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

The role of epidermal growth factor (EGF) receptor autophosphorylation sites in the regulation of receptor functions has been studied using cells transfected with mutant EGF receptors. Simultaneous point mutation of 4 tyrosines (Y1068, Y1086, Y1148, Y1173) to phenylalanine, as well as removal of these sites by truncation of the carboxyl-terminal 123 amino acid residues, resulted in reduced receptor phosphorylation of an in vivo specific substrate phospholipase C-gamma 1 to less than 50% compared to the wild-type receptor. The internalization rate constant Ke was also significantly lower in these mutants (0.15/min) compared to cells transfected with wild-type receptor (0.27/min). Additional mutation of tyrosine 992 to phenylalanine in the truncated receptor mutant (Dc-123F) further decreased the receptor internalization rate to a minimal level (ke = 0.07-0.10/min), equivalent to the ke measured for cells expressing kinase-negative receptor (A721). Moreover, tyrosine kinase activity of the Dc-123F receptor toward phospholipase C-gamma 1, compared to wild-type receptor, was reduced by 90%. Taken together, these results show that EGF receptor lacking five autophosphorylation sites functions similar to a kinase-negative receptor. Mutation of tyrosine residue Y992 alone in the context of full length EGF receptor, however, did not affect receptor internalization or kinase activity toward phospholipase C-gamma 1. These data indicate that tyrosine 992 is critical for substrate phosphorylation and internalization only in the context of the truncated receptor, and that minor autophosphorylation sites, such as Y992, may act as compensatory regulatory sties in the absence of the major EGF receptor autophosphorylation sites.
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PMID:Multiple autophosphorylation sites of the epidermal growth factor receptor are essential for receptor kinase activity and internalization. Contrasting significance of tyrosine 992 in the native and truncated receptors. 131 35

Autophosphorylation of gp185erbB-2 in vivo is confined to its carboxy terminus and is required for optimal erbB-2 transforming activity under conditions of receptor overexpression. It remains unresolved, however, to what extent autophosphorylation regulates erbB-2 mitogenic signaling in normal cells, nor is the biochemical basis for such a regulatory function known. To address these issues, we utilized a chimeric molecule encompassing the extracellular domain of the epidermal growth factor (EGF) receptor (EGFR) fused to the transmembrane and intracellular domains of the erbB-2 product. In this EGFR/erbB-2 chimera, erbB-2 kinase activity is regulated by EGF binding. An EGFR/erbB-2 mutant bearing multiple Tyr----Phe substitutions at erbB-2 autophosphorylation sites (EGFR/erbB-2 5P) displayed markedly reduced phosphotyrosine content following EGF stimulation in comparison with the non-mutated chimera. When expressed in NR6 cells, the EGFR/erbB-2 5P mutant was unable to deliver a sizeable mitogenic signal when activated by EGF at physiological levels. In intact cells, the 5P mutant was still able to stimulate phosphorylation of the gamma isozyme of phospholipase C (PLC-gamma), a prototype erbB-2 substrate, although with a delayed time course, indicating that the 5P mutation decreased the affinity of the erbB-2 kinase for this substrate. This conclusion was further supported by the inability of the 5P mutant to associate with PLC-gamma in co-immunoprecipitation experiments. We infer that a major role of autophosphorylation is to increase the affinity of the erbB-2 kinase for its cellular substrates, so that, under physiological conditions, autophosphorylation is absolutely required for erbB-2 mitogenic signaling.
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PMID:erbB-2 autophosphorylation is required for mitogenic action and high-affinity substrate coupling. 135 97

