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

The major sites of serine and threonine phosphorylation of the human epidermal growth factor (EGF) receptor observed in intact cells are Thr654, Thr669, Ser1046, and Ser1047. Phosphorylation of the EGF receptor is increased at these sites in cells treated with platelet-derived growth factor or phorbol ester. This increase in EGF receptor phosphorylation is associated with an inhibition of the high affinity binding of EGF to cell surface receptors and an inhibition of the receptor tyrosine protein kinase activity. In order to test the hypothesis that the phosphorylation of the EGF receptor is mechanistically related to the modulation of EGF receptor function, we replaced the major sites of serine and threonine phosphorylation with alanine residues. EGF receptors containing single point mutations or multiple mutations were expressed in Chinese hamster ovary cells. Analysis of the regulation of the EGF receptor tyrosine protein kinase activity demonstrated that phorbol ester caused an inhibition of the tyrosine phosphorylation of wild-type receptors and receptors lacking Thr669, Ser1046, or Ser1047. In contrast, the inhibition of EGF receptor tyrosine phosphorylation caused by phorbol ester was not observed for any of the mutated EGF receptors that lacked Thr654. These data are consistent with the hypothesis that the phosphorylation of the EGF receptor at Thr654 is required for the inhibition of the receptor tyrosine protein kinase activity caused by phorbol ester. Investigation of the apparent affinity of the EGF receptor demonstrated that treatment with phorbol ester caused an inhibition of the high affinity binding of 125I-EGF to cells expressing wild-type EGF receptors and each of the mutated EGF receptors examined. We conclude that the regulation of the apparent affinity of the EGF receptor is independent of the major sites of serine and threonine phosphorylation of the EGF receptor.
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PMID:Multisite phosphorylation of the epidermal growth factor receptor. Use of site-directed mutagenesis to examine the role of serine/threonine phosphorylation. 210 48

The epidermal growth factor (EGF) receptor contains multiple sites of phosphorylation on serine, threonine, and tyrosine residues. Because the biological responsiveness of the EGF receptor is regulated by phosphorylation at several of these sites, we studied the functional consequences of removal of the Thr669 and Ser671 phosphorylation sites using site-directed mutagenesis. The mutant EGF receptor expressed in mouse B82 cells displayed normal EGF binding and in vivo autophosphorylation and was fully active in biological signal transduction as measured by EGF-stimulated gene transcription. However, the EGF-dependent phosphorylation of an 85-kDa cellular substrate by the mutant receptor was impaired relative to the wild type receptor, indicating that the mutated region may specifically interact with this substrate. Endocytosis of the mutant receptor was also impaired as measured by both receptor down-regulation and ligand internalization studies. This was due to impaired uptake of the mutant receptor by the saturable, high affinity endocytic system. Several aspects of mutant receptor function were regulated normally by TPA, indicating a lack of interaction between the mutated phosphorylation sites and the nearby protein kinase C phosphorylation site Thr654. These results suggest that phosphorylation of the EGF receptor at Thr669 and Ser671 mediates interaction of the receptor with a specific tyrosine kinase substrate and is required for efficient ligand-induced receptor internalization.
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PMID:Mutational removal of the Thr669 and Ser671 phosphorylation sites alters substrate specificity and ligand-induced internalization of the epidermal growth factor receptor. 211 82

The addition of gangliosides to tissue culture cells causes a decrease in the tyrosine protein kinase activity of the epidermal growth factor (EGF) receptor and an inhibition of EGF-stimulated growth. Based on these data, the hypothesis that the EGF receptor is physiologically regulated by gangliosides has been proposed by E.G. Bremer, J. Schlessinger, and S. Hakomori (J. Biol. Chem. (1986) 261, 2434-2440). To test this hypothesis, a mutant Chinese hamster ovary cell line (clone Idl D) that has a reversible defect in the biosynthesis of gangliosides (Kingsley, D.M., Kozarsky, K. F., Hobbie, L., and Krieger, M. (1986) Cell 44, 749-759) was investigated. The human EGF receptor cDNA was expressed in the mutant cells, and the properties of the EGF receptor were examined using cells grown under permissive and nonpermissive conditions. Changes in ganglioside expression were not observed to cause any significant alterations in the affinity or number of EGF receptors detected at the cell surface. However, decreased levels of ganglioside expression were associated with 1) increased EGF receptor autophosphorylation on tyrosine residues, and 2) increased EGF-stimulated cellular proliferation. The inverse correlation observed between the level of ganglioside expression and signal transduction by the EGF receptor is consistent with the hypothesis that the function of the EGF receptor is physiologically regulated by gangliosides.
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PMID:Regulation of epidermal growth factor receptor signal transduction. Role of gangliosides. 214 59

