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

Previous studies have shown that lysine- and arginine-rich proteins can enhance the activity of tyrosine and serine/threonine protein kinases. However, the kinetics and mechanism of this activation are not fully understood. Therefore we investigated the ability of poly(amino acids) and the arginine-rich protein, protamine, to alter the kinetic properties of epidermal growth factor (EGF) receptor protein-tyrosine kinase activity using immunoaffinity-purified receptor isolated from human epidermoid carcinoma (A431) cells. Poly(L-lysine), poly(L-arginine) and protamine stimulated EGF receptor kinase activity by 3-5-fold at non-saturating doses of ATP and peptide substrate, while poly(L-glutamate) had no effect. Initial kinetic studies demonstrated an increase in the maximum velocity and a decrease in the apparent Km for the peptide substrate angiotensin II in the presence of the basic effectors. Further analysis of the kinetic mechanism by product inhibition revealed that protamine altered the pattern of ADP inhibition towards the peptide substrate but not towards ATP. The change was indicative of the receptor's ability to form an enzyme-angiotensin II-ADP ternary complex in the presence of protamine but not in its absence. In addition, the basic effectors had a substantially decreased influence on the kinase activity of a C-terminally truncated form of the EGF receptor. Thus the changes in kinase activity may be partially mediated by the C-terminal region of the receptor, which contains the sites of receptor self-phosphorylation. These results suggest that the basic domains of proteins can interact with the EGF receptor to induce changes in its kinetic properties, especially with regard to reactant recognition and binding.
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PMID:Alteration of the kinetic properties of the epidermal growth factor receptor tyrosine kinase by basic proteins. 137 Jun 7

To study the activity of the epidermal growth factor (EGF) receptor during EGF-directed internalization, liver epithelial cells were exposed to EGF at 37 degrees C for various periods of time, washed, and homogenized at 0 degrees C. EGF receptor autophosphorylation was assessed in homogenates using [gamma-32P]ATP. Autophosphorylation was stimulated 3- to 6-fold in homogenates of cells incubated with EGF (100 ng/ml) for 15 min but was at or below basal levels in homogenates of cells treated with EGF for 2.5-5 min. This was surprising because immunoblotting revealed that EGF receptor phosphotyrosine (P-Tyr) content in intact cells was near maximal from 30 s to 5 min after EGF treatment. Excess EGF (1 microgram/ml), added after homogenization but prior to the assay, increased autophosphorylation in homogenates of cells that had not been treated with EGF, but failed to increase activity in homogenates of cells treated with EGF in culture for 2.5-5 min. Suppression of tyrosine phosphorylation of an exogenous kinase substrate was also observed at times paralleling the suppression of EGF receptor autophosphorylation. The transient suppression of receptor autophosphorylation in the cell-free assay was not explained by persistent occupation of autophosphorylation sites by phosphate added in the intact cells. The sites were greater than 80% dephosphorylated during the homogenization. Additionally phosphatase inhibition that prevented the normal loss of EGF receptor P-Tyr in intact cells at 15 min did not affect the pattern of early (2.5-5 min) suppression and later (15 min) stimulation of autophosphorylation measured in the cell-free assay. The suppression was not explained by activation of protein kinase C in that depletion of greater than 95% of cellular protein kinase C activity by an 18-h incubation of cells with 10 microM 12-O-tetradecanoylphorbol 13-acetate (TPA) did not affect the early suppression of autophosphorylation in EGF-treated cells. Moreover, under the conditions tested, activation of protein kinase C by short-term treatment (0.5-10 min) with TPA or angiotensin II did not appreciably alter subsequent autophosphorylation in the cell-free assay. In contrast, a 30 degrees C preincubation of homogenates from cells with suppressed EGF receptor autophosphorylation led to the recovery of the ability of EGF to stimulate EGF receptor autophosphorylation. These results suggest that a rapid reversible protein kinase C-independent process prevents detection of EGF receptor kinase activity during an early phase of EGF-dependent receptor internalization.
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PMID:Transient epidermal growth factor (EGF)-dependent suppression of EGF receptor autophosphorylation during internalization. 169 15

