Purified EGF receptor-kinase interacts specifically with antibodies to Rous sarcoma virus transforming protein

Transformation by several RNA tumour viruses seems to be mediated by virally coded protein kinases which specifically phosphorylate tyrosine. A tyrosine-specific protein kinase also seems to be involved in the mitogenic action of epidermal growth factor (EGF). This EGF-stimulated kinase activity is closely associated with the EGF receptor, with which it copurifies during EGF-affinity chromatography. Because both the virus- and EGF-stimulated tyrosine kinases may be involved in stimulation of cell growth, and because the viral kinases may be antigenically related to normal cell proteins, we examined the interaction of antibodies to viral tyrosine kinases with the affinity-purified EGF receptor-kinase preparation. We report here that the receptor-kinase specifically phosphorylates antibodies directed against the transforming protein kinase pp60src of Rous sarcoma virus. However, none of these antibodies, including those which cross-react with the normal cellular homologue of pp60src (pp60sarc), precipitate the receptor-kinase. These results suggest that the EGF receptor-kinase is related to, but probably not identical with, pp60sarc.     

正常人口腔粘膜中表皮生长因子及其受体的表达

目的：研究人口腔粘膜中表皮生长因子（EGF）及其受体（EGF－R）的分布状态,方法：采用免疫组织化学方法,结果：EGF主要分布于口腔上皮底层细胞中,并随细胞向上分化而逐渐减少,其免疫染色出现在细胞浆中,EGF－R也主要在基底层细胞表达,除存在于细胞浆外,在细胞核中亦见表达,结论：表皮生长因子及其受体对口腔上皮细胞生长,分化的调节有重要作用,但EGF－R在细胞核中的表达,其作用尚需深入研究。     

Biologically active phorbol esters specifically alter affinity of epidermal growth factor membrane receptors

TPA (12-O-tetradecanoyl-phorbol-13-acetate) reversibly inhibits the binding of (125)I-labelled epidermal growth factor (EGF) to treated mouse and human cells, but does not affect the binding of various other ligands to their membrane receptors. It alters the affinity of the receptors for EGF without changing the total number of available receptors per cell. Those phorbol esters which stimulate cell growth in culture and have tumour-promoting activity in vivo alter the EGF-receptor affinity, while the biologically inactive derivatives fail to change the affinity of EGF for its receptors.     

ATP-stimulated interaction between epidermal growth factor receptor and supercoiled DNA

The receptor for epidermal growth factor (EGF) has been identified as a transmembrane glycoprotein that has tyrosine-specific kinase activity. The kinase activity of the receptor is enhanced in the presence of EGF (or related peptides), and the phosphorylation of a number of substrates, as well as autophosphorylation of the receptor, has been reported. Analogous findings have been described for the insulin receptor and the receptor for platelet-derived growth factor (PDGF). Thus, a number of hormone receptors and several viral transforming proteins appear to share the highly unusual property of tyrosine-specific kinase activity. Nevertheless, the specific relationship between tyrosine kinase activity and the control of cell growth and replication is unknown. It is known that after the initial binding of EGF to the plasma membrane, the hormone together with its receptor is rapidly internalized in endocytic vesicles and the hormone is eventually degraded in lysosomes. It is possible that the function of EGF is simply to stimulate internalization of its receptor, and that as a result of its altered location the receptor is able to phosphorylate a cytoplasmic component or even interact directly with a nuclear component. We now report that the purified receptor for EGF is able to interact with and nick supercoiled double-stranded DNA in an ATP-stimulated manner.     

Epidermal growth factor-dependent phosphorylation of lipocortin

Lipocortin-like proteins are a family of steroid-induced inhibitors of phospholipase activity with potential anti-inflammatory activity. Related proteins have been detected