The mutant c-erbB-2 protein with Glu instead of Val-659 exhibited transforming activity in NIH 3T3 cells. This protein showed enhanced tyrosine kinase activity in vitro and enhanced autophosphorylation at Tyr-1248 located proximal to the carboxyl terminus. Enhanced tyrosine phosphorylation of several cellular proteins was detected in cells expressing the Glu-659 c-erbB-2 protein. Introduction of an additional mutation at the ATP-binding site (Lys-753 to Met) of this protein resulted in abolition of its transforming ability. These data indicate that the transforming potential of c-erbB-2 is closely correlated with elevated tyrosine kinase activity of the gene product. To investigate the role of autophosphorylation in cell transformation, we introduced an additional mutation at the autophosphorylation site of the Glu-659 c-erbB-2 protein (Tyr-1248 to Phe). This mutant protein exhibited lower tyrosine kinase activity and lower transforming activity. On the other hand, when the carboxyl-terminal 230 amino acid residues were deleted from the c-erbB-2 protein, the tyrosine kinase activity and cell-transforming activity of the protein were enhanced. Thus, the carboxyl-terminal domain, which contains the major autophosphorylation site, Tyr-1248, may regulate cellular transformation negatively and autophosphorylation may eliminate this negative regulation.
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PMID:The transforming potential of the c-erbB-2 protein is regulated by its autophosphorylation at the carboxyl-terminal domain. 167 Dec 96

An epidermal growth factor (EGF) receptor monoclonal antibody (mAb), mAb LA22, was used to analyze the covalent coupling of human EGF receptors to mouse EGF by the amine-reactive cross-linking agent disuccinimidyl suberate. A soluble Mr 105,000 truncated form of the receptor secreted by A-431 epidermoid carcinoma cells and consisting of the ligand-binding extracellular domain was cross-linked to 125I-labeled EGF. Digestion of this complex with an endoproteinase that specifically cleaves at the COOH side of glutamyl residue released a single radiolabeled glycosylated fragment of Mr 18,000 that reacted with mAb LA22. As the epitope for mAb LA22 resided between Ala-351 and Asp-364 of the mature receptor, this result localized the cross-linked receptor residue(s) to the 47-amino acid interval from Phe-321 to Glu-367. The receptor residue(s) involved in the covalent coupling of rat 125I-labeled transforming growth factor alpha was similarly localized to this region of the receptor. This receptor interval, which included two glycosylated asparaginyl residues at positions 328 and 337, contained but three amino acid residues that were potentially reactive with disuccinimidyl suberate: Lys-332, Lys-333, and Lys-336. Characterization of mAb LA22-reactive 125I-EGF-labeled receptor fragments generated by an endoproteinase specific for the COOH side of lysyl residue placed the NH2 termini of the two smallest fragments between the glycosylated residues Asn-328 and Asn-337. These results indicated that disuccinimidyl suberate cross-linked the NH2 group of EGF residue Asn-1 to the human EGF receptor residue Lys-336. Our results further suggest that EGF and transforming growth factor alpha, two members of the EGF family of peptide growth factors, interact with closely apposed or identical features of the receptor.
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PMID:Human epidermal growth factor receptor residue covalently cross-linked to epidermal growth factor. 169 2

Signals that can mediate ligand-induced receptor internalization and calcium regulation are present in a 48-amino acid "calcium-internalization" domain in the C' terminus of the epidermal growth factor (EGF) receptor. The basis of calcium and internalization regulation signalled by this 48-amino acid sequence was analyzed using deletion and substitution mutant receptors. Cells expressing truncated receptors containing either the NH2- or COOH-terminal portion of the 48-residue domain displayed high affinity EGF-dependent endocytosis and receptor down-regulation. These endocytosis-competent EGF receptor mutants that lacked any autophosphorylation site were unable to increase the concentration of intracellular calcium. To investigate the role of self-phosphorylation in EGF-induced calcium mobilization, phenylalanine was substituted for the single autophosphorylated tyrosine residue in this region of an internalization-competent truncated receptor. The receptor-mediated calcium response was abolished, while ligand-dependent receptor internalization was unimpaired. These results demonstrate that EGF-dependent receptor endocytosis and calcium mobilization are separate events. Tyrosine self-phosphorylation is required for increased [Ca2+]i, while structural features distinct from autophosphorylation are required for receptor internalization.
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PMID:Ligand-induced internalization and increased cell calcium are mediated via distinct structural elements in the carboxyl terminus of the epidermal growth factor receptor. 174 39

