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

One of the immediate cellular responses to stimulation by various growth factors is the activation of a phosphatidylinositol (PI) 3-kinase. We recently cloned the 85-kDa subunit of PI 3-kinase (p85) from a lambda gt11 expression library, using the tyrosine-phosphorylated carboxy terminus of the epidermal growth factor (EGF) receptor as a probe (E. Y. Skolnik, B. Margolis, M. Mohammadi, E. Lowenstein, R. Fischer, A. Drepps, A. Ullrich, and J. Schlessinger, Cell 65:83-90, 1991). In this study, we have examined the association of p85 with EGF and platelet-derived growth factor (PDGF) receptors and the tyrosine phosphorylation of p85 in 3T3 (HER14) cells in response to EGF and PDGF treatment. Treatment of cells with EGF or PDGF markedly increased the amount of p85 associated with EGF and PDGF receptors. Binding assays with glutathione S-transferase (GST) fusion proteins demonstrated that either Src homology region 2 (SH2) domain of p85 is sufficient for binding to EGF and PDGF receptors and that receptor tyrosine autophosphorylation is required for binding. Binding of a GST fusion protein expressing the N-terminal SH2 domain of p85 (GST-N-SH2) to EGF and PDGF receptors was half-maximally inhibited by 2 and 24 mM phosphotyrosine (P-Tyr), respectively, suggesting that the N-SH2 domain interacts more stably with PDGF receptors than with EGF receptors. The amount of receptor-p85 complex detected in HER14 cells treated with EGF or PDGF. Growth factor treatment also increased the amount of p85 found in anti-PDGF-treated HER14 cells, suggesting that the vast majority of p85 in the anti-P-Tyr fraction is receptor associated but not phosphorylated on tyrosine residues. Only upon transient overexpression of p85 and PDGF receptor did p85 become tyrosine phosphorylated. These are consistent with the hypothesis that p85 functions as an adaptor molecule that targets PI 3-kinase to activated growth factor receptors.
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PMID:Interaction of phosphatidylinositol 3-kinase-associated p85 with epidermal growth factor and platelet-derived growth factor receptors. 137 91

Conflicting results concerning the ability of the epidermal growth factor (EGF) receptor to associate with and/or activate phosphatidylinositol (PtdIns) 3-kinase have been published. Despite the ability of EGF to stimulate the production of PtdIns 3-kinase products and to cause the appearance of PtdIns 3-kinase activity in antiphosphotyrosine immunoprecipitates in several cell lines, we did not detect EGF-stimulated PtdIns 3-kinase activity in anti-EGF receptor immunoprecipitates. This result is consistent with the lack of a phosphorylated Tyr-X-X-Met motif, the p85 Src homology 2 (SH2) domain recognition sequence, in this receptor sequence. The EGF receptor homolog, ErbB2 protein, also lacks this motif. However, the ErbB3 protein has seven repeats of the Tyr-X-X-Met motif in the carboxy-terminal unique domain. Here we show that in A431 cells, which express both the EGF receptor and ErbB3, PtdIns 3-kinase coprecipitates with the ErbB3 protein (p180erbB3) in response to EGF. p180erbB3 is also shown to be tyrosine phosphorylated in response to EGF. In contrast, a different mechanism for the activation of PtdIns 3-kinase in response to EGF occurs in certain cells (PC12 and A549 cells). Thus, we show for the first time that ErbB3 can mediate EGF responses in cells expressing both ErbB3 and the EGF receptor.
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PMID:ErbB3 is involved in activation of phosphatidylinositol 3-kinase by epidermal growth factor. 751 47

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

Src homology regions 2 (SH2) and 3 (SH3) are noncatalytic domains that are conserved among several proteins implicated in the regulation of cell proliferation. Using bacterially expressed fusion proteins containing the SH2 domain of the abl tyrosine kinase, we have quantitated the binding of these domains to the activated epidermal growth factor (EGF) receptor (EGFR). A 35S-labeled abl SH2 fusion protein binds to the human EGFR immunoprecipitated from EGF-treated NIH3T3 cells that overexpress the receptor. This binding is totally dependent on the pretreatment of cells with EGF. The interaction is rapid, reaching 50% of maximum within 1 min, and attaining apparent equilibrium by 10 min. Dissociation of the complex is biphasic with a rapidly dissociating component (t1/2 of less than 1 min), as well as a slowly dissociable component. The 35S-labeled abl SH2 fusion protein specifically binds to the EGFR in a saturable manner and is differentially inhibited by unlabeled fusion proteins containing SH2 domains from phospholipase C, the p85 subunit of phosphatidylinositol-3 kinase, and the GTPase activation protein of ras. To identify residues critical for abl SH2-EGFR binding, six point mutants were constructed in the highly conserved FLVRES motif. Three mutants (V170L, E172Q, and E174Q) display binding affinities similar to that of wild type. However, three other mutants (R171K, S173C, and S175C) have greatly reduced affinity. Interestingly, the binding affinity to the EGFR determined by the in vitro assay directly correlates with the transforming ability of the corresponding v-abl constructs in vivo (Mayer, B. J., Jackson, P. K., Etten, R. A. V., and Baltimore, D. (1992) Mol. Cell. Biol. 12, 609-618). These data indicate that the Arg-171, Ser-173, and Ser-175 are critical for both transformation and abl SH2 domain binding to phosphotyrosine-containing proteins.
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PMID:Direct analysis of the binding of the abl Src homology 2 domain to the activated epidermal growth factor receptor. 767 9

c-erbB-3 is a member of the type I (EGF receptor-related) family of growth factor receptors for which no ligand has been identified. To facilitate ligand stimulation we have constructed a chimeric receptor which possesses an activatable kinase and promotes the growth of NIH 3T3 fibroblasts. In this study we have shown that SHC and phosphatidylinositol 3'-kinase bind to the activated EGF receptor/c-erbB-3 chimera. Whereas p85 is not phosphorylated to a significant extent, SHC appears to be a major substrate for phosphorylation on tyrosine. In contrast to EGF receptor and c-erbB-2, we were unable to detect binding of activated c-erbB-3 to GRB2. Using synthetic peptides corresponding to each of 13 potential phosphorylation sites on c-erbB-3, we have shown that tyrosine 1309 is responsible for SHC binding. Peptides containing the motif YXXM inhibit p85 association. By comparison with recently reported SHC binding sites on Middle T antigen and Trk we have identified a SHC binding motif, NPXY.
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PMID:Identification of c-erbB-3 binding sites for phosphatidylinositol 3'-kinase and SHC using an EGF receptor/c-erbB-3 chimera. 802 68

Protein kinases share a number of highly conserved or invariant amino acid residues in their catalytic domains, suggesting that these residues are necessary for kinase activity. In p180erbB3, a receptor tyrosine kinase belonging to the epidermal growth factor (EGF) receptor subfamily, three of these residues are altered, suggesting that this protein might have an impaired protein tyrosine kinase activity. To test this hypothesis, we have expressed human EGF receptor and bovine p180erbB3 in insect cells via baculovirus infection and have compared their autophosphorylation and substrate phosphorylation activities. We have found that, while the EGF receptor readily undergoes EGF-stimulated autophosphorylation and catalyzes the incorporation of phosphate into the model substrates (E4Y1)n (random 4:1 copolymer of glutamic acid and tyrosine) and GST-p85 (glutathione S-transferase fusion protein with the 85-kDa subunit of phosphatidylinositol 3-kinase), p180erbB3 autophosphorylation and substrate phosphorylation are at least 2 orders of magnitude less efficient. However, p180erbB3 is capable of binding the ATP analog 5'-p-fluorosulfonylbenzoyladenosine, indicating that the lack of observed kinase activity is probably not due to nonfunctional or denatured receptors expressed by the insect cells. On the basis of these results, we propose that p180erbB3 possesses an impaired intrinsic tyrosine kinase activity.
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PMID:Insect cell-expressed p180erbB3 possesses an impaired tyrosine kinase activity. 805 68

To determine whether individual autophosphorylation sites in the epidermal growth factor (EGF) receptor define specific interaction sites for the in vivo association of signal transduction proteins that contain src homology 2 (SH2) domains, the capacity of wild-type and mutant EGF receptors to associate with several SH2 domain-containing proteins has been assayed. Mutants included receptors with single autophosphorylation site mutations at each of five autophosphorylation sites and receptors in which multiple autophosphorylation sites were removed by point mutation or deletion of carboxyl-terminal residues. Receptor association, as measured by coimmunoprecipitation, has been determined for phospholipase C-gamma 1, the ras GTPase-activating protein, the p85 subunit of phosphatidylinositol 3-kinase, and the src homology and collagen protein. In contrast to data obtained with single autophosphorylation site mutants of other receptor tyrosine kinases, none of the EGF receptor single site mutants was dramatically impaired in its capacity to associate with any of these SH2-containing proteins. However, association was completely abrogated when all five autophosphorylation sites were mutated or removed by deletion. These results indicate that individual autophosphorylation sites in the EGF receptor are not stringently required for the recognition and association of different SH2-containing substrates. Thus, EGF receptor autophosphorylation sites seem to be flexible and/or compensatory in their capacity to mediate association with these four SH2-containing substrates.
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PMID:Individual epidermal growth factor receptor autophosphorylation sites do not stringently define association motifs for several SH2-containing proteins. 816 37

Heregulins (HRGs) induce tyrosine phosphorylation of several members of the erb-B family of receptors. Although originally isolated as the ligands for p185c-erb-2, recent evidence suggests that other receptors of the erbB family, including p180erbB-3 and p180erbB-4, are their true cognate receptors. Stimulation of MDA MB-453 cells with HRG beta 2 resulted in the tyrosine phosphorylation of p185c-erbB-2 and p180erbB-4 in a time- and dose-dependent fashion. This event was accompanied by the formation of multimeric complexes between the activated receptors and SH2-containing proteins. Ligand caused p120-rasGTPase activating protein (GAP), SHC and the p85 subunit of phosphatidylinositol-3'-kinase (PI3K) to be associated with both p185c-erbB-2 and p180erbB-4. In addition, tyrosine phosphorylation of p85-PI3K and SHC, but not of GAP or of its associated p62 and p190 proteins, was also detected. HRG also induced the association of GRB2 with tyrosine phosphorylated p185c-erbB-2, p180erbB-4 and SHC. Activation of mitogen-activated protein kinase (MAPK) ( > 30-fold over untreated controls) was observed upon receptor(s) activation, as it was the induction of the immediate early gene c-fos ( > 200-fold). These observations suggest that p21ras activation plays a role in the HRG pathway. Furthermore, comparative analysis of the binding of p85-PI3K to 185c-erbB-2 and p180erbB-4, revealed a preferential association with activated p180erbB-4. These findings might suggest a model of HRG action in which the relative expression of the various erb-B family members and the partitioning of signal transduction molecules between each type of receptor might determine the nature of the signal elicited by the ligand and the biological response attained.
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PMID:Signal transduction pathways induced by heregulin in MDA-MB-453 breast cancer cells. 862 88

We and others have shown that Cbl, the protein product of the c-cbl proto-oncogene, is an early target of tyrosine phosphorylation upon stimulation through the immune cell surface receptors, which signal through noncovalently associated cytoplasmic tyrosine kinases. Using human mammary epithelial cells that express a natural epidermal growth factor (EGF) receptor and require EGF as an essential growth factor, we demonstrate here that Cbl is a prominent target of tyrosine phosphorylation upon stimulation through the EGF receptor tyrosine kinase. Phosphorylation of Cbl was EGF dose-dependent, rapid (detectable as early as 5 s and maximal by 2 min), and relatively sustained (detectable even after 1 h). Co-immunoprecipitation studies demonstrated that Cbl became associated with the EGF receptor in an EGF-dependent manner. Cbl was basally associated with the adaptor protein growth factor receptor-binding protein 2 (Grb2), and this interaction was further enhanced by EGF stimulation; however, the interaction was entirely mediated via the Grb2 Src homology 3 (SH3) domains, suggesting that binding of Grb2 SH2 domain to EGF receptor provides one mechanism of Cbl's association with the EGF receptor. EGF stimulation also induced the association of Cbl with Src homology and collagen (Shc) protein, p85 subunit of the phosphatidylinositol 3-kinase and Crk proteins, in particular with the CrkL isoform. Interactions of Cbl with the EGF receptor and multiple downstream signaling proteins suggest a role for this proto-oncogene product in mitogenic signaling through growth factor receptor kinases.
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PMID:Tyrosine phosphorylation of Cbl upon epidermal growth factor (EGF) stimulation and its association with EGF receptor and downstream signaling proteins. 866 98

Amplification and overexpression of the c-erbB-2 gene in 21MT-2 and 21MT-1 human breast carcinoma cells results in progressively elevated levels of constitutively tyrosine-phosphorylated p185erbB-2 and is associated with progressive insulin-like growth factor (IGF) and combined IGF/epidermal growth factor (EGF) independence in culture. In addition, the neu differentiation factor/heregulins (HRGs), a family of ligands that activate p185erbB-2 through direct binding to erbB-3 or erbB-4, are potent mitogens for various nonneoplastic mammary epithelial cells and carcinoma cell lines in the absence of both IGF and EGF in culture. We have investigated the ability of ligand induction with HRGs or the constitutive activation of p185erbB-2 in the 21MT breast carcinoma cells to induced the recruitment of phosphatidylinositol 3-kinase (PI3K) by p185erbB-2 and erbB-3. HRG was found to potently induce the recruitment of the M(r) 85,000 regulatory subunit of PI3K by phosphotyrosine proteins in both nonneoplastic H16N-2 mammary epithelial cells (which express normal c-erbB-2 levels) and in the 21MT-2 and 21MT-1 cell lines, which were all isolated from a single patient with intraductal and invasive ductal carcinoma of the breast and express c-erbB-3 but not c-erbB-4 in culture. The activation of PI3K in these cells was also associated with high-level mitogenic responsiveness to HRG, as well as the IGF/EGF-independent proliferation of the 21MT cell lines in culture. The recruitment of PI3K by phosphotyrosine protein during ligand-induced activation, or that seen constitutively in the 21MT tumor cells, did not involve detectable tyrosine phosphorylation of p85. The HRG-induced recruitment of p85 and the constitutive recruitment of p85 in the 21MT cell lines involved direct association with both p185erbB-2 and erbB-3, although greater levels were recruited directly by erbB-3. Wortmannin, a potent inhibitor of PI3K enzymatic activity, also blocked the autonomous proliferation of the 21MT cells, and this effect was reversible in long-term cultures. These data indicate that PI3K may be an especially important mediator of HRG-induced proliferation in mammary epithelial cells and is involved in the autonomous proliferation of growth factor-independent breast carcinoma cells with c-erbB-2 gene amplification.
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PMID:Phosphatidylinositol 3-kinase recruitment by p185erbB-2 and erbB-3 is potently induced by neu differentiation factor/heregulin during mitogenesis and is constitutively elevated in growth factor-independent breast carcinoma cells with c-erbB-2 gene amplification. 873 65


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