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 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

The mechanism by which the protein kinase activity of the epidermal growth factor (EGF) receptor is activated by binding of growth factor was investigated. Detergent-solubilized receptor in monomeric form was isolated by sucrose density gradient centrifugation and both its kinase and autophosphorylation activities monitored. In a low ionic strength medium and with MnCl2 as an activator, the activity of the monomeric receptor was EGF-independent. However, with 0.25 M ammonium sulfate present, the MnCl2-stimulated kinase activity was strikingly EGF-dependent. In contrast, the kinase activity expressed in the presence of MgCl2 showed growth factor control in the absence of added salt. Under the conditions of these experiments there was apparently little tendency for growth factor to induce aggregation of the receptor, indicating that the allosteric activation of the receptor kinase by EGF occurred via an intramolecular mechanism. Whereas detergent-solubilized receptor was the subject of these studies, the kinase activity of cell surface receptors might also be controlled by an intramolecular mechanism. These results indicate that an individual receptor molecule has the potential to function as a transmembrane signal transducer.
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PMID:Growth factor control of epidermal growth factor receptor kinase activity via an intramolecular mechanism. 325 89

The physiochemical properties of the purified cytoplasmic domain of the epidermal growth factor (EGF) receptor, its self-phosphorylation and peptide phosphorylation activities, and its activation by ammonium sulphate have been studied. Highly efficient purification procedures for the isolation of the recombinant cytoplasmic domain (Met644-Ala1186) of the EGF receptor, expressed in the baculovirus/insect cell system, are described. Physicochemical characterization of the protein included investigation of its isoelectric and hydrodynamic properties, stability, oligomeric status, and secondary structure using far-u.v. circular dichroism. The recombinant protein was not recognized by anti-phosphotyrosine antibodies, unless first self-phosphorylated in vitro. Tryptic phosphopeptide maps of self-phosphorylated recombinant cytoplasmic domain and the EGF-stimulated A431-membrane receptor were very similar, suggesting that the recombinant had similar self-phosphorylation capacity and specificity. The preparations were characterized by high specific activity towards peptide tyrosine phosphorylation. Although the cytoplasmic domain was isolated as a homogeneously monomeric protein, storage at 4 degrees C led to slow, spontaneous aggregation with reduction in specific activity. Both high activity and monomeric state were maintained by storage below 0 degree C. The dependence of the initial rate of self-phosphorylation on protein concentration was consistent with cross-phosphorylation but not with the known oligomerization-induced activation of holoreceptor. The peptide phosphorylation activity was stimulated by Mn2+, Mg2+ and (NH4)2SO4 at high concentrations. The substrate specificity of (NH4)2SO4 activation was studied using synthetic peptides. Self-phosphorylation was inhibited by (NH4)2SO4 in the range 0-0.25 M but activated at 1.0-1.5 M, possibly as a result of ionic and hydrophobic protein interactions respectively. Phosphopeptide maps of cytoplasmic domain phosphorylated in the presence of high (NH4)2SO4 showed that the protein was more extensively phosphorylated than in the absence of salt, or than the native receptor. Far-u.v. circular-dichroism spectra of the cytoplasmic domain changed dramatically at 1 M (NH4)2SO4, raising the possibility that (NH4)2SO4 activates the kinase catalytic domain by inducing conformational changes.
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PMID:Physicochemical characterization of the cytoplasmic domain of the epidermal growth factor receptor and evidence for conformational changes associated with its activation by ammonium sulphate. 770 58