The protein-tyrosine kinase activity of the epidermal growth factor (EGF) receptor is critical for EGF-stimulated cell growth, although little is known about the molecular details of its enzymatic activity. Previous studies have found that EGF receptor kinase activity can be stimulated by factors such as ammonium sulfate ((NH4)2SO4), but the manner in which (NH4)2SO4 induces this effect is unclear. Therefore, we have explored the processes by which (NH4)2SO4 potentiated tyrosine kinase activity to better understand not only the molecular events involved in (NH4)2SO4 activation, but also the kinetic properties and mechanism of the EGF receptor. In this study, the addition of an optimum concentration of (NH4)2SO4 (250 mM) resulted in a 5-fold stimulation of kinase activity toward the peptide substrate, angiotensin II. The sulfate group is primarily involved in this action, since other salts containing SO4(2-) increased kinase activity similarly, whereas salts containing Cl- and F- had less of an effect, and divalent salts such as HPO4(2-) and NaVO4(2-) were inhibitory at doses of 1 mM or more. In addition, EGF receptor kinase activation by (NH4)2SO4 did not strictly correlate with changes in the ionic strength or conductivity of the solution. However, several lines of evidence suggest that SO4(2-) directly alters the kinetic properties of the EGF receptor kinase: (1) the maximum velocity (Vmax) and Km (ATP) for EGF receptor phosphorylation of angiotensin II were substantially higher in the presence of (NH4)2SO4. (2) EGF receptor kinase activity in the absence of (NH4)2SO4 required either Mn2+ or Mg2+, yet in the presence of (NH4)2SO4, only Mn2+ supported the increase in kinase activity. (3) Ammonium sulfate addition altered the product inhibition pattern of ADP versus angiotensin II, suggesting that an enzyme-angiotensin II-ADP complex can form in the presence of (NH4)2SO4 but not in its absence. (4) The near-maximal rate of self-phosphorylation was not affected by (NH4)2SO4 but the apparent Km (ATP) was greatly increased. From these results, we propose a model for (NH4)2SO4 stimulation of EGF receptor kinase activity in which SO4(2-) interacts directly with the receptor or receptor-Mn(2+)-ATP complex and alters reactant binding and the catalytic efficiency of the tyrosine kinase.
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PMID:Potentiation of epidermal growth factor receptor protein-tyrosine kinase activity by sulfate. 173 63

Estrogen-stimulated growth of the human mammary adenocarcinoma cell line MCF-7 is significantly inhibited by monoclonal antibodies to the epidermal growth factor (EGF) receptor that act as antagonists of EGF's mitogenic events by competing for high-affinity EGF receptor binding sites. These antibodies likewise inhibit the EGF or transforming growth factor-alpha (TGF-alpha)-stimulated growth of these MCF-7 cells. An analogous pattern of specific EGF or TGF-alpha growth inhibitory activity was obtained using a synthetic peptide analog encompassing the third disulfide loop region of TGF-alpha, but containing additional modifications designed for increased membrane affinity [( Ac-D-hArg(Et)2(31),Gly32,33]HuTGF-alpha(31-43)NH2). The growth factor antagonism by this synthetic peptide was specific in that it inhibited EGF, TGF-alpha, or estrogen-stimulated growth of MCF-7 cells but did not inhibit insulin-like growth factor-1 (IGF-1)-stimulated cell growth. Altogether, these results suggest that a significant portion of the estrogen-stimulated growth of these MCF-7 cells is mediated in an autocrine/paracrine manner by release of EGF or TGF-alpha-like growth factors. The TGF-alpha peptide likewise inhibited EGF- but not fibroblast growth factor (FGF)- or platelet-derived growth factor (PDGF)-stimulated growth of NIH-3T3 cells in completely defined media; but had no effect on growth or DNA synthesis of G0-arrested cells, nor did it effect growth of NR-6 cells, which are nonresponsive to EGF. Although this synthetic peptide did not directly compete with EGF for cell surface receptor binding, it exhibited binding to a cell surface component (followed by internalization), which likewise was not competed by EGF. The peptide did not directly inhibit EGF-stimulated phosphorylation of the EGF receptor, nor did it inhibit phosphorylation of an exogenous substrate, angiotensin II, by activated EGF receptor. The TGF-alpha peptide did, however, affect the structure of laminin as manifested by laminin self-aggregation; this affect on laminin may, in turn, have a modulatory effect on EGF-mediated cell growth.
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PMID:Inhibition of epidermal growth factor/transforming growth factor-alpha-stimulated cell growth by a synthetic peptide. 253 Feb 43

Two retroviral protein-tyrosine kinases, v-src and v-ros, have been reported to possess phosphatidylinositol (PtdIns) kinase activity. Because the epidermal growth factor (EGF) receptor is a protein-tyrosine kinase with structural homology to p60v-src and because EGF stimulates PtdIns turnover in A431 cells, the EGF receptor has been examined for PtdIns kinase activity. Preparations of the EGF receptor, isolated from A431 cells and purified by two different methods of affinity chromatography, possessed an associated PtdIns kinase activity. This activity which co-purified with the EGF receptor represented only about 2% of the total PtdIns kinase activity of A431 membranes, and there was no correlation between the number of EGF receptors and the amount of PtdIns kinase activity in membranes from various cell types. A peptide substrate, angiotensin II, and PtdIns did not compete with each other as substrates for the protein-tyrosine and PtdIns kinase activities of the EGF receptor. When self-phosphorylated EGF receptor was fractionated by Sephacryl S-300 gel permeation chromatography, the peak of PtdIns kinase activity was separated from the comigrating peak of protein-tyrosine kinase activity and the self-phosphorylated EGF receptor. These results indicate that the protein-tyrosine kinase and PtdIns kinase activities which co-purify with the EGF receptor reside on different molecules. Angiotensin II and PtdIns did not compete as substrates for p60v-src isolated by immunoabsorption with a monoclonal antibody, suggesting that PtdIns kinase activity may also not be intrinsic to p60v-src.
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PMID:Separation and characterization of a phosphatidylinositol kinase activity that co-purifies with the epidermal growth factor receptor. 299 Dec 21

