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 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

To investigate the possible functional role of epidermal growth factor (EGF) receptor-phospholipase C-gamma 1 (PLC-gamma 1) complexes, we have measured PLC-gamma 1 activity in vitro in the absence or presence of purified EGF receptor. Immunoprecipitates of PLC-gamma 1 from control A-431 cells were incubated without or with purified EGF receptor in the absence or presence of ATP. Under these conditions the EGF receptor increased non-tyrosine-phosphorylated PLC-gamma 1 activity 3-4-fold in the absence or presence of ATP, but increased tyrosine-phosphorylated and activated PLC-gamma 1 by only 20-50%. Both basal and autophosphorylated forms of the purified EGF receptor increased the activity of the non-tyrosine-phosphorylated PLC-gamma 1, and stoichiometric levels of purified receptor were required to increase PLC activity. Other tyrosine kinases such as the platelet-derived growth factor receptor and erbB-2, but not the insulin receptor, also stimulated PLC-gamma 1 activity. PLC-gamma 1 activity could be activated with the kinase-negative EGF receptor, but a C-terminal truncated receptor was much less effective. Purified EGF receptor could also activate PLC-beta 1, but with a much decreased potency compared with PLC-gamma 1. Our results suggest that in vitro the EGF receptor can increase PLC-gamma 1 activity independently of tyrosine phosphorylation.
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PMID:Non-catalytic activation of phospholipase C-gamma 1 in vitro by epidermal growth factor receptor. 834 30

The earliest substrates to the transmembrane insulin receptor tyrosine kinase, that would function in insulin signalling, are likely to be associated with the plasma membrane. Rat liver plasma membrane 180,000 M(r) protein (p180) is a substrate to the insulin receptor in vitro [Goren et al. (1990) Cellular Signalling 2, 537-555]. The question as to whether p180 is a substrate in vivo was addressed. Half ml 0.9% NaCl or 500 micrograms insulin was injected into rat livers. Purified plasma membrane glycoproteins from the livers were assayed for in vitro phosphorylation reaction products and endogenous tyrosine-phosphorylated proteins. Membranes from insulin-injected rat livers contained phosphorylated p180 and phosphorylated insulin receptor beta-subunit, whereas saline-injected rat liver membranes contained neither. These data suggested that p180 is an in vivo substrate to the insulin receptor. In vitro p180 is tyrosine-phosphorylated in the absence of insulin. p180, therefore, may be the epidermal growth factor (EGF) receptor or another tyrosine kinase that could be part of a phosphorylation cascade initiated by insulin. Two different experiments suggested that p180 is not the EGF receptor: (i) two-dimensional gel electrophoresis (first dimension--non-equilibrium pH-gradient gel electrophoresis) indicated that p180 is a more basic glycoprotein than EGF receptor; and (ii) based on reverse-phase high pressure liquid chromatography, the tryptic-phosphopeptides of carboxymethyl-Sepharose-purified phosphorylated-p180 were different from those of A431 cell phosphorylated-EGF receptor. Similarly, two different experiments demonstrated that p180 is not a tyrosine kinase: (i) gel-permeation chromatography separated the insulin receptor from p180 and only insulin receptor was autophosphorylated in vitro; and (ii) membrane proteins not bound to immobilized ATP contained p180. Thus, p180 can associate with the insulin receptor and be phosphorylated in vitro and in vivo; however, p180 does not function in an insulin receptor-mediated phosphorylation cascade.
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PMID:Plasma membrane p180, which insulin receptor phosphorylates in vivo, is not a tyrosine kinase. 834 20

In the course of a screening program for tyrosine kinase inhibitors, the chloroform extract of a tropical plant, Desmos chinensis, strongly inhibited the enzyme activity. The active substance was purified by silica gel, gel filtration, and finally crystallized. The structure was elucidated by mass spectrometry and X-ray crystallography to be 8-formyl-2,5,7-trihydroxy-6- methylflavanone, and we named it desmal. Desmal competed with peptide substrate and non-competed with ATP. It inhibited tyrosine kinase in situ in epidermal growth factor (EGF) receptor-overexpressing NIH3T3 (ER12) cells. It also inhibited EGF-induced inositol phosphate formation and morphological changes.
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PMID:Isolation of a novel substrate-competitive tyrosine kinase inhibitor, desmal, from the plant Desmos chinensis. 845 34