Phosphatidylinositol-specific phospholipase C isozyme gamma (PLC-gamma, Mr 145,000) is an excellent substrate for the epidermal growth factor (EGF) receptor both in vivo and in vitro. PLC-beta-1, another PLC isozyme, is a poor substrate for the EGF receptor. We examined the relative phosphorylation of PLC-gamma and PLC-beta-1 by the 170-kDa native EGF receptor molecule, the 66-kDa cytoplasmic kinase domain of the EGF receptor (Arg647-Ala1186), the alpha 2 beta 2 native insulin receptor, and the 48-kDa cytoplasmic kinase domain of the insulin receptor beta subunit (Gly947-Ser1343). Similar to the intact EGF receptor, the cytoplasmic kinase domain of the EGF receptor preferentially phosphorylated PLC-gamma. High-performance liquid chromatographic comparison of tryptic phosphopeptides from PLC-gamma phosphorylated by both forms of the EGF receptor kinase indicated similar patterns of multiple tyrosine phosphorylations. These results imply that substrate selectivity, at least in terms of PLC isozymes, is independent of the extracellular ligand-binding and membrane anchor domains of the EGF receptor. In comparison, neither the intact insulin receptor nor the beta-chain kinase domain was able to phosphorylate PLC-gamma to a significant extent. Also, insulin failed to stimulate the phosphorylation of PLC-gamma in NIH 3T3/HIR cells, which overexpress the human insulin receptor. Thus PLC-gamma is not a phosphorylation substrate for the insulin receptor in vitro or in the intact cell.
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PMID:Selectivity of phospholipase C phosphorylation by the epidermal growth factor receptor, the insulin receptor, and their cytoplasmic domains. 215 2

The abilities of different GTP-binding proteins to serve as phosphosubstrates for the epidermal growth factor (EGF) receptor/tyrosine kinase have been examined in reconstituted phospholipid vesicle systems. During the course of these studies we discovered that a low molecular mass, high affinity GTP-binding protein from bovine brain (designated as the 22-kDa protein) served as an excellent phosphosubstrate for the tyrosine-agarose-purified human placental EGF receptor. The EGF-stimulated phosphorylation of the purified 22-kDa protein occurs on tyrosine residues, with stoichiometries approaching 2 mol of 32Pi incorporated/mol of [35S]guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-binding sites. The EGF-stimulated phosphorylation of the brain 22-kDa protein requires its reconstitution into phospholipid vesicles. No phosphorylation of this GTP-binding protein is detected if it is simply mixed with the purified EGF receptor in detergent solution or if detergent is added back to lipid vesicles containing the EGF receptor and the 22-kDa protein. The EGF-stimulated phosphorylation of this GTP-binding protein is also markedly attenuated by guanine nucleotides, i.e. GTP, GTP gamma S, or GDP, suggesting that maximal phosphorylation occurs when the GTP-binding protein is in a guanine nucleotide-depleted state. Purified preparations of the 22-kDa phosphosubstrate do not cross-react with antibodies against the ras proteins. However, they do cross-react against two different peptide antibodies generated against specific sequences of the human platelet (and placental) GTP-binding protein originally designated Gp (Evans, T., Brown, M. L., Fraser, E. D., and Northrup, J. K. (1986) J. Biol. Chem. 261, 7052-7059) and more recently named G25K (Polakis, P. G., Synderman, R., and Evans, T. (1989) Biochem. Biophys. Res. Commun. 160, 25-32). When highly purified preparations of the human platelet Gp (G25K) protein are reconstituted with the purified EGF receptor into phospholipid vesicles, an EGF-stimulated phosphorylation of the platelet GTP-binding protein occurs with a stoichiometry approaching 2 mol of 32Pi incorporated/mol of [35S]GTP gamma S-binding sites. As is the case for the brain 22-kDa protein, the EGF-stimulated phosphorylation of the platelet GTP-binding protein is attenuated by guanine nucleotides. Overall, these results suggest that the brain 22-kDa phosphosubstrate for the EGF receptor is very similar, if not identical, to the Gp (G25K) protein. Although guanine nucleotide binding to the brain 22-kDa protein or to the platelet. GTP-binding protein inhibits phosphorylation, the phosphorylated GTP-binding proteins appear to bind [35S]GTP gamma S slightly better than their nonphosphorylated counterparts.
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PMID:The identification and characterization of an epidermal growth factor-stimulated phosphorylation of a specific low molecular weight GTP-binding protein in a reconstituted phospholipid vesicle system. 215 36