Activation of several protein kinases is mediated, at least in part, by phosphorylation of conserved Thr or Tyr residues located in a variable loop region, near the active site. In certain kinases, this activation loop also controls access of peptide substrates to the active site. In the corresponding region of the epidermal growth factor (EGF) receptor, a potential phosphorylation site, Tyr-845, does not appear to have a major regulatory role. In order to find out whether this variable loop can modulate the peptide phosphorylation and self-phosphorylation activities of the EGF receptor kinase, we investigated the role of residues around Tyr-845, using site-directed mutagenesis. Multiple sequence alignment showed that residues Glu-842, Glu-844 and His-846 are conserved or nearly conserved in eight members of the EGF receptor family. Mutants Glu-842-->Ser, Glu-844-->Gln and His-846-->Ala were expressed in the baculovirus/insect cell system, purified to near-homogeneity and characterized with respect to their peptide phosphorylation and self-phosphorylation activities. All three mutants were active, and these changes did not affect ATP binding directly. However, all mutations increased the Km(app.) for peptide substrates and MnATP in peptide phosphorylation reactions. The Vmax. for the phosphorylation of peptide RREELQDDYEDD was unaltered, but the Vmax. for self-phosphorylation (with variable [MnATP]) decreased 4-, 2- and 7-fold for mutants Glu-842-->Ser, Glu-844-->Gln and His-846-->Ala respectively, compared with the wild-type. These results suggest that binding of this peptide restored an optimal conformation at the active site that might be impaired by the mutations. A study of the dependence of initial rates of self-phosphorylation on cytoplasmic domain concentration showed that the order of reaction increased with the progress of self-phosphorylation. Both pre-phosphorylation and high concentrations of ammonium sulphate restored maximal or near-maximal levels of self-phosphorylation in the mutants, possibly through compensating conformational changes. A plausible homology model, based on the cyclic AMP-dependent protein kinase catalytic subunit, accommodated the sequence Glu-841-Glu-Lys-Glu as an insertion in the peptide binding loop at the edge of the active site cleft. The model suggests that Glu-844 and His-846 may participate in H-bonding interactions, thus stabilizing the active site region, while Glu-842 does not appear to interact with regions of the catalytic core.
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PMID:An investigation of the role of Glu-842, Glu-844 and His-846 in the function of the cytoplasmic domain of the epidermal growth factor receptor. 775 68

The effect of the pineal indole hormone melatonin on the life span of mice, rats and fruit flies has been studied using various approaches. It has been observed that in female CBA, SHR, SAM and transgenic HER-2/neu mice long-term administration of melatonin was followed by an increase in the mean life span. In rats, melatonin treatment increased survival of male and female rats. In D. melanogaster, supplementation of melatonin to nutrient medium during developmental stages produced contradictory results, but and increase in the longevity of fruit flies has been observed when melatonin was added to food throughout the life span. In mice and rats, melatonin is a potent antioxidant both in vitro and in vivo. Melatonin alone turned out neither toxic nor mutagenic in the Ames test and revealed clastogenic activity at high concentration in the COMET assay. Melatonin has inhibited mutagenesis and clastogenic effect of a number of indirect chemical mutagens. Melatonin inhibits the development of spontaneous and 7-12-dimethlbenz(a)anthracene (DMBA)- or N-nitrosomethylurea-induced mammary carcinogenesis in rodents; colon carcinogenesis induced by 1,2-dimethylhydrazine in rats, N-diethylnitrosamine-induced hepatocarcinogenesis in rats, DMBA-induced carcinogenesis of the uterine cervix and vagina in mice; benzo(a)pyrene-induced soft tissue carcinogenesis and lung carcinogenesis induced by urethan in mice. To identify molecular events regulated by melatonin, gene expression profiles were studied in the heart and brain of melatonin-treated CBA mice using cDNA gene expression arrays (15,247 and 16,897 cDNA clone sets, respectively). It was shown that genes controlling the cell cycle, cell/organism defense, protein expression and transport are the primary effectors for melatonin. Melatonin also increased the expression of some mitochondrial genes (16S, cytochrome c oxidases 1 and 3 (COX1 and COX3), and NADH dehydrogenases 1 and 4 (ND1 and ND4)), which agrees with its ability to inhibit free radical processes. Of great interest is the effect of melatonin upon the expression of a large number of genes related to calcium exchange, such as Cul5, Dcamkl1 and Kcnn4; a significant effect of melatonin on the expression of some oncogenesis-related genes was also detected. Thus, we believe that melatonin may be used for the prevention of premature aging and carcinogenesis.
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PMID:Melatonin as antioxidant, geroprotector and anticarcinogen. 1667 84