein kinases can apparently phosphorylate the receptor and these may exert additional controls on EGF receptor/kinase function.
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PMID:Regulation of the epidermal growth factor receptor by phosphorylation. 300 Nov 10

A method was devised that allows the identification of proteins related to the Mr 36,000 tyrosine kinase substrate calpactin based on their ability to interact with actin and phospholipid in a calcium-dependent manner. Two distinct proteins, detected in human A431 cells and fibroblasts, were resolved by two-dimensional gel electrophoresis. One of these proteins (calpactin I) appears identical to the Mr 34,000-39,000 substrate of the pp60src tyrosine kinase and the second (calpactin II) reacts with antibodies to the Mr 35,000 substrate of the epidermal growth factor receptor. Both proteins interact with phospholipid and actin, are rather basic, and share structural and antigenic determinants. A major difference between the two proteins is noted in their state of association with the Mr 10,000 light chain; i.e., calpactin I is associated with the light chain while calpactin II is not.
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PMID:Two related but distinct forms of the Mr 36,000 tyrosine kinase substrate (calpactin) that interact with phospholipid and actin in a Ca2+-dependent manner. 301 61

Site-specific antibodies were generated against the erbB protein and epidermal growth factor (EGF) receptor by immunizing rabbits with a synthetic peptide corresponding to amino acid residues 285-296 of the predicted AEV-H erbB protein sequence. This peptide region lies within the tyrosine kinase domain of erbB and EGF receptor. Antibodies directed against this region readily identified native and denatured forms of the erbB gene product and EGF receptor as demonstrated by immuneprecipitation and immunoblot analysis. The anti-peptide antibody immuneprecipitated a functional EGF binding receptor molecule. Scatchard analysis demonstrated a KD for 125I-labeled EGF binding of 40 nM, a value consistent with that of detergent solubilized EGF receptor. Immuneprecipitates, though able to bind EGF, were unable to transfer phosphate from gamma-labeled ATP in a standard phosphorylation reaction. In detergent solubilized extracts of crude A431 microsomes, the anti-peptide antibody inhibited in a dose dependent manner the autophosphorylation of EGF receptor as well as receptor mediated phosphorylation of exogenously added substrates. In addition, this anti-peptide antibody reduced the overall level of tyrosine kinase activity present in microsomes prepared from AEV-transformed erythroblasts. This site-specific antisera should be useful for understanding the role of EGF receptor and erbB tyrosine kinase activity and their link with cell proliferation.
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PMID:Characterization of site-specific antibodies to the erbB gene product and EGF receptor: inhibition of tyrosine kinase activity. 301 25

A region of the primary amino acid sequence of the epidermal growth factor receptor (EGF) protein-tyrosine kinase, which is involved in ATP binding, was identified using chemical modification and immunological techniques. EGF receptor was 14C-labelled with the ATP analogue 5'-p-fluorosulphonylbenzoyladenosine and from a tryptic digest a single radiolabelled peptide was isolated. The amino acid sequence was determined to be residues 716-724 and hence lysine residue 721 is located within the ATP-binding site. Antisera were elicited in rabbits to a synthetic peptide identical to residues 716-727 of the EGF receptor and the homologous sequence in v-erb B transforming protein from avian erythroblastosis virus. The affinity-purified antibodies precipitated human ECF receptor from A431 cells and placenta, and the v-erb B protein from erythroblasts. The antibodies inhibited EGF-stimulated receptor protein-tyrosine kinase autophosphorylation and phosphorylation of an exogenous peptide substrate containing tyrosine. The antibodies did not immunoprecipitate the transforming proteins pp60v-src or P120gag-abl or cAMP-dependent protein kinase, proteins which have homologous but not identical sequences surrounding the lysine residue within the ATP-binding site, nor did they react with the platelet-derived growth factor receptor. The antibodies had no effect on the kinase activity of purified v-abl protein in solution. The antibodies may therefore be a specific inhibitor of the tyrosine kinase of the EGF receptor.
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PMID:Antibodies to the ATP-binding site of the human epidermal growth factor (EGF) receptor as specific inhibitors of EGF-stimulated protein-tyrosine kinase activity. 301 11

A431 cells have been used as an immunogen for generating monoclonal antibodies against the epidermal growth factor (EGF) receptor. Two immunoglobulin M and eight immunoglobulin G3 anti-EGF receptor antibodies were cloned. All ten antibodies immunoprecipitated biosynthetically labeled mature A431 cell EGF receptor and were able to recognize the receptor in Western blotting. However, none of the antibodies immunoprecipitated precursor polypeptides of the A431 cell EGF receptor, neither did they recognize EGF receptors from human foreskin fibroblasts, human placenta, nor a human-mouse hybrid cell expressing EGF receptor. The antibodies were found to bind to glycolipids from A431 cells and it was shown that the determinant involved was the blood group A antigen. It appears that this determinant is present on both the EGF receptor and glycolipids of A431 cells but is not expressed on EGF receptors from other human cells tested. One of the monoclonal antibodies raised was used for immunoaffinity purification of the EGF receptor. The procedure took advantage of the carbohydrate nature of the antigenic determinant by employing sugar-specific elution. The mild conditions permitted the purification of A431 cell EGF receptor (70-80% pure) that possessed an intrinsic EGF-stimulated tyrosine kinase activity with a specific activity of about 20 nmol/min/mg.
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PMID:Monoclonal antibodies against the human epidermal growth factor receptor from A431 cells. Isolation, characterization, and use in the purification of active epidermal growth factor receptor. 608 58


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