Previous work identified a protein kinase activity that phosphorylates the epidermal growth factor (EGF) receptor at Thr669. An assay for this protein kinase activity present in homogenates prepared from A431 human epidermoid carcinoma cells was developed using a synthetic peptide substrate corresponding to residues 663-681 of the EGF receptor (peptide T669). Here we report that a greater initial rate of T669 phosphorylation was observed in experiments using homogenates prepared from EGF- or phorbol ester-treated cells compared with control cells. EGF and 4 beta-phorbol 12-myristate 13-acetate (PMA) caused a 6-fold and a 2-fold increase in protein kinase activity, respectively. A kinetic analysis of T669 phosphorylation demonstrated that the increase in protein kinase activity observed was accounted for by an increase in Vmax. To examine the interaction between protein kinase C and signal transduction by the EGF receptor, the effect of pretreatment of cells with PMA on the subsequent response to EGF was investigated. Treatment of cells with PMA caused greater than 90% inhibition of the EGF-stimulated tyrosine phosphorylation of the EGF receptor and abolished the EGF-stimulated formation of soluble inositol phosphates. In contrast, PMA was not observed to inhibit the stimulation of T669 protein kinase activity caused by EGF. Thus, the apparent functional desensitization of the EGF receptor caused by PMA does not inhibit signal transduction mediated by the T669 protein kinase. Our results demonstrate that EGF receptor transmodulation alters the pattern of signal-transduction pathways that are utilized by the EGF receptor.
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PMID:Signal transduction by the epidermal growth factor receptor after functional desensitization of the receptor tyrosine protein kinase activity. 216 44

Various derivatives of thiazolidine-diones have been identified as tyrosine protein kinase inhibitors. The epidermal growth factor (EGF) receptor kinase and c-src kinase were inhibited in vitro with IC50 values in the range of 1-7 microM. The v-abl tyrosine protein kinase was not inhibited by thiazolidine-diones. Inhibition was found to be specific for tyrosine protein kinases. Inhibition of serine/threonine protein kinases was not observed. The active derivatives were shown to inhibit EGF-induced receptor autophosphorylation, either in vitro or in intact cells, and were also found to inhibit growth of the EGF-dependent BALB/MK and A431 cell lines (IC50 1-3 microM). Growth of the interleukin-3-dependent myeloid cell line FDC-P1 was inhibited with equal efficiency. Thus, in these cell lines, members of the c-src kinase family are also potential targets for inhibition by the compounds.
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PMID:Thiazolidine-diones. Biochemical and biological activity of a novel class of tyrosine protein kinase inhibitors. 217 10