GTPase-activating protein (GAP) stimulates the ability of p21ras to hydrolyze GTP to GDP. Since GAP is phosphorylated by a variety of activated or oncogenic protein-tyrosine kinases, it may couple tyrosine kinases to the Ras signaling pathway. The epidermal growth factor (EGF) receptor cytoplasmic domain phosphorylated human GAP in vitro within a single tryptic phosphopeptide. The same GAP peptide was also apparently phosphorylated on tyrosine in EGF-stimulated rat fibroblasts. Circumstantial evidence suggested that residue 460 might be the site of GAP tyrosine phosphorylation. This possibility was confirmed by phosphorylation of a synthetic peptide corresponding to the predicted tryptic peptide containing Tyr-460. Alteration of Tyr-460 to phenylalanine by site-directed mutagenesis diminished the in vitro phosphorylation of a bacterial GAP polypeptide by the EGF receptor. We conclude that Tyr-460 is a site of GAP tyrosine phosphorylation by the EGF receptor in vitro and likely in vivo. GAP Tyr-460 is located immediately C terminal to the second GAP SH2 domain, suggesting that its phosphorylation might have a role in regulating protein-protein interactions.
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PMID:The epidermal growth factor receptor phosphorylates GTPase-activating protein (GAP) at Tyr-460, adjacent to the GAP SH2 domains. 185 98

The product of the erbB-2 gene is a 185-kD receptor-like glycoprotein. erbB-2 gp185 displays constitutive tyrosine kinase activity and transforms NIH 3T3 cells when expressed 100-fold over the normal levels. We have analyzed the role of tyrosine kinase function and of receptor autophosphorylation in the regulation of erbB-2 biological activity. Abolition of erbB-2 gp185 tyrosine kinase function resulted in complete loss of its transforming activity and the absence of in vivo tyrosine phosphorylation. The steady-state content of phosphotyrosine in erbB-2 gp185 was found to be solely dependent on receptor autophosphorylation and to be dependent on the specific enzymatic activity of the erbB-2 protein. The major sites of erbB-2 autophosphorylation were shown to be in its COOH-terminal domain. Biological analysis of erbB-2 mutants containing either individual or multiple Tyr----Phe substitutions at the potential sites of autophosphorylation revealed that autophosphorylation upregulates erbB-2 gp185 transforming activity. Autophosphorylation did not modulate receptor turnover. A Tyr----Phe substitution of erbB-2 Tyr-877 homologous to pp60c-src Tyr-416 did not alter erbB-2 biological and biochemical properties, thus excluding the possibility that phosphorylation of this residue, located in the kinase domain, modulates erbB-2 gp185 catalytic function. Hence, autophosphorylation of tyrosine residues localized in its COOH terminus appears to be required for optimal coupling of erbB-2 gp185 with its mitogenic pathway.
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PMID:The role of autophosphorylation in modulation of erbB-2 transforming function. 198 72

The epidermal growth factor (EGF) receptor, which exhibits intrinsic protein tyrosine kinase activity, undergoes a rapid, intramolecular self-phosphorylation reaction following EGF activation. The primary sites of tyrosine self-phosphorylation in vivo are located in the extreme carboxyl-terminal region of the molecule, principally Tyr-1173. To test the biological and biochemical consequences of this EGF receptor self-phosphorylation, we made the mutation Tyr----Phe-1173. Membranes containing the mutated receptor exhibited an ED50 for EGF activation of tyrosine kinase activity equivalent to control receptor at both high and low substrate levels, but exhibited reduced basal and EGF-stimulated tyrosine kinase activity at low, non-saturating substrate levels. The Tyr----Phe-1173 mutant possessed high affinity EGF binding and could still self-phosphorylate other tyrosine sites in an intramolecular fashion with a low Km for ATP (200 nM), suggesting that this alteration did not grossly change receptor structure. When EGF-dependent growth of Chinese hamster ovary cells expressing comparable levels of control or mutant EGF receptor was measured, the ability of the mutant receptor to mediate cell growth in response to EGF was reduced by approximately 50%, yet both receptors exhibited a similar affinity and ED50 for EGF. These results support the concept that this self-phosphorylation site can act as a competitive/alternate substrate for the EGF receptor, and that this region of the molecule is important in modulating its maximal biological activity.
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PMID:Alteration of epidermal growth factor receptor activity by mutation of its primary carboxyl-terminal site of tyrosine self-phosphorylation. 334 10