The nucleotide binding properties of the epidermal growth factor (EGF) receptor protein-tyrosine kinase were investigated with the fluorescent nucleotide analog 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP). TNP-ATP was found to be an active substrate for the autophosphorylation reaction of the recombinant EGF receptor protein-tyrosine kinase domain (TKD). Whereas the Vmax for the TNP-ATP-dependent autophosphorylation reaction was approximately 200-fold lower than that of ATP, the Km for this reaction was similar to that observed with ATP. The nucleotide analog was also shown to be an inhibitor of the ATP-dependent autophosphorylation and substrate phosphorylation reactions of the TKD. Spectroscopic studies demonstrated both a high affinity binding of TNP-ATP to the recombinant TKD and a markedly enhanced fluorescence of the bound nucleotide analog. The fluorescence of enzyme-bound TNP-ATP was attenuated in the presence of ATP, which enabled determination of the dissociation constants for both ATP and the Mn2+ complex of ATP. A truncated form of the EGF receptor TKD lacking the C-terminal autophosphorylation domain exhibited an enhanced affinity for TNP-ATP, which indicated that the autophosphorylation domain occupied the peptide substrate binding site of the TKD and modulated the binding of the nucleotide substrates.
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PMID:Nucleotide binding by the epidermal growth factor receptor protein-tyrosine kinase. Trinitrophenyl-ATP as a spectroscopic probe. 855 May 78

We have found that the epidermal growth factor (EGF) receptor kinase can utilize the fluorescent ATP derivative, methylanthraniloyl ATP, as a substrate. On the basis of this observation, together with our previous studies that showed that 5'-(p-fluorosulfonylbenzoyl)adenosine (5'-FSBAdo) is a highly specific affinity label for the ATP site of the kinase domain of the EGF receptor, we prepared new derivatives of 5'-FSBAdo, 5'-(p-fluorosulfonyl)-2'(or 3')-(methylanthraniloyl)adenosine (FSBMantAdo), as fluorescent affinity labels for adenine nucleotide binding sites, and in particular for the ATP site of the EGF receptor. The two products were purified by HPLC and were characterized by UV-Vis absorbance spectroscopy, mass spectrometry, nuclear magnetic resonance spectroscopy, and fluorescence spectroscopy. Incubation of membrane vesicles containing the EGF receptor with either the 2' or 3' derivative resulted in irreversible inhibition of the receptor kinase activity, as assessed by autophosphorylation assays. Preincubation of vesicles with AMP imidodiphosphate (AMPPNP), a hydrolysis-resistant ATP analog, prior to treatment with FSBMantAdo resulted in the protection of the receptor kinase activity' from FSBMantAdo inactivation. Steady state fluorescence spectra (with excitation at 360 nm) revealed a blue shift in the emission maximum of partially purified FSBMantAdo-labeled receptor (426 nm), as compared with the emission maximum of free FSBMantAdo (441 nm) in aqueous solution, suggesting that the receptor-bound label is in a relatively low polarity environment. These studies show that FSBMantAdo is a specific affinity label for the ATP site of the EGF receptor. FSBMantAdo may also prove useful as a fluorescent affinity label for other ATP binding sites.
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PMID:5'-(p-fluorosulfonylbenzoyl)-2'(or 3')-(methylanthraniloyl)adenosine, fluorescent affinity labels for adenine nucleotide binding sites: interaction with the kinase active site of the receptor for epidermal growth factor. 870 25

We report the use of structure-based drug design to create a selective erbB-1 (a.k.a. epidermal growth factor receptor) and erbB-2 (a.k.a. neu/her2 growth factor receptor) tyrosine kinase inhibitor. Using the X-ray crystal structure of the ternary complex of the cAMP-dependent Ser/Thr kinase together with a sequence alignment of the catalytic domains of a representative set of Ser/Thr and Tyr protein kinases, we have examined the nucleotide binding site for potential positions to attach an irreversible inhibitor. This information, combined with homology modeling of the erbB-1 and erbB-2 tyrosine kinase catalytic domains, has led to the identification of Cys797 of erbB1 and Cys805 of erbB2, which are structurally equivalent to Glu127 in the cAMP dependant Ser/Thr kinase as potential target residues. The X-ray structure of the cAMP Ser/Thr kinase shows Glu127 to be involved in a hydrogen-bonding interaction with the 2'-OH of the ribose portion of ATP. Using molecular modeling, it was predicted that the Cys side chains in erbB-1 and erbB-2 performed an analogous role, and it was postulated that the replacement of the 2'-OH of adenosine with a thiol might allow for a covalent bond to form. Since only erbB-1 and erbB-2 have a Cys at this position, the inhibitor should be selective. This model was subsequently tested experimentally by chemical synthesis of 2'-thioadenosine and assayed against the full length erbB-1 receptor and the catalytic domains of erbB-2, insulin receptor, beta-PDGF receptor, and the FGF receptor. Our results show that thioadenosine covalently inactivates erbB-1 with a second-order rate constant of k(max)/K(S) = 2000 +/- 500 M(-1) s(-1). Inactivation is fully reversed by 1 mM dithiothreitol, suggesting that inactivation involves the modification of a cysteine residue at the active site, presumably Cys797. The rate of inactivation saturates with increasing thioadenosine concentrations, suggesting that inactivation occurs through initial formation of a noncovalent complex with K(D) = 1.0 +/- 0.3 microM, followed by the slow formation of a disulfide bond with a rate constant of k(max) = (2.3 +/- 0.2) x 10(-3) s(-1). This approach may have application in the design of selective irreversible inhibitors against other members of the kinase family.
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PMID:Structure-based design of a potent, selective, and irreversible inhibitor of the catalytic domain of the erbB receptor subfamily of protein tyrosine kinases. 908 34

PD 089828, a novel protein tyrosine kinase inhibitor of a new structural class, the 6-aryl-pyrido-[2,3-d]pyrimidines, was identified by screening a compound library with assays that measured protein tyrosine kinase activity. PD 089828 was found to inhibit human full-length fibroblast growth factor (FGF) receptor-1 (FGFR-1), platelet-derived growth factor (PDGF) receptor beta subunit (PDGFR-beta), Src nonreceptor tyrosine kinase (c-Src) and epidermal growth factor (EGF) receptor (EGFR) tyrosine kinases with half-maximal inhibitory potencies (IC50 values) of 0.15 +/- 0.02 (n = 4), 0.18 +/- 0.04 (n = 3), 1.76 +/- 0.28 (n = 4) and 5.47 +/- 0.78 (n = 6) microM, respectively. PD 089828 was further characterized as an ATP competitive inhibitor of the growth factor receptor tyrosine kinases (FGFR-1, PDGFR-beta and EGFR) but a noncompetitive inhibitor of c-Src tyrosine kinase with respect to ATP. In addition, PD 089828 inhibited PDGF- and EGF-stimulated receptor autophosphorylation in vascular SMC (VSMC) and basic FGF-mediated tyrosine phosphorylation in A121 cells with IC50 values similar to the potencies observed for inhibition of receptor tyrosine kinase activity. The inhibition of PDGF receptor autophosphorylation in VSMC by PD 089828 occurred rapidly, with maximal effects reached within 5 min of drug exposure. Inhibition after single exposure was long lasting but also rapidly reversible, occurring within 5 min after drug removal. The PDGF-induced association of downstream signaling proteins, including phosphoinositide-3-kinase (PI-3K), growth factor receptor binding protein-2 (GRB2), SH-2 domain and collagen like (Shc) and phospholipase Cgamma (PLCgamma), with VSMC PDGF receptors was also blocked as a result of the inhibition of PDGF-stimulated receptor autophosphorylation by PD 089828. PD 089828 also inhibited the PDGF-induced tyrosine phosphorylation of the 44- and 42-kDa mitogen-activated protein kinase isoforms. Moreover, the effects of PD 089828 were demonstrated in functional assays in which PDGF-stimulated DNA synthesis, PDGF-directed migration and serum-stimulated growth of VSMC were all inhibited to the same extent as PDGF receptor autophosphorylation (IC50 = 0.8, 4.5 and 1.8 microM, respectively). These results highlight the biological characteristics of PD 089828 as a novel, broadly active protein tyrosine kinase inhibitor with long-lasting but reversible cellular effects. The potential therapeutic use of these broadly acting, nonselective inhibitors as antiproliferative and antimigratory agents could extend to such diseases as cancer, atherosclerosis and restenosis in which redundancies in growth-signaling pathways are known to exist.
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PMID:Inhibition of growth factor-mediated tyrosine phosphorylation in vascular smooth muscle by PD 089828, a new synthetic protein tyrosine kinase inhibitor. 919 Aug 82

Earlier reports have indicated that epidermal growth factor (EGF) receptor autophosphorylation, thought to be a key step in receptor transmembrane signaling, can be inactivated with the relatively sulfhydryl-specific reagent N-ethylmaleimide (NEM); however, no Cys residue has been implicated in the catalytic mechanism of the kinase. In an effort to address the mechanism of inhibition by NEM, we have investigated effects of several sulfhydryl-modifying reagents on EGF receptor autophosphorylation and on the kinase activity of the receptor toward an exogenous peptide substrate. Kinase activity is relatively insensitive to iodoacetic acid (IAAcid) and iodoacetamide (IAAmide), though IAAmide-treated receptor displays a higher Km(app) with respect to ATP, relative to untreated receptor. In contrast, even low concentrations of the very specific sulfhydryl reagent p-chloromercuribenzoic acid (PCMB) inactivate the receptor kinase. Pretreatment of the receptor with IAAmide, but not IAAcid, provides substantial protection of the kinase from subsequent treatment with NEM and a degree of protection from subsequent treatment with PCMB. Further, inactivation by NEM, and to a lesser extent PCMB, is inhibited by coincubation of the receptor with the hydrolysis-resistant ATP analog AMP-PNP. The protective effect of IAAmide from inactivation by NEM is also lost when AMP-PNP is present during the IAAmide treatment. Pretreatment of receptor with IAAcid has no effect on subsequent modification by IAAmide. These results, taken together, suggest that NEM, PCMB, and IAAmide, but not IAAcid, modify a Cys residue of the EGF receptor kinase that is inaccessible when nucleotide is bound. Modification of this residue by a bulky reagent (NEM, PCMB) inactivates the kinase by a steric mechanism, while modification with the smaller reagent (IAAmide) results in an active enzyme with altered affinity for ATP. Further, PCMB appears to react with an additional Cys residue (or residues), also resulting in steric inactivation.
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PMID:Effects of sulfhydryl modification reagents on the kinase activity of the epidermal growth factor receptor. 924 24

The tyrosine kinase inhibitors PD 69896, 153717, and 158780, which belong to the chemical class 4-[ar(alk)ylamino]pyridopyrimidines, have been characterized with respect to enzymology, target specificity, and antiproliferative effects in tumor cells. These compounds were competitive inhibitors with respect to ATP against purified epidermal growth factor (EGF) receptor tyrosine kinase and inhibited EGF receptor autophosphorylation in A431 human epidermoid carcinoma with IC50 values of 2085, 110, and 13 nM, respectively. Onset of inhibition was immediate once cells were exposed to these compounds, whereas recovery of receptor autophosphorylation activity after the cells were washed free of the compound was dependent on inhibitory potency. Thus, full activity returned immediately after removal of PD 69896 but required 8 hr after exposure to PD 158780. PD 158780 was highly specific for the EGF receptor in Swiss 3T3 fibroblasts, inhibiting EGF-dependent receptor autophosphorylation and thymidine incorporation at low nanomolar concentrations while requiring micromolar levels for platelet-derived growth factor- and basic fibroblast growth factor-dependent processes. PD 158780 inhibited heregulin-stimulated phosphorylation in the SK-BR-3 and MDA-MB-453 breast carcinomas with IC50 values of 49 and 52 nM, respectively, suggesting that the compound was active against other members of the EGF receptor family. The antiproliferative effects of this series of compounds against A431 cells correlated precisely with the inhibitory potency against EGF receptor autophosphorylation. PD 158780 reduced clone formation in soft agar of fibroblasts transformed by EGF, EGF receptor, or the neu oncogene but not ras or raf, further demonstrating its high degree of specificity. Finally, this compound was active against clone formation in several breast tumors having different expression patterns of the erbB family, indicating an anticancer utility in tumors expressing these receptors.
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PMID:Biochemical and antiproliferative properties of 4-[ar(alk)ylamino]pyridopyrimidines, a new chemical class of potent and specific epidermal growth factor receptor tyrosine kinase inhibitor. 935 88


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