The erbB2 oncogene encodes a 185-kilodalton transmembrane protein whose sequence is similar to the epidermal growth factor receptor (EGFR). A 30-kilodalton factor (gp30) secreted from MDA-MB-231 human breast cancer cells was shown to be a ligand for p185erbB2. An antibody to EGFR abolished the tyrosine phosphorylation induced by EGF and transforming growth factor-alpha (TGF-alpha) but only partially blocked that produced by gp30 in SK-BR-3 breast cancer cells. In two cell lines that overexpress erbB2 but do not expresss EGFR (MDA-MB-453 breast cancer cells and a Chinese hamster ovary cell line that had been transfected with erbB2), phosphorylation of p185erbB2 was induced only by gp30. The gp30 specifically inhibited the growth of cells that overexpressed p185erbB2. An antibody to EGFR had no effect on the inhibition of SK-BR-3 cell colony formation obtained with gp30. Thus, it appeared that gp30 interacted directly with the EGFR and erbB2. Direct binding of gp30 to p185erbB2 was confirmed by binding competition experiments, where gp30 was found to displace the p185erbB2 binding of a specific antibody to p185erbB2. The evidence described here suggests that gp30 is a ligand for p185erbB2.
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PMID:Direct interaction of a ligand for the erbB2 oncogene product with the EGF receptor and p185erbB2. 221 96

Cytoplasmic proteins that regulate signal transduction or induce cellular transformation, including cytoplasmic protein-tyrosine kinases, p21ras GTPase-activating protein (GAP), phospholipase C gamma, and the v-crk oncoprotein, possess one or two copies of a conserved noncatalytic domain, Src homology region 2 (SH2). Here we provide direct evidence that SH2 domains can mediate the interactions of these diverse signaling proteins with a related set of phosphotyrosine ligands, including the epidermal growth factor (EGF) receptor. In src-transformed cells GAP forms heteromeric complexes, notably with a highly tyrosine phosphorylated 62-kDa protein (p62). The stable association between GAP and p62 can be specifically reconstituted in vitro by using a bacterial polypeptide containing only the N-terminal GAP SH2 domain. The efficient phosphorylation of p62 by the v-Src or v-Fps tyrosine kinases depends, in turn, on their SH2 domains and correlates with their transforming activity. In lysates of EGF-stimulated cells, the N-terminal GAP SH2 domain binds to both the EGF receptor and p62. Fusion proteins containing GAP or v-Crk SH2 domains complex with similar phosphotyrosine proteins from src-transformed or EGF-stimulated cells but with different efficiencies. SH2 sequences, therefore, form autonomous domains that direct signaling proteins, such as GAP, to bind specific phosphotyrosine-containing polypeptides. By promoting the formation of these complexes, SH2 domains are ideally suited to regulate the activation of intracellular signaling pathways by growth factors.
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PMID:Src homology region 2 domains direct protein-protein interactions in signal transduction. 223 73

Endogenous substrates of the EGF receptor have been described in transformed cells; however, little is known about substrates in normal tissue. To characterize epidermal growth factor (EGF) receptor phosphorylation and search for endogenous substrates in normal rat hepatocytes, cells were labeled with [32P]orthophosphate, and phosphotyrosine-containing proteins were sought by using a high-affinity, specific anti-phosphotyrosine antibody. Exposure of 32P-labeled freshly isolated hepatocytes to 1 microgram/mL EGF caused phosphorylation of several proteins of Mr 185K, 160K, and 120K. The 185- and 160-kDa proteins (pp185 and pp160) were identified as the intact and proteolyzed forms of the EGF receptor by virtue of their immunoprecipitation with anti-EGF receptor antibody. This antibody failed to recognize the 120-kDa phosphoprotein (pp120). The phosphopeptide map derived from pp120 was by trypsinization and HPLC separation different from that of pp185, further indicating that pp120 is distinct from the EGF receptor. This pp120 was also immunologically distinct from the pp120 substrate of the insulin receptor kinase and from ATP-citrate lyase. Phosphoamino acid analysis revealed pp120 to be phosphorylated on both tyrosine and serine residues. Autophosphorylation of EGF receptor and phosphorylation of pp120 were almost maximal within 1 min of EGF stimulation. The dose-response curves for phosphorylation of the EGF receptor and pp120 were identical (ED50 = 30 ng/mL) and were superimposable with the fractional occupancy of the EGF receptor. In A431 cells, a transformed cell line whose growth is inhibited by EGF, EGF produced a decrease in pp120 phosphorylation. These data suggest that pp120 is an endogenous substrate for the EGF receptor in hepatocytes whose phosphorylation may be closely related to EGF stimulation of cell growth.
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PMID:Epidermal growth factor stimulated phosphorylation of a 120-kilodalton endogenous substrate protein in rat hepatocytes. 224 61


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