Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q9UL75 (A431)
 5,640 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Epidermal growth factor (EGF) receptor protein has been purified in a single high-yield step by immunoaffinity chromatography of extracts of A431 cells. A monoclonal antibody directed against the EGF binding site of the receptor was immobilized to Sepharose 4B as a specific immune absorbent and competitive elution with EGF was used to obtain purified EGF receptor protein with tyrosine kinase activity. The stoichiometry of EGF binding was determined by comparing 125I-EGF binding to A431 cells with the mass of EGF receptor protein in those cells as measured by immunoaffinity chromatography, radioimmunoassay, and immune precipitation. Each measurement indicated one EGF binding site/EGF receptor protein molecule. Study of the kinetics of autophosphorylation revealed rapid incorporation of 1 mol of phosphate/mol of enzyme followed by slower incorporation of additional phosphate groups. The autophosphorylation reaction has a Km for ATP (0.2 microM) which is about 10-fold lower than that for phosphorylation of exogenous substrates. The kinetically preferred autophosphorylation is an intramolecular reaction.
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PMID:Immunoaffinity purification of the epidermal growth factor receptor. Stoichiometry of binding and kinetics of self-phosphorylation. 609 67

Protein kinases that phosphorylate the hydroxyl group of tyrosine residues of proteins have been implicated in cell transformation by some retroviruses and in regulation of normal cell growth by some polypeptide growth factors. To facilitate the identification of tyrosine kinase substrates, we developed monoclonal antibodies to the hapten azobenzylphosphonate. One of these antibodies, MA-2G8, proved to be especially attractive in that it bound a derivative of aminophenylphosphate, a close phosphotyrosine analog, with higher affinity than it bound the corresponding derivative of aminobenzylphosphonate; however, its affinity for phosphoserine was negligible. In this paper we describe the optimal conditions for using this antibody to isolate phosphotyrosine proteins, emphasizing particularly that its interaction with phosphotyrosyl proteins is sensitive to ionic detergents and to antibody density on the immunosorbent matrix. The antibody also bound ATP citrate lyase; this enzyme lacks phosphotyrosine but contains phosphohistidine, which is similar structurally to phosphotyrosine. By attaching the antibody at high density to Sepharose beads and omitting ionic detergents from the buffers, it was possible by microbatch immunoadsorption (followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to isolate the 120,000-dalton transforming protein and several other phosphotyrosyl proteins from cells transformed by Abelson murine leukemia virus. Under the same conditions, phosphotyrosyl proteins were also isolated from human epidermal carcinoma cells (A431) that had been stimulated with epidermal growth factor; most prominent among these proteins was the 170,000-dalton receptor for epidermal growth factor.
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PMID:Characterization and use of monoclonal antibodies for isolation of phosphotyrosyl proteins from retrovirus-transformed cells and growth factor-stimulated cells. 619 25

Growth of a selected variant of A431 cells (clone 29) is stimulated by epidermal growth factor (EGF) in contrast to the growth inhibition caused by EGF in an unselected clone, A431(8). Twelve phosphoproteins from each clone were compared to determine whether unique EGF-dependent substrate phosphorylations might explain the cells' differing growth responses to EGF. Treatment of both clone 29 and A431(8) cells with EGF increased phosphorylation of the EGF receptor/kinase and six cellular proteins identified on 2-dimensional polyacrylamide gels. Four of these proteins (the EGF receptor/kinase and proteins of 36, 70, and 81 kd) contained phosphotyrosine in both clone 29 and A431(8) cells, indicating that the same modification of several proteins occurred in cells which have totally different growth responses to EGF. Two proteins were identified whose phosphorylation was EGF dependent and which were unique to clone 29 cells; however, EGF increased phosphorylation of only serine residues in these proteins. This indicates that these proteins are not primary targets of the EGF-dependent tyrosine-specific protein kinase, but rather are substrates for serine-specific kinase(s) activated as a consequence of EGF:receptor interaction. cAMP, which inhibited growth of both clones, was utilized to compare the effects of EGF when the growth response of both cell lines was similar. In the presence of cAMP, EGF increased A431(8) cellular phosphotyrosine content and the phosphorylation of the same phosphotyrosine-containing proteins of both clone 29 and A431(8) cells. The in vivo activity of a second tyrosine-specific protein kinase, p60V -src in B77 Rous sarcoma virus (RSV)-transformed newborn rat kidney (NRK) cells, was also unaffected by cAMP. Thus cAMP did not block the in vivo activity of two tyrosine-specific kinases or the tyrosine phosphorylation of three specific protein substrates. A threshold model of tyrosine kinase activity is proposed as an alternative explanation for the differing growth responses to EGF.
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PMID:Comparison of protein phosphorylations in variant A431 cells with different growth responses to epidermal growth factor. 620 5