Phosphorylation of two newly identified epidermal growth factor (EGF) receptor substrates, eps8 and eps15, which do not possess Src homology (SH2) domains, was investigated using EGF receptor mutants of the autophosphorylation sites and deletion mutants of the carboxyl-terminal region. Two mutants, F5, in which all five tyrosine autophosphorylation sites substituted by phenylalanine, and Dc 123F, in which four tyrosines were removed by deletion and the fifth (Tyr-992) was mutated into phenylalanine, phosphorylated eps8 and eps15 as efficiently as the wild-type receptor. In contrast, SH2-containing substrates, phospholipase C gamma, the GTPase-activating protein of Ras, the p85 subunit of phosphatidylinositol 3 kinase, and the Src and collagen homology protein, are not phosphorylated by the F5 and Dc 123F mutants. A longer EGF receptor deletion mutant, Dc 214, lacking all five autophosphorylation sites, was unable to phosphorylate eps15 but phosphorylated eps8 13-fold more than the wild-type receptor. To determine the EGF receptor region important for phosphorylation of eps8 and eps15, progressive deletion mutants lacking the final 123, 165, 196, and 214 COOH-terminal residues were used. eps8 phosphorylation was progressively increased in Dc 165, Dc 196, and Dc 214 EGF receptor mutants, indicating that removal of the final 214 COOH-terminal residues increases the phosphorylation of this substrate by the EGF receptor. In contrast, eps15 was phosphorylated by Dc 123 and Dc 165 EGF receptor mutants but not by Dc 196 and Dc 214 mutants. This indicates that a region of 30 residues located between Dc 165 and Dc 196 is essential for eps15 phosphorylation. This is the first demonstration of structural requirements in the EGF receptor COOH terminus for efficient phosphorylation of non-SH2-containing substrates. In addition, enhanced eps8 phosphorylation correlates well with the increased transforming potential of EGF receptor deletion mutants Dc 196 and Dc 214, suggesting that this substrate may be involved in mitogenic signaling.
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PMID:Structural requirements of the epidermal growth factor receptor for tyrosine phosphorylation of eps8 and eps15, substrates lacking Src SH2 homology domains. 760 94

An expression cloning method which allows direct isolation of cDNAs encoding substrates for tyrosine kinases was applied to the study of the epidermal growth factor (EGF) receptor (EGFR) signaling pathway. A previously undescribed cDNA was isolated and designated eps15. The structural features of the predicted eps15 gene product allow its subdivision into three domains. Domain I contains signatures of a regulatory domain, including a candidate tyrosine phosphorylation site and EF-hand-type calcium-binding domains. Domain II presents the characteristic heptad repeats of coiled-coil rod-like proteins, and domain III displays a repeated aspartic acid-proline-phenylalanine motif similar to a consensus sequence of several methylases. Antibodies specific for the eps15 gene product recognize two proteins: a major species of 142 kDa and a minor component of 155 kDa, both of which are phosphorylated on tyrosine following EGFR activation by EGF in vivo. EGFR is also able to directly phosphorylate the eps15 product in vitro. In addition, phosphorylation of the eps15 gene product in vivo is relatively receptor specific, since the erbB-2 kinase phosphorylates it very inefficiently. Finally, overexpression of eps15 is sufficient to transform NIH 3T3 cells, thus suggesting that the eps15 gene product is involved in the regulation of mitogenic signals.
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PMID:eps15, a novel tyrosine kinase substrate, exhibits transforming activity. 768 53


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