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

The rat neu oncogene product is a member of the epidermal growth factor (EGF) receptor subgroup of the superfamily of growth factor receptor tyrosine kinases. The oncogenic activation of the neu protein occurs by a point mutation within its transmembrane region which results in an increase in its tyrosine kinase activity. Using three different forms of neu expressed in insect cells via baculovirus infection, we have examined the biochemical differences between the normal and transforming forms of neu and investigated the role of the transmembrane domain in its tyrosine kinase activity. One form of neu which was expressed in insect cells consisted of the complete tyrosine kinase domain but lacked the extracellular and transmembrane regions (designated NTK). The other two forms consisted of the tyrosine kinase domain, the transmembrane domain, and 40 amino acids of the extracellular domain. One of these transmembrane forms of neu contained the normal valine residue at position 664 within the transmembrane region (MS-N), while the other contained the oncogenic glutamic acid residue at this position (MS-T). Direct comparisons of NTK, MS-N, and MS-T have shown that the NTK protein is capable of the highest extents of both autophosphorylation activity and the tyrosine phosphorylation of exogenous substrate, suggesting that the presence of the transmembrane region of neu suppresses the tyrosine kinase activity of this receptor. In addition, we have found that the oncogenic point mutation within the transmembrane region stimulates the tyrosine kinase activity of the neu protein by allowing it to more effectively utilize Mg2+. Overall, the results of these studies suggest that the valine to glutamic acid substitution at position 664 may at least partially relieve a negative constraint imparted by the membrane-spanning domain on the tyrosine kinase activity of neu and enables a more effective use of Mg2+ in the catalysis of tyrosine phosphorylation of exogenous substrates.
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PMID:Biochemical comparisons of the normal and oncogenic forms of insect cell-expressed neu tyrosine kinases. 135 72

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 activation of the epidermal growth factor (EGF) receptor tyrosine kinase activity is thought to represent a key initial step in EGF-mediated mitogenesis. The mechanisms underlying the regulation of the EGF receptor tyrosine kinase activity were examined through comparisons of the holoreceptor, purified from human placenta, and a soluble 42 kDa tyrosine kinase domain (TKD), generated by the limited trypsin proteolysis of the holoreceptor. The results of these studies highlight the importance of divalent metal ions (Me2+), i.e., Mn2+ and Mg2+, as activators of the tyrosine kinase activity. Manganese is an extremely effective activator of the holoreceptor tyrosine kinase, and under some conditions (low ionic strength) it completely alleviates the need for EGF to stimulate activity. In contrast, Mg2+ only weakly stimulates the holoreceptor tyrosine kinase activity in the absence of EGF, but promotes essentially full activity in the presence of the growth factor. Like the holoreceptor, the soluble TKD is highly active in the presence of Mn2+. However, the isolated TKD is completely inactive in the presence of Mg2+, and, in fact, Mg2+ inhibits the Mn2(+)-stimulated tyrosine kinase activity. The differences in the effects of Mn2+ and Mg2+ on the isolated TKD were further demonstrated by monitoring the effects of Me2+ on the modification of a reactive cysteine residue(s) on the TKD. While Mn2+ potentiates the inhibition by cysteine-directed reagents of the tyrosine kinase activity, Mg2+ has no effect on either the rate or the extent of the inhibition. Both the regulation by Mn2+ of the kinase activity of the TKD and the potentiation by Mn2+ of the cysteine reactivity of the TKD occur over a millimolar concentration range, which implicates a direct binding interaction by the metal ion. Overall, these results demonstrate that there are two key activator sites on the EGF receptor, i.e., the EGF binding site on the extracellular domain and a Me2+ binding site on the cytoplasmic TKD. Me2+ interactions with the cytoplasmic kinase domain apparently result in conformational changes which regulate the levels of tyrosine kinase activity, influence the degree to which this activity is responsive to EGF, and probably account for the effects of Me2+ on the aggregation state of the receptor (Carraway, K.L., III, Koland, J.G. and Cerione, R.A. (1989) J. Biol. Chem. 264, 8699-8707). In general, Mg2(+)-induced conformation changes prime the receptor for activation by EGF, while Mn2+ can fully activate the receptor tyrosine kinase and thereby short-circuit growth factor control.
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PMID:Activation of the EGF receptor tyrosine kinase by divalent metal ions: comparison of holoreceptor and isolated kinase domain properties. 235 10

The specific tyrosine phosphorylation of glucose-6-phosphate dehydrogenase (G6PDH) by the epidermal growth factor (EGF) receptor in vitro is demonstrated. The Km values of the substrate G6PDH and of ATP for the receptor tyrosine kinase were ca. 1 and 10 microM, respectively. The rate of phosphorylation was EGF dependent, with a four-fold increase in Vmax in the presence of EGF. The phosphorylation was stimulated maximally by 0.2 microM or greater EGF, with an ED50 of ca. 20 nM which is consistent with the affinity of the solubilized receptor for EGF. Using conditions of 5 microM G6PDH, 100 microM ATP, 5 mM Mg2+, and 1 mM Mn2+, up to 0.3 mol phosphate was incorporated into 1 mol of the 55-kDa subunit of Baker's yeast G6PDH. Tryptic peptide mapping revealed several unique phosphopeptides for both Baker's yeast and bovine adrenal G6PDH. The patterns of phosphopeptides for a given enzyme were identical for basal and EGF-stimulated phosphorylation.
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PMID:Epidermal growth factor receptor tyrosine kinase phosphorylation of glucose-6-phosphate dehydrogenase in vitro. 312 60

Purified preparations of epidermal growth factor (EGF) receptor were used to test hen oviduct progesterone receptor subunits as substrates for phosphorylation catalyzed by EGF receptor. Both the 80-kilodalton (kDa) (A) and the 105-kDa (B) progesterone receptor subunits were phosphorylated in a reaction that required EGF and EGF receptor. No phosphorylation of progesterone receptor subunits was observed in the absence of EGF receptor, even when Ca2+ was substituted for Mg2+ and Mn2+. Phospho amino acid analysis revealed phosphorylation at tyrosine residues, with no phosphorylation detectable at serine or threonine residues. Two-dimensional maps of phosphopeptides generated from phosphorylated 80- or 105-kDa subunits by tryptic digestion revealed similar patterns, with resolution of two major, several minor, and a number of very minor phosphopeptides. The Km of progesterone receptor for phosphorylation by EGF-activated EGF receptor was 100 nM and the Vmax was 2.5 nmol/min per mg of EGF receptor protein at 0 degrees C. The stoichiometry of phosphorylation/hormone binding for progesterone receptor subunits was 0.31 at ice-bath temperature and approximately 1.0 at 22 degrees C.
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PMID:Progesterone receptor subunits are high-affinity substrates for phosphorylation by epidermal growth factor receptor. 620 Aug 81

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