The transforming proteins (p21) of Harvey and Kirsten sarcoma viruses threonine kinase activity, which phosphorylates threonine 59 of the p21 proteins themselves. A tridecapeptide: Arg-Arg-Leu56-Asp-Thr-Thr59-Gly-Gln-Glu-Tyr-Ser-Ala66 containing residues 56-66 of p21 is phosphorylated solely on tyrosine by the epidermal growth factor (EGF)-stimulated tyrosine kinase of A431 cell membranes. Km-Values of 240 and 80 microM and Vmax values of 1.7 and 0.1 nmol.min-1.mg-1 were obtained in the presence and absence of EGF, respectively.
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PMID:A synthetic peptide containing the autophosphorylation site of the transforming protein of Harvey sarcoma virus is phosphorylated by the EGF-stimulated tyrosine kinase. 631 15

The Ca2+- and phospholipid-dependent protein kinase (C-kinase) binds tightly in the presence of Ca2+ to purified membranes of A431 human epidermoid carcinoma cells. The major membrane substrate for C-kinase is the epidermal growth factor (EGF) receptor. Phosphorylation of the EGF receptor is Ca2+-dependent and occurs at threonine and serine residues. After tryptic digestion of the receptor, three major phosphothreonine-containing peptides were identified. These are identical with three new phosphopeptides present in the EGF receptor isolated from A431 cells treated with either of the tumor promoters 12-O-tetradecanoylphorbol 13-acetate or teleocidin. C-kinase catalyzes phosphorylation at these same sites in purified EGF receptor protein. These results indicate that, in A431 cells exposed to tumor promoters, C-kinase catalyzes phosphorylation of a significant population of EGF receptor molecules. This phosphorylation of EGF receptors results in decreased self-phosphorylation of the EGF receptor at tyrosine residues both in vivo and in vitro and in decreased EGF-stimulated tyrosine kinase activity in vivo.
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PMID:C-kinase phosphorylates the epidermal growth factor receptor and reduces its epidermal growth factor-stimulated tyrosine protein kinase activity. 632 73

Four mouse monoclonal antibodies specific for human epidermal growth factor (EGF) receptors have been prepared using EGF receptor protein from human A431 epidermoid carcinoma cells as immunogen. We have determined the effect of these antibodies on two known functions of the EGF receptor: EGF binding and tyrosine kinase. Three of these antibodies (225, 528, and 579) are inhibitors of EGF binding, whereas the fourth (455) does not compete for binding but immunoprecipitates the EGF receptor. Inhibition is of the mixed competitive and noncompetitive type. The three competing monoclonal antibodies are antagonists of EGF-stimulated tyrosine protein kinase activity assayed both in intact cells and using an exogenous peptide substrate in solubilized membranes. These immunoglobulins are partial agonists in self-phosphorylation of the EGF receptor in solubilized membranes but exhibit only antagonist activity for this reaction in intact cells. The three competing monoclonal immunoglobulins recognize receptors in variant A431 cells with the same efficiency as in parental A431 cells. Such antagonist monoclonal antibodies can be used to control the concentration of receptors which can be activated by EGF.
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PMID:Monoclonal anti-epidermal growth factor receptor antibodies which are inhibitors of epidermal growth factor binding and antagonists of epidermal growth factor binding and antagonists of epidermal growth factor-stimulated tyrosine protein kinase activity. 633 79

Tyrosine phosphorylation seems to be a key event in the control of cellular growth. Several viral transforming proteins, including the src protein of Rous sarcoma virus, the p120 protein of Abelson leukaemia virus and the middle T antigen of polyoma virus, are phosphorylated by associated tyrosine kinases. The levels of kinase activity correlate with the transforming efficiency of the virus. The receptors for epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and insulin are also phosphorylated by associated tyrosine kinase activities, which are stimulated by EGF, PDGF and insulin, respectively. The EGF-stimulated kinase and the src protein share similar substrate specificity for tyrosines immediately C-terminal to a sequence of acidic amino acids. Such a sequence is also found adjacent to the phosphotyrosine of middle T antigen, and in the homologous region of the hormone gastrin, adjacent to a tyrosine which is sulphated in approximately half the gastrin isolated from gastric mucosa. Reports that gastrin acts as a growth factor for cells of the gastrointestinal tract suggested that phosphorylation of this tyrosine might be physiologically more relevant than sulphation. We report here that synthetic human gastrin 17 is phosphorylated by the EGF-stimulated tyrosine kinase of A431 cell membranes. The Km values of 53-87 and 223-547 microM obtained in the presence and absence of EGF, respectively, are the lowest reported so far for this enzyme.
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PMID:Phosphorylation of gastrin-17 by epidermal growth factor-stimulated tyrosine kinase. 660 May 11

In the present study, we have demonstrated that human growth hormone (hGH) can be phosphorylated by the epidermal growth factor (EGF)-stimulated tyrosine kinase of A431 cell membranes. Phosphotyrosine was the predominant phosphoamino acid released from phosphorylated hGH on partial acid hydrolysis. All five tyrosine-containing tryptic peptides of hGH are also phosphorylated by the EGF-stimulated tyrosine kinase. The highest phosphate incorporation was found for peptide T4 (residues 20-38), which is distinguished by a high frequency of acidic amino acids. The phosphorylated peptides have been characterized by HPLC and two-dimensional mapping on paper. Comparison with the labeled peptides obtained on tryptic digestion of phosphorylated hGH suggests that tyrosine phosphorylation is restricted to two tryptic peptides, T4 (tyrosine-28 or -35) and T6 (tyrosine-42). It is suggested that the absence of early insulin-like activity in the naturally occurring Mr 20,000 variant of hGH, which has an internal deletion spanning residues 32-46, may be a consequence of the loss of the tyrosine phosphorylation sites at residues 35 and 42.
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PMID:Phosphorylation of human growth hormone by the epidermal growth factor-stimulated tyrosine kinase. 660 72

Epidermal growth factor (EGF) increases 12-lipoxygenase mRNA by about 2-fold with a lag period of 4 to 8 hr, which precedes the increase in 12-lipoxygenase activity by 2 to 4 hr in human epidermoid carcinoma A431 cells. Induction of 12-lipoxygenase expression in human erythroleukemia cells by phorbol 12-myristate 13-acetate (PMA) has been reported previously. The present report describes a study of the involvement of protein kinase C (PKC) in EGF-induced 12-lipoxygenase expression in A431 cells. EGF-induced 12-lipoxygenase expression was inhibited by methyl 2,5-dihydroxycinnamate, a tyrosine kinase inhibitor. Staurosporine and calphostin C, which are two PKC inhibitors, inhibited EGF-induced enzyme activity and mRNA expression of 12-lipoxygenase. 1,2-Dioctanoyl-sn-glycerol (a membrane-permeant diacylglycerol) and PMA significantly induced enzyme activity and mRNA expression. Simultaneous treatment of cells with EGF and PMA did not exhibit an additive effect, suggesting that EGF and PMA share a common biochemical pathway in 12-lipoxygenase induction. Expression of mRNA for PKC alpha, delta and zeta was detected in A431 cells, whereas no mRNA expression for PKC beta 1, gamma and epsilon was observed. Taken together, these results suggest that EGF-induced 12-lipoxygenase expression is at least in part mediated by the PKC signal transduction pathway.
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PMID:Induction of 12-lipoxygenase expression by epidermal growth factor is mediated by protein kinase C in A431 cells. 752 31

We describe nonspecific and moderate activation of cellular protein tyrosine phosphorylation by a chemical compound, vesnarinone, which results in enhanced synthesis and/or assembly of cytoskeletal proteins and morphological alterations in several transformed cells. In A431 cells, vesnarinone induced tyrosine phosphorylation of the overexpressed epidermal growth factor receptors (EGFR) as well as other unidentified proteins, increased the synthesis of cytokeratins, and caused amplification of the intermediate filament networks and cell flattening. The drug effects were abolished by tyrphostin, a protein tyrosine kinase inhibitor. Two other cell lines responded to the drug with increased synthesis of a cell type-specific cytoskeletal protein: vimentin in QG56 human lung carcinoma cells and alpha-tubulin in NIH3T3 cells transformed with v-src. In all cell lines tested, the drug-induced tyrosine phosphorylation was localized in cell-cell and cell-substrate contacts as detected by immunofluorescent staining. Responsive protein substrates and their sensitivity to the drug varied from one cell line to another as observed by immunoblot analysis. Vesnarinone exerted neither activating nor inhibitory effect on in vitro enzyme reactions including EGFR tyrosine kinase, v-src kinase, and protein tyrosine phosphatases. This suggests that vesnarinone indirectly activates tyrosine phosphorylation of membrane proteins related to cell adhesion, which influences a signaling pathway linked to the stress fiber assembly in certain cell lines. The possible mechanism by which vesnarinone induces the cellular responses is discussed.
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PMID:Alterations of the cytoskeletal organization in tumor cell lines by a cardiotonic drug, vesnarinone, through protein tyrosine phosphorylation. 754 53


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