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)

Several cytoplasmic tyrosine kinases contain a conserved, non-catalytic stretch of approximately 100 amino acids called the src homology 2 (SH2) domain, and a region of approximately 50 amino acids called the SH3 domain. SH2/SH3 domains are also found in several other proteins, including phospholipase C-gamma (PLC gamma). Recent studies indicate that SH2 domains promote association between autophosphorylated growth factor receptors such as the epidermal growth factor (EGF) receptor and signal transducing molecules such as PLC gamma. Because SH2 domains bind specifically to protein sequences containing phosphotyrosine, we examined their capacity to prevent tyrosine dephosphorylation of the EGF and other receptors with tyrosine kinase activity. For this purpose, various SH2/SH3 constructs of PLC gamma were expressed in Escherichia coli as glutathione-S-transferase fusion proteins. Our results show that purified SH2 domains of PLC gamma are able to prevent tyrosine dephosphorylation of the EGF receptor and other receptors with tyrosine activity. The inhibition of tyrosine dephosphorylation paralleled the capacity of various SH2-containing constructs to bind to the EGF receptor, suggesting that the tyrosine phosphatase and the SH2 domain compete for the same tyrosine phosphorylation sites in the carboxy-terminal tail of the EGF receptor. Analysis of the phosphorylation sites protected from dephosphorylation by PLC gamma-SH2 revealed substantial inhibition of dephosphorylation of Tyr992 at 1 microM SH2. This indicates that Tyr992 and its flanking sequence is the high-affinity binding site for SH2 domains of PLC gamma.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:SH2 domains prevent tyrosine dephosphorylation of the EGF receptor: identification of Tyr992 as the high-affinity binding site for SH2 domains of phospholipase C gamma. 153 35

In the present study, utilizing anti-phosphotyrosine monoclonal antibodies, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) as sources of NO and murine fibroblasts expressing the human epidermal growth factor (EGF) receptor (HER14 cells), we showed that tyrosine phosphorylation of a set of proteins (126, 56 and 43 kDa) was stimulated when cells were incubated with either SNP or SNAP and abolished by Methylene Blue and oxyhaemoglobin. Inhibition by Methylene Blue suggested an involvement of cyclic GMP in the process, which was evidenced by the effects of 8-bromo cyclic GMP. This analogue of cyclic GMP stimulated tyrosine phosphorylation of the same set of proteins phosphorylated after incubation with the NO source. Tyrosine phosphorylation of the same set of proteins was stimulated when cells were incubated simultaneously with SNP and EGF, showing that NO also potentiates EGF-evoked tyrosine kinase activity in HER14 cells. However, stimulation of the autophosphorylation of the EGF receptor, above the levels obtained for EGF alone, was not observed under those conditions. Additionally, we investigated the effects of NO on EGF-receptor tyrosine phosphatase activities in HER14 cells. Increasing concentrations of NO correlate with a gradual inhibition of these activities in HER14 cells, either in intact cells or in cell lysates. Taken together, these observations suggest that NO modulates tyrosine phosphorylation in HER14 cells.
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PMID:Nitric oxide stimulates tyrosine phosphorylation in murine fibroblasts in the absence and presence of epidermal growth factor. 753 Apr 47

The intrinsic protein tyrosine kinase activity of the epidermal growth factor (EGF) receptor is necessary for ligand-induced signaling. To determine whether cellular protein tyrosine kinases are substrates for EGF-activated receptors, phosphotyrosine-containing proteins were isolated from EGF-treated cells and assayed for tyrosine kinase activity using peptide substrates. A tyrosine kinase activity that is distinct from the EGF receptor was adsorbed to monoclonal anti-phosphotyrosine antibody columns and eluted with phenyl phosphate. Near-maximal tyrosine phosphorylation of this kinase occurred within 1 min of cell stimulation with an ED50 for EGF of 2.5 nM. The kinase was deactivated by incubation with purified CD45 tyrosine phosphatase in vitro, but activity could be restored by incubation with purified EGF receptor and Mn2+ ATP. These results suggest a cascade of tyrosine kinase signaling analogous to well characterized serine/threonine kinase cascades.
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PMID:Epidermal growth factor stimulates a protein tyrosine kinase which is separable from the epidermal growth factor receptor. 826 33

Breast carcinomas are frequently characterized by hyperactivated c-erbB receptor tyrosine kinase signaling. Combination of anti-proliferative retinoids with growth-inhibitory c-erbB-specific agents might induce therapeutic benefit. We demonstrate close interactions between the c-erbB and the retinoic acid receptor system in SK-BR-3 breast cancer cells. Epidermal growth factor and heregulin-beta1 activate c-erbB receptors and dose- and time-dependently up-regulate retinoic acid receptor-alpha (RAR-alpha) mRNA. Similar effects have been found for the growth-inhibitory c-erbB-2 receptor tyrosine kinase-activating antibody 4D5 and the tyrosine phosphatase inhibitor orthovanadate. In contrast, the tyrosine kinase-inhibitor herbimycin A reduces tyrosine-specific protein phosphorylation and down-regulates RAR-alpha. Our data demonstrate that the expression of RAR-alpha, which represents a key mediator of the anti-proliferative effects of retinoids in breast cancer cells, is regulated by modulators of tyrosine kinase signaling. The levels of RAR-beta and -gamma mRNAs, however, are not affected by such agents.
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PMID:Tyrosine kinase signaling pathways control the expression of retinoic acid receptor-alpha in SK-BR-3 breast cancer cells. 909 80

Ligands that bind to the epidermal growth factor (EGF) receptor are initially synthesized as integral membrane proteins that are released from the cell surface by regulated proteolysis. To study the role of the membrane-anchoring domain in ligand release, we made two artificial ligands. The first possessed the membrane-anchoring domain from EGF whereas the second had the corresponding domain from heparin binding EGF-like growth factor (HB-EGF). Both ligands lacked amino-terminal extensions, and were epitope-tagged at the carboxyl terminus. Following stable expression in human mammary epithelial cells, their cellular localization and rate of proteolytic release were examined. We found that constructs with the membrane-anchoring domain from EGF were found primarily at the cell surface and displayed a relatively high rate of constitutive release. Constructs with the HB-EGF membrane-anchoring domain displayed a higher internalized fraction and a very low rate of constitutive release. The two ligand constructs also displayed different patterns of stimulated release. Proteolysis of the chimera with the HB-EGF membrane-anchoring domain was stimulated by activation of protein kinase C, but release of EGF from constructs with the EGF membrane-anchoring domain was insensitive to this. Calcium ionophores, calmodulin antagonists, and tyrosine phosphatase inhibitors stimulated the release of both ligands. Furthermore, the release of the two constructs showed different sensitivity to metalloprotease inhibitors. Despite a large fold-increase in ligand proteolysis following cell stimulation, only a small fraction of total cell-associated ligand was released per hour. Our results show that the membrane-anchoring domain of EGF-like ligands can specify both their localization and proteolytic processing. The structures of the membrane-anchoring region of this class of ligands can thus regulate their activity.
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PMID:Trafficking and proteolytic release of epidermal growth factor receptor ligands are modulated by their membrane-anchoring domains. 1061 51

Interleukin (IL)-6, a multifunctional regulator of immune response, hematopoiesis, and acute phase reactions, has also been shown to regulate cancer cell proliferation. We have investigated IL-6 signaling pathways and cellular responses in the T47D breast carcinoma cell line. The IL-6-type cytokines, IL-6 and oncostatin M, simultaneously inhibited cell proliferation and increased cell migration. In T47D cells, IL-6 stimulated the activation of Janus-activated kinase 1 tyrosine kinase and signal transducers and activators of transcription (STAT) 1 and STAT3 transcription factors. Expression of dominant negative STAT3 in the cells strongly reduced IL-6-mediated growth inhibition but did not prevent IL-6-induced cell migration. IL-6 treatment led to activation of the mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3'-kinase (PI3K) pathways. Inhibition of MAPK or PI3K activity reversed IL-6- and oncostatin M-stimulated migration. Because cross-talk between cytokine receptors and members of the ErbB family of receptor tyrosine kinases has been described previously, we have examined their interaction in T47D cells. Down-regulation of ErbB receptor activity, through the use of specific pharmacological inhibitors or dominant negative receptor constructs, revealed that IL-6-induced MAPK activation was largely dependent on epidermal growth factor (EGF) receptor activity, but not on ErbB-2 activity. Using a monoclonal antibody that interferes with EGF receptor-ligand interaction, we have shown that in T47D cells, IL-6 cooperates with an EGF receptor autocrine activity loop for signaling through the MAPK and PI3K pathways and for cell migration. Both the tyrosine phosphatase SHP-2 and the multisubstrate docking molecule Gab1, which are potential links between IL-6 and the MAPK/PI3K pathways, were constitutively associated with the active EGF receptor. On IL-6 stimulation, SHP-2 and Gab1 were recruited to the gp130 subunit of the IL-6 receptor and tyrosine phosphorylated, allowing downstream signaling to the MAPK and PI3K pathways. Thus, in T47D breast carcinoma cells, IL-6 acts in synergy with EGF receptor autocrine activity to signal through the MAPK/PI3K pathways. Cooperation between IL-6 and the EGF receptor in T47D breast carcinoma cells illustrates how a combination of multiple stimuli, either exogenous or endogenous, may result in synergistic cellular responses.
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PMID:Interleukin 6 inhibits proliferation and, in cooperation with an epidermal growth factor receptor autocrine loop, increases migration of T47D breast cancer cells. 1119 91

The adaptor protein Grb2-associated binder-1 (Gab1) is known to bind to the SHP-2 tyrosine phosphatase on epidermal growth factor (EGF) receptor stimulation. To clarify the roles of these two proteins in EGF receptor (EGFR) signaling and determine their possible alteration during neoplastic cell progression, we studied these proteins in a Syrian hamster embryo (SHE) cell line model of neoplastic progression. Specifically, we used asbestos-transformed SHE fibroblasts: the 10W+8 clone, which is immortal but nontumorigenic; and the 10W2T clone, which is tumorigenic. Gab1 was detected, and the EGF-dependent formation of the EGFR-Gab1-SHP-2 complex was observed in 10W+8 cells. After cloning hamster Gab1 cDNA, exogenous expression of Gab1 significantly enhanced EGF-dependent mitogenic activity in 10W+8 cells. On the other hand, Gab1 was not detected in 10W2T cells, and the EGF-dependent association of SHP-2 with EGFR was also absent. Exogenous Gab1 expression in transfected 10W2T cells restored the EGF-dependent association of SHP-2 with EGFR, although it only showed a marginal effect on EGF-dependent mitogenic activity. Thus, Gab1 plays a pivotal role in the EGFR signaling pathway via the formation of the EGFR-Gab1-SHP-2 complex, and alteration in the expression and function of Gab1 is implicated in the neoplastic progression of SHE cells.
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PMID:Identification of epidermal growth factor receptor- Grb2-associated binder-1-SHP-2 complex formation and its functional loss during neoplastic cell progression. 1143 5

Using stable isotope labeling and mass spectrometry, we performed a sensitive, quantitative analysis of multiple phosphorylation sites of the epidermal growth factor (EGF) receptor. Phosphopeptide detection efficiency was significantly improved by using the tyrosine phosphatase inhibitor sodium pervanadate to boost the abundance of phosphorylation of the EGF receptor. Nine phosphorylation sites (pT669, pS967, pS1002, pY845, pY974, pY1045, pY1086, pY1148, and pY1173) of EGF receptor were quantified from EGF-stimulated cells in suspension and adherent conditions. Our data sets revealed that EGF stimulation of adherent cells induced higher levels of tyrosine phosphorylation relative to EGF stimulation of suspended cells. In contrast, EGF stimulation of adherent cells induced lower levels of serine and threonine phosphorylation relative to EGF stimulation of suspended cells. These findings are consistent with the hypothesis that cellular adhesion modulates phosphorylation of plasma membrane receptor tyrosine kinases relevant for EGF-induced signal transduction processes. Furthermore, our results suggest that strong phosphatase inhibitors should be used to generate reference datasets in comparative phosphoproteomics experiments.
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PMID:Quantitation of multisite EGF receptor phosphorylation using mass spectrometry and a novel normalization approach. 1752 11

Endogenous sphingolipid metabolites such as ceramides and sphingosines have been increasingly recognized as lipid mediators of cell growth, differentiation and apoptosis. We have previously studied the ability of sphingosine (Sph) and N,N-dimethylsphingosine (DMS) to induce apoptosis in a variety of solid tumor cell lines. Here we report that in tumor cell lines displaying high mitogen-activated protein kinase activity (MAPK), treatment with 5 mu M of these sphingolipids significantly inhibited MAPK activity within 2-5 min (p < 0.005-0.01 as compared to controls) and induced apoptosis within hours. In contrast, untransformed cells and those tumor cell lines with low MAPK activity showed no significant change in activity and no apoptosis. High concentrations of C2-ceramide (50-100 mM), which induced apoptosis in the solid tumor cells, did not show significant effect on MAPK activity. MAPK activity was not directly inhibited in vitro, but tyrosine phosphatase activity was increased 2-4 fold in solid tumor cells by Sph or DMS (p < 0.01-0.05), suggesting that a phosphatase may play an important role in sphingolipid-directed MAPK regulation. Sph/DMS-induced apoptosis, but not MAPK inhibition, was blocked by protease inhibitors, indicating that MAPK inhibition is an earlier step of Sph/DMS-induced apoptosis than proteolysis. Furthermore, in human breast carcinoma MDA468 cells and human epidermal carcinoma A431 cells, both of which overexpress the epidermal growth factor (EGF) receptor, 20-200 nM EGF inhibited MAPK (p < 0.005-0.01) and induced apoptosis. These observations suggest that inhibition of the MAPK cascade may be involved in apoptotic signaling by Sph/DMS in some solid tumor cells, or by EGF in some cancer cells which overexpress the EGF receptor. Finally, the PKC-specific inhibitor, calphostin C, under conditions in which PKC is completely suppressed, inhibited MAPK activity and induced apoptosis only weakly in these solid tumor cells, whereas the non-specific PKC inhibitor staurosporine induced both apoptosis and MAPK inhibition significantly, suggesting that MAPK inhibition and apoptosis by Sph/DMS occurs independently of PKC in these cell lines, although these pathways may act cooperatively in other cell types. This study provides insight into possible mechanisms involved in sphingolipid-induced apoptosis in solid cancer tumor cell lines.
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PMID:Inhibition of MAP kinase by sphingosine and its methylated derivative, N,N-dimethylsphingosine. 2152 77