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)

gamma RF-1 is a recently identified transcription factor induced by interferon-gamma (IFN-gamma) which binds to a unique palindromic enhancer, gamma RE-1, in the promoter of the mig gene. This paper describes the ligand-dependent and ligand-independent activation of gamma RF-1 in a cell-free system. gamma RF-1 activity was induced by IFN-gamma in a time-dependent manner from 5 to 60 min in lysates prepared from the human monocytic leukaemia line THP-1 and the human epidermoid carcinoma line A431. The activation of gamma RF-1 in vitro required both ATP and an inhibitor of tyrosine phosphatases (sodium orthovanadate or pervanadate). In the presence of limiting concentrations (micromolar) of ATP, activation was also dependent upon stimulation with IFN-gamma, whereas at millimolar concentrations of ATP, gamma RF-1 was activated by either sodium orthovanadate or pervanadate in the absence of ligand. Based on cell fractionation studies, both membrane and cytosol components were essential for activation of gamma RF-1 in vitro. Consistent with a role for one or more tyrosine kinases in the activation of gamma RF-1, its DNA binding activity was blocked by monoclonal anti-phosphotyrosine antibodies and by the tyrosine kinase inhibitors genistein, lavendustin A and herbimycin A. A comparison with recently described pathways of IFN-mediated transcription factor regulation indicates that the in vitro activation of gamma RF-1 is unique, requiring both membrane and cytosol fractions and inhibition of endogenous tyrosine phosphatase activity.
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PMID:Ligand-dependent and -independent activation of the transcription factor gamma RF-1 in a cell-free system. 754 74

The generation of oxygen-derived free radicals has been implicated in the disordered vascular regulation of inflammation and reperfusion. In the vasculature, oxygen-derived free radicals are vasodilatory. The mechanisms underlying this effect remain unclear. To examine the cellular processes involved, we studied the effects of hydrogen peroxide (H2O2) on adenylyl cyclase activity in A10 cells, a murine vascular smooth muscle cell line. Pretreatment with H2O2 caused a dose-dependent enhancement of forskolin-stimulated adenylyl cyclase activity (ED50, 44 mumol/L to a maximum of 166% of control activity; n = 4). This enhancement was attenuated by iron chelation with deferoxamine and by the intracellular hydroxyl scavenger dimethylthiourea and mimicked by preincubation with purine/xanthine oxidase either alone or in the presence of superoxide dismutase. The effects of H2O2 were completely blocked by the tyrosine kinase inhibitors genistein and tyrphostin A9 but not by its inactive analogue tyrphostin A1 (H2O2 alone, 149 +/- 13%; H2O2 + tyrphostin A9, 100 +/- 9%; H2O2 + tyrphostin A1, 171 +/- 21%; n = 4). H2O2 comparably enhanced adenylyl cyclase activity stimulated by isoproterenol (166 +/- 17% of control, n = 5) and sodium fluoride (177 +/- 18% of control, n = 5). Thus oxygen-derived free radicals enhance adenylyl cyclase activation, probably via tyrosine kinase-mediated effects on the catalytic subunit of adenylyl cyclase. Sensitization of adenylyl cyclase activation may be an important mechanism by which free radicals modulate hormone-mediated vasodilation.
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PMID:Oxidant stress enhances adenylyl cyclase activation. 755 17

Proliferation of some cultured human tumor cell lines bearing high numbers of epidermal growth factor (EGF) receptors is paradoxically inhibited by EGF in nanomolar concentrations. In the present study, we have investigated the biochemical mechanism of growth inhibition in A431 human squamous carcinoma cells exposed to exogenous EGF. In parallel, we studied a selected subpopulation, A431-F, which is resistant to EGF-mediated growth inhibition. We observed a marked reduction in cyclin-dependent kinase-2 (CDK2) activity when A431 and A431-F cells were cultured with 20 nM EGF for 4 h. After further continuous exposure of A431 cells to EGF, the CDK2 activity remained at a low level and was accompanied by persistent G1 arrest. In contrast, the early reduced CDK2 activity and G1 accumulation in A431-F cells was only transient. We found that, at early time points (4-8 h), EGF induces p21Cip1/WAF1 mRNA and protein expression in both EGF-sensitive A431 cells and EGF-resistant A431-F cells. But only in A431 cells, was p21Cip1/WAF1 expression sustained at a significantly increased level for up to 5 d after addition of EGF. Induction of p21Cip1/WAF1 by EGF could be inhibited by a specific EGF receptor tyrosine kinase inhibitor, tyrphostin AG1478, suggesting that p21Cip1/WAF1 induction was a consequence of receptor tyrosine kinase activation by EGF. We also demonstrated that the increased p21Cip1/WAF1 was associated with both CDK2 and proliferating cell nuclear antigen (PCNA). Taken together, our results demonstrate that p21Cip1/WAF1 is an important mediator of EGF-induced G1 arrest and growth inhibition in A431 cells.
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PMID:Prolonged induction of p21Cip1/WAF1/CDK2/PCNA complex by epidermal growth factor receptor activation mediates ligand-induced A431 cell growth inhibition. 755 80

Differentiation therapy is an attractive option for the treatment of superficial, localized neoplastic lesions of the skin. Topical application of agents that induce differentiation could selectively inhibit tumor cell growth, inducing a program of cell death with the production of cross-linked protein envelopes as the terminal event of this process at the skin surface, effectively eliminating the neoplastic phenotype. The nonspecific kinase inhibitor staurosporine induces cornified envelope assembly in neoplastic keratinocytes and causes tumor regression (A. A. Dlugosz and S. H. Yuspa, Cancer Res., 51: 4677-4684, 1991). In pursuit of less toxic agents, specific tyrosine kinase inhibitors were tested for the ability to induce differentiation in keratinocyte-derived cells. Of a range of inhibitors tested, only MC was able to induce cross-linked protein and consequent cell death in mouse and human primary normal keratinocytes, 308 neoplastic mouse keratinocytes, HPV-18-infected immortalized human keratinocytes, and human lines SQCC-Y1 (squamous carcinoma) and A431 (epidermoid carcinoma). MC increased cross-linked protein in a dose-dependent manner (0.05-1 mM). To confirm differentiation, MC-treated mouse primary normal keratinocytes were tested for activation of the endogenous cross-linking enzyme transglutaminase, but no association was found between transglutaminase activity and MC-induced protein cross-linking. MC also induced protein cross-linking in the fibroblast cell line NIH3T3 and in B16 melanoma cells, in which cornified envelope assembly is not part of the differentiation process. This cross-linking occurred at 4 degrees C, suggesting a nonphysiological process. Western blot analysis of an in vitro assay with purified EGF receptor showed that MC was able to cross-link the receptor. As in NIH3T3 cells, DTT inhibited cross-linking, suggesting that oxidation of MC or an acceptor group may be required for this effect. Thus, MC does not induce differentiation by a physiological mechanism in epithelial cells but causes chemical protein cross-linking into cornified envelope-like structures at high concentration.
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PMID:The erbstatin analogue methyl 2,5-dihydroxycinnamate cross-links proteins and is cytotoxic to normal and neoplastic epithelial cells by a mechanism independent of tyrosine kinase inhibition. 758 35

The FER gene encodes a cytoplasmic tyrosine kinase with a single SH2 domain and an extensive amino terminus. In order to understand the cellular function of the FER kinase, we analyzed the effect of growth factor stimulation on the phosphorylation and activity of FER. Stimulation of A431 cells and 3T3 fibroblasts with epidermal growth factor or platelet-derived growth factor results in the phosphorylation of FER and two associated polypeptides. The associated polypeptides were shown to be the epidermal growth factor receptor or the platelet-derived growth factor receptor and a previously identified target, pp120. Since pp120 had previously been shown to interact with components of the cadherin-catenin complex, these results implicate FER in the regulation of cell-cell interactions. The physical association of FER with pp120 was found to be constitutive and was mediated by a 400-amino-acid sequence in the amino terminus of FER. Analyses of that sequence revealed that it has the ability to form coiled coils and that it oligomerizes in vitro. The identification of a coiled coil sequence in the FER kinase and the demonstration that the sequence mediates association with a potential substrate suggest a novel mechanism for signal transduction by cytoplasmic tyrosine kinases.
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PMID:The cytoplasmic tyrosine kinase FER is associated with the catenin-like substrate pp120 and is activated by growth factors. 762 46

Addition of epidermal growth factor to A431 cells results in dramatic changes in cell morphology. Initially the cells form membrane ruffles accompanied by increased actin polymerization, followed by cell rounding. Activation of the tyrosine kinase of the receptor by binding epidermal growth factor leads also to phosphorylation and activation of phospholipase C-gamma 1, a key enzyme in the phosphoinositide pathway. In this study we have investigated the localization of phospholipase C-gamma 1 during cell activation by epidermal growth factor. It is shown that addition of the growth factor to A431 cells leads to a translocation of phospholipase C-gamma 1 from the cytosol to the membrane fraction. Interestingly, this relocation is exclusively directed to the membrane ruffles. Most of the phospholipase C-gamma 1 associates to the membrane and a small fraction to the underlying skeleton. Immunocytochemical studies demonstrated that phospholipase C-gamma 1 co-localizes with the epidermal growth factor receptor and also filamentous actin at the membrane ruffles. Moreover, using anti-phosphotyrosine antibodies we found that the membrane ruffles are significantly enriched in phosphotyrosyl proteins. Between 5 and 10 minutes after stimulation the membrane ruffles disappear and also the co-localization of phospholipase C-gamma 1 with the epidermal growth factor receptor and filamentous actin. These results support the notion that activation of A431 cells by epidermal growth factor leads to the formation of a signalling complex of its receptor, phospholipase C-gamma 1 and filamentous actin which is primarily localized at membrane ruffles.
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PMID:Epidermal growth factor induces rapid and transient association of phospholipase C-gamma 1 with EGF-receptor and filamentous actin at membrane ruffles of A431 cells. 767 64

A putative mitogen-activated protein kinase (MAPK) has recently been identified, which potentially phosphorylates the human epidermal growth factor (EGF) receptor at a physiological site (Thr-669) and is distinguished from other MAPKs/extracellular signal-regulated protein kinases (ERKs) on the basis of chromatographic, immunological, and kinetic data. Here we report that this newly discovered MAPK is physically associated with the EGF receptor in A431 cells and with the related receptor/tyrosine kinase HER2 (encoded by c-neu) in enzyme preparations obtained from Wilm's tumors. This human EGF receptor-associated kinase is characterized as a 40-kDa Thr-669 kinase that exists in a high molecular mass complex with the respective growth factor receptor. EGF treatment of A431 cells stimulates the tyrosine phosphorylation of p40 and increases Thr-669 kinase activity in p40-containing fractions. The 40-kDa kinase is recognized by affinity-purified polyclonal antibodies directed against the sea star p44mpk and a Pan-ERK antibody directed against the conserved subdomain VIII of MAPKs/ERKs, but is not recognized by antibodies selective for the rat p44erk1 and/or the p42mapk/erk2 isoforms, thus identifying the EGF receptor-associated kinase as a novel MAPK that may regulate receptor function in vivo.
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PMID:Identification of a human epidermal growth factor receptor-associated protein kinase as a new member of the mitogen-activated protein kinase/extracellular signal-regulated protein kinase family. 768 42

A431 cells, a human epidermoid carcinoma, possess specific [3H]platelet-activating factor (PAF) and [3H]WEB 2086 binding sites indicating the presence of PAF receptors. PAF-stimulated PLC as determined by the increase in inositol phosphate levels. Pretreatment of A431 cells with genistein, a putative tyrosine kinase inhibitor, abolished the ability of PAF to activate PLC, whereas pretreatment with staurosporine, a protein kinase C inhibitor, potentiated the ability of PAF to activate PLC. Pretreatment of A431 cells with phorbol-12-myristate-13-acetate, a protein kinase C activator, blocked PAF-stimulated PLC. Overnight exposure of cells to pertussis toxin (PT) partially blocked the ability of PAF to stimulate PLC. Based on these observations the involvement of PT-sensitive and -insensitive guanine nucleotide-binding protein(s) (G-protein) as well as the role of tyrosine kinase in the activation of PLC by PAF was considered further. PT treatment of A431 cell membranes obliterated PAF-stimulated GTPase and indicated that PT-insensitive membrane-associated G-proteins were not involved in PAF actions. In alpha-toxin permeabilized cells, PT blocked GTP-gamma-S potentiation of PLC activation by PAF, thus suggesting that PT-insensitive G-proteins were not involved in PAF activation of PLC in A431 cells. PAF stimulated tyrosine kinase activity as observed with the increase in radioactivity associated with proteins immunoprecipitated with polyclonal antibodies to phosphotyrosine residues. This increase was blocked by PAF receptor antagonists, CV 6209 and TCV 309, and by pretreatment with genistein. PAF also activated the phosphorylation of pp60c-src and Src associated proteins in A431 cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Role of guanine nucleotide-binding protein and tyrosine kinase in platelet-activating factor activation of phospholipase C in A431 cells: proposal for dual mechanisms. 768

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

Epidermal growth factor (EGF) induces rapid actin filament assembly in the membrane skeleton of A431 cells, leading to a approximately 30% rise in cellular filamentous actin levels. EGF-induced actin polymerization depends upon EGF receptor (EGFR) tyrosine kinase activity, since the selective tyrosine kinase inhibitor AG213 abolishes EGF-induced actin polymerization. In accordance, confocal laser scanning microscopy shows that newly assembled actin filaments localize selectively to the tyrosine-phosphorylated EGFR in the plasma membrane, since actin polymerization is not observed at the internalized tyrosine-phosphorylated EGFR. Actin binding proteins (ABP's) are generally believed to regulate actin filament assembly. Ca2+ is known as one of the important regulatory factors for the activity of ABP's in vitro [15]. Therefore, we investigated the importance of the EGF-induced transient rise in [Ca2+]i for the regulation of actin polymerization in vivo. Continuous high [Ca2+]i in the millimolar range induces a prominent rise in cellular filamentous actin levels to approximately 50% over control cells. However, actin polymerization is unimpaired under conditions which effectively block the EGF-induced [Ca2+]i transient. These data demonstrate that EGF-induced actin polymerization localizes to the activated EGFR in the membrane skeleton, independent of the cytosolic free calcium transient.
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PMID:Actin polymerization localizes to the activated epidermal growth factor receptor in the plasma membrane, independent of the cytosolic free calcium transient. 773 61


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