Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P04626 (erbB-2)
 5,251 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The ATP substrate site in the epidermal growth factor (EGF) receptor was mapped by using a series of 26 ATP derivatives with modifications at the base, ribose or triphosphate moiety. Ki values for these derivatives were determined by competition with [gamma-32P]ATP. The enzyme seems to interact specifically with the beta-phosphate in an ion-pair bond with the N-6 amino group at the adenine in a hydrogen bond. With ribosyl-2-aminopurine triphosphate and GTP, the enzyme most likely recognizes the 2-amino group in a hydrogen bond. This high specificity for ATP and GTP is unique for the ATP site in the EGF receptor among all investigated protein kinases. The available data on the interaction between ATP derivatives and protein kinases were used to assign conserved amino acid residues found in diverse protein kinases to the ATP site in this type of enzyme.
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PMID:Assignment of conserved amino acid residues to the ATP site in the protein kinase domain of the receptor for epidermal growth factor. 300 81

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

The possible role of epidermal growth factor (EGF) receptor phosphorylation at threonine 654 in modulating the protein-tyrosine kinase activity of EGF-treated A431 cells has been studied. It has been suggested that EGF could indirectly activate a protein-serine/threonine kinase, protein kinase C, that can phosphorylate the EGF receptor at threonine 654. Protein kinase C is known to be activated, and threonine 654 is phosphorylated, when A431 cells are exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA). The protein-tyrosine kinase activity of EGF receptors is normally evidenced in EGF-treated cells by phosphorylation of the receptor at tyrosine. This is inhibited when TPA-treated cells are exposed to EGF. We now show that receptor phosphorylation at threonine 654 can also be detected in EGF-treated A431 cells, presumably due to indirect stimulation of protein kinase C or a similar kinase. Some receptor molecules are phosphorylated both at threonine 654 and at tyrosine. Since prior phosphorylation at threonine 654 inhibits autophosphorylation, we propose that protein kinase C can phosphorylate the threonine 654 of autophosphorylated receptors. This provides evidence for models in which protein kinase C activation, consequent upon EGF binding, could reduce the protein-tyrosine kinase activity of the EGF receptor. Indeed, we find that 12-O-tetradecanoylphorbol-13-acetate, added 10 min after EGF, further increases threonine 654 phosphorylation and induces the loss of tyrosine phosphate from A431 cell EGF receptors.
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PMID:Effects of protein kinase C activation after epidermal growth factor binding on epidermal growth factor receptor phosphorylation. 301 18

Treatment of cells with tumor-promoting phorbol diesters, which causes activation of protein kinase C, leads to phosphorylation of the epidermal growth factor (EGF) receptor at threonine-654. Addition of phorbol diesters to intact cells causes inhibition of the EGF-induced tyrosine-protein kinase activity of the EGF receptor and it has been suggested that this effect of phorbol diesters is mediated by the phosphorylation of the receptor by protein kinase C. We measured the activity of protein kinase C in A431 cells by determining the incorporation of [32P]phosphate into peptides containing threonine-654 obtained by trypsin digestion of EGF receptors. After 3 h of exposure to serum-free medium, A431 cells had no detectable protein kinase C activity. Addition of EGF to these cells resulted in [32P] incorporation into threonine-654 as well as into tyrosine residues. This indicates that EGF promotes the activation of protein kinase C in A431 cells. The phosphorylation of threonine-654 induced by EGF was maximal after only 5 min of EGF addition and the [32P] incorporation into threonine-654 reached 50% of the [32P] in a tyrosine-containing peptide. This indicates that a significant percentage of the total EGF receptors are phosphorylated by protein kinase C. A variety of external stimuli activate Na+/H+ exchange, including EGF, phorbol diesters, and hypertonicity. To ascertain whether activation of protein kinase C is an intracellular common effector of all of these systems, we measured the activity of protein kinase C after exposure of A431 cells to hyperosmotic conditions and observed no effect on phosphorylation of threonine-654, therefore, activation of Na+/H+ exchange by hypertonic medium is independent of protein kinase C activity. Since stimulation of protein kinase C by phorbol diesters results in a decrease in EGF receptor activity, the stimulation of protein kinase C activity by addition of EGF to A431 cells contributes to a feedback mechanism which results in the attenuation of EGF receptor function.
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PMID:Epidermal growth factor (EGF) promotes phosphorylation at threonine-654 of the EGF receptor: possible role of protein kinase C in homologous regulation of the EGF receptor. 302 81

Tyrosine-specific protein kinase activity of the epidermal growth factor (EGF) receptor, pp60v-src and pp110gag-fes was inhibited in vitro by an isoflavone genistein. The inhibition was competitive with respect to ATP and noncompetitive to a phosphate acceptor, histone H2B. By contrast, genistein scarcely inhibited the enzyme activities of serine- and threonine-specific protein kinases such as cAMP-dependent protein kinase, phosphorylase kinase, and the Ca2+/phospholipid-dependent enzyme protein kinase C. When the effect of genistein on the phosphorylation of the EGF receptor was examined in cultured A431 cells, EGF-stimulated serine, threonine, and tyrosine phosphorylation was decreased. Phosphoamino acid analysis of total cell proteins revealed that genistein inhibited the EGF-stimulated increase in phosphotyrosine level in A431 cells.
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PMID:Genistein, a specific inhibitor of tyrosine-specific protein kinases. 310 39

We have tested the hypothesis that the mechanism of platelet-derived growth factor (PDGF) and phorbol diester action to decrease the apparent affinity of the epidermal growth factor (EGF) receptor is the phosphorylation of the EGF receptor at the Ca2+/phospholipid-dependent protein kinase (protein kinase C) phosphorylation site, threonine 654. Protein kinase C-deficient cells were prepared by prolonged incubation of human fibroblasts with phorbol diester. Addition of phorbol diesters to these cells fails to regulate EGF receptor affinity or threonine 654 phosphorylation. In contrast, PDGF treatment of both control and protein kinase C-deficient fibroblasts causes a decrease in the apparent affinity of the EGF receptor and an increase in threonine 654 phosphorylation. Thus, the ability of PDGF or phorbol diester to modulate EGF receptor affinity occurs only when threonine 654 phosphorylation is increased. The stoichiometry of threonine 654 phosphorylation associated with a 50% decrease in the binding of 125I-EGF to high affinity sites was 0.15 versus 0.3 mol of phosphate per mole of EGF receptor when 32P-labeled fibroblasts are treated with PDGF or phorbol diester, respectively. It is concluded that EGF receptor phosphorylation at threonine 654 can be regulated by PDGF independently of protein kinase C, substoichiometric phosphorylation of the total EGF receptor pool at threonine 654 is caused by maximally effective concentrations of PDGF, and different extents of phosphorylation of EGF receptors at threonine 654 are observed for maximally effective concentrations of PDGF and phorbol diester, respectively. The data are consistent with the hypothesis that a specific subpopulation of EGF receptors that exhibit high affinity for EGF are regulated by threonine 654 phosphorylation.
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PMID:Stimulation of epidermal growth factor receptor threonine 654 phosphorylation by platelet-derived growth factor in protein kinase C-deficient human fibroblasts. 310 61

The specific tyrosine phosphorylation of glucose-6-phosphate dehydrogenase (G6PDH) by the epidermal growth factor (EGF) receptor in vitro is demonstrated. The Km values of the substrate G6PDH and of ATP for the receptor tyrosine kinase were ca. 1 and 10 microM, respectively. The rate of phosphorylation was EGF dependent, with a four-fold increase in Vmax in the presence of EGF. The phosphorylation was stimulated maximally by 0.2 microM or greater EGF, with an ED50 of ca. 20 nM which is consistent with the affinity of the solubilized receptor for EGF. Using conditions of 5 microM G6PDH, 100 microM ATP, 5 mM Mg2+, and 1 mM Mn2+, up to 0.3 mol phosphate was incorporated into 1 mol of the 55-kDa subunit of Baker's yeast G6PDH. Tryptic peptide mapping revealed several unique phosphopeptides for both Baker's yeast and bovine adrenal G6PDH. The patterns of phosphopeptides for a given enzyme were identical for basal and EGF-stimulated phosphorylation.
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PMID:Epidermal growth factor receptor tyrosine kinase phosphorylation of glucose-6-phosphate dehydrogenase in vitro. 312 60

The epidermal growth factor (EGF) receptor is regulated by EGF-stimulated autophosphorylation and by phorbol ester-stimulated, protein kinase C (Ca2+/phospholipid-dependent enzyme) mediated phosphorylation at identified sites. The EGF receptor contains additional phosphorylation sites including a prominent phosphothreonine and several phosphoserines which account for the majority of phosphate covalently bound to the receptor in vivo. We have identified three of these sites in EGF receptor purified from 32P-labeled A431 cells. The major phosphothreonine was identified as threonine 669 in the EGF receptor sequence. Phosphoserine residues were identified as serines 671 and 1046/1047 of the EGF receptor. Two other phosphoserine residues were localized to tryptic peptides containing multiple serine residues located carboxyl-terminal to the conserved protein kinase domain. The amino acid sequences surrounding the three identified phosphorylation sites are highly conserved in the EGF receptor and the protein products of the v-erb B and neu oncogenes. Analysis of predicted secondary structure of the EGF receptor reveals that all of the phosphorylation sites are located near beta turns. In A431 cells phosphorylation of the serine residues was dependent upon serum. In mouse B82 L cells transfected with a wild type human EGF receptor. EGF increased the 32P content in all tryptic phosphopeptides. A mutant EGF receptor lacking protein tyrosine kinase activity was phosphorylated only at threonine 669. Regulated phosphorylation of the EGF receptor at these threonine and serine residues may influence aspects of receptor function.
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PMID:Epidermal growth factor receptor threonine and serine residues phosphorylated in vivo. 313 33

In this report, we demonstrate a novel post-translational modification of the epidermal growth factor (EGF) receptor. This modification involves the presence of phosphate, previously thought to exist only on amino acid residues in the EGF receptor, on oligosaccharides of the receptor. We have utilized several independent approaches to determine that mannose phosphate is present on the EGF receptor in A-431 cells. Following metabolic labeling with 32P, immunoisolation of the EGF receptor, and digestion with Pronase radioactivity was determined to be present on high mannose type oligosaccharides by concanavalin A chromatography. Also, after acid hydrolysis of in vivo 32P-labeled EGF receptor, radioactivity was detected that co-migrated with mannose 6-phosphate on two-dimensional thin layer electrophoresis. This radiolabeled material co-eluted with a mannose 6-phosphate standard from a high pressure liquid chromatography anion exchange column. Last, an acid hydrolysate of [3H]mannose-labeled EGF receptor contained two radiolabeled fractions, as analyzed by thin layer electrophoresis, and the radioactivity in one of these fractions was substantially reduced by alkaline phosphatase treatment prior to electrophoresis. These experiments indicate that the mature EGF receptor in A-431 cells contains mannose phosphate. This is a novel modification for membrane receptors and has only been reported previously for lysosomal enzymes and a few secreted proteins.
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PMID:Presence of mannose phosphate on the epidermal growth factor receptor in A-431 cells. 319 17

The epidermal growth factor (EGF) receptor is a transmembrane glycoprotein of relative molecular mass 170,000 with intrinsic ligand-dependent protein tyrosine kinase activity. Binding of EGF to its receptor activates a number of immediate biochemical processes, such as alterations of intracellular free calcium, pH, and increased transcription of several responsive genes, which usually culminate many hours later in DNA replication and cell division. Abolishing the tyrosine kinase activity of three related oncogenes, v-src, v-mos, and v-fps, eliminates their capacity to transform cell. Several reports have suggested that specific aspects of EGF receptor function are independent of the intrinsic tyrosine kinase activity; however, these studies used an antibody against EGF receptor which failed to activate phosphorylation of exogenous substrates and an insertional mutation in the EGF receptor tyrosine kinase domain which had not been shown to abolish protein kinase activity in cells. Because many transmembrane receptors interact with intrinsic membrane proteins to activate second messenger systems, it is important to resolve experimentally whether mechanisms, in addition to activation of the intrinsic tyrosine kinase activity, mediate some EGF actions. From functional analyses of an EGF receptor containing a single amino-acid mutation at a site required for phosphate transfer from ATP, we conclude that the tyrosine kinase activity of the EGF receptor is essential for the diverse biochemical effects of EGF, including rapid alterations in intracellular calcium, activation of gene transcription, receptor down-regulation and the ultimate stimulatory effects on cell proliferation.
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PMID:Requirement for intrinsic protein tyrosine kinase in the immediate and late actions of the EGF receptor. 349 22


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