Previous reports have shown that the epidermal growth factor (EGF) receptor is associated with the detergent-insoluble actin cytoskeleton. To assess how this association can influence receptor function, EGF-stimulated protein-tyrosine kinase activity was examined in the detergent-soluble and -insoluble (cytoskeletal) fractions of human A431 epidermoid carcinoma cells. EGF receptor extraction was optimal using 0.15% Triton X-100, and higher detergent concentrations did not significantly increase the amount of solubilized receptor as assessed by immunoblotting. Normalization of EGF-stimulated tyrosine kinase activity on the basis of receptor mass revealed that the specific activity of the cytoskeletal (0.15% Triton-insoluble) fraction is nearly 3-fold greater than that of the soluble receptor when using angiotensin II as the peptide substrate. The increased specific activity of the Triton-insoluble receptor suggests that interaction with the cytoskeleton can facilitate maximal kinase activity. This hypothesis is supported by the observation that, when compared with the soluble EGF receptor, the receptor in the cytoskeletal fraction demonstrates a 15-fold more favorable apparent Michaelis-Menten constant for ATP and a 4-fold more favorable Michaelis-Menten constant for angiotensin II. Although the cytoskeletal EGF receptor seems to represent less than 10% of the total receptor mass in cells not exposed to EGF, these data indicate that it comprises a highly active receptor pool. To examine the regulation of receptor association with the detergent-insoluble fraction, A431 cells were treated at 37 C with EGF for up to 5 h, or with the phorbol ester 12-phorbol 12-myristate 13-acetate for 1 h, and total receptor mass and distribution were determined. In these studies, total immunodetectable receptor decreased significantly after 20 min of EGF administration, whereas the population of Triton-insoluble receptors increased within 40 min to greater than four times that observed before EGF addition and remained at that level for the full 5 h of EGF treatment. Conversely, 12-phorbol 12-myristate 13-acetate treatment, which is known to down-regulate high affinity EGF binding, had little effect on receptor association with the cytoskeletal fraction. In sum, these data indicate the presence of a highly active subpopulation of cytoskeletally associated EGF receptors that can be up-regulated during long-term (5 h) ligand exposure.
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PMID:Modulation of epidermal growth factor receptor interaction with the detergent-insoluble cytoskeleton and its effects on receptor tyrosine kinase activity. 772 Jun 69

The ligand-stimulated tyrosine kinase activity of the normal human epidermal growth factor (EGF) receptor and a truncated EGF receptor lacking 164 carboxy-terminal (C-terminal) amino acids was examined in intact cells and after Triton X-100 extraction into Triton-soluble and -insoluble (cytoskeletal) preparations. Detergent extraction of the intact and truncated receptors appeared complete using 0.3% Triton as demonstrated by anti-EGF receptor immunoblots, tyrosine kinase assays, and marker enzyme (alkaline phosphatase) solubilization. Higher Triton concentrations yielded no additional EGF receptor extraction and began to inhibit EGF-stimulated kinase activity toward angiotensin II (AII). Furthermore, the tyrosine kinase activity of the truncated EGF receptor exhibited increased sensitivity to Triton extraction, suggesting a lower affinity or a more labile association of this receptor with the cytoskeleton. However, both EGF receptor forms had altered catalytic activity when associated with the cytoskeletal fraction, as evidenced by the increased phosphorylation of the exogenous substrates: AII, src-peptide, and [Val5]AII. Kinetic analyses of both receptor types revealed that the cytoskeletal fractions obtained using 0.3% Triton contain EGF receptor activity that exhibits a Michaelis-Menten constant (Km) for AII that is 2- to 3-fold more favorable than that calculated for the soluble receptor forms. EGF treatment of intact cells containing either the intact or truncated receptor revealed similar phosphorylated proteins in the soluble fraction of both cell types, although there was evidence for the enhanced phosphorylation of certain proteins (e.g. 115 and 50 kilodalton proteins) in cells containing the truncated receptor. There was also a greater number of tyrosine-phosphorylated proteins in the Triton-insoluble fraction of cells containing the truncated receptor, suggesting an altered specificity of this receptor toward selected cytoskeletal proteins. This work indicates that EGF receptor-cytoskeletal interaction may be an important consideration in the control of receptor-kinase activity and has examined the detergent sensitivity of this association. These studies also suggest that the C-terminal domain of the EGF receptor may affect cytoskeletal interaction in addition to influencing the receptor's catalytic capacity.
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PMID:Evidence for the potentiation of epidermal growth factor receptor tyrosine kinase activity by association with the detergent-insoluble cellular cytoskeleton: analysis of intact and carboxy-terminally truncated receptors. 824 11

We have recently reported that angiotensin II (Ang II)-induced mitogen-activated protein kinase (MAPK) activation is mainly mediated by Ca2+-dependent activation of a protein tyrosine kinase through Gq-coupled Ang II type 1 receptor in cultured rat vascular smooth muscle cells (VSMC). In the present study, we found Ang II rapidly induced the tyrosine phosphorylation of the epidermal growth factor (EGF) receptor and its association with Shc and Grb2. These reactions were inhibited by the EGF receptor kinase inhibitor, AG1478. The Ang II-induced phosphorylation of the EGF receptor was mimicked by a Ca2+ ionophore and completely inhibited by an intracellular Ca2+ chelator. Thus, AG1478 abolished the MAPK activation induced by Ang II, a Ca2+ ionophore as well as EGF but not by a phorbol ester or platelet-derived growth factor-BB in the VSMC. Moreover, Ang II induced association of EGF receptor with catalytically active c-Src. This reaction was not affected by AG1478. These data indicate that Ang II induces Ca2+-dependent transactivation of the EGF receptor which serves as a scaffold for pre-activated c-Src and for downstream adaptors, leading to MAPK activation in VSMC.
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PMID:Calcium-dependent epidermal growth factor receptor transactivation mediates the angiotensin II-induced mitogen-activated protein kinase activation in vascular smooth muscle cells. 953 70

We have reported that angiotensin II induces the epidermal growth factor (EGF) receptor transactivation leading to extracellular signal-regulated kinase (ERK) activation in rat vascular smooth muscle cells. Here, we report that the EGF receptor kinase inhibitor AG1478 and the ERK kinase inhibitor PD98059 markedly inhibited angiotensin II-induced c-Fos expression and protein synthesis but not c-Jun expression in these cells. These data suggest that the EGF receptor transactivation and subsequent ERK activation are indispensable for angiotensin II-mediated growth promotion of vascular smooth muscle cells providing a new mechanistic insight whereby angiotensin II contributes abnormal vascular remodeling.
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PMID:Epidermal growth factor receptor is indispensable for c-Fos expression and protein synthesis by angiotensin II. 1044 Jan 5

In adrenal glomerulosa cells, the stimulation of aldosterone biosynthesis by angiotensin II (Ang II) involves the activation of a capacitative Ca(2+) influx through calcium release-activated calcium (CRAC) channels. In various mammalian cell systems, it has been shown that CRAC channel activation and Ca(2+) entry require tyrosine kinase activity. We have therefore examined in this work whether similar mechanisms contribute to Ang II-induced mineralocorticoid biosynthesis. In fluo-3-loaded isolated bovine glomerulosa cells, two inhibitors of tyrosine kinases, genistein and methyl-2, 5-dihydroxycinnamate (MDHC) (100 microM) prevented capacitative Ca(2+) entry elicited by Ang II (by 54 and 62% respectively), while the inhibitor of epidermal growth factor (EGF) receptor tyrosine kinase, lavendustin A, was without effect. Similar results were observed on Ca(2+) influx triggered by thapsigargin, an inhibitor of microsomal Ca(2+) pumps. The inhibitors blocked Ang II-stimulated pregnenolone and aldosterone production in the same rank order. In addition to its specific effect on capacitative Ca(2+) influx, genistein also affected the late steps of the steroidogenic pathway, as shown by experiments in which the rate-limiting step (intramitochondrial cholesterol transfer) was bypassed with 25-OH-cholesterol (25-OH-Chol), cytosolic calcium was clamped at stimulated levels or precursors of the late enzymatic steps were supplied. In contrast, genistin, a structural analogue of genistein devoid of tyrosine kinase inhibitory activity, was almost without effect on pregnenolone or 11-deoxycorticosterone (DOC) conversion to aldosterone. These results suggest that, in bovine adrenal glomerulosa cells, Ang II promotes capacitative Ca(2+) influx and aldosterone biosynthesis through tyrosine kinase activation.
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PMID:The role of tyrosine kinases in capacitative calcium influx-mediated aldosterone production in bovine adrenal zona glomerulosa cells. 1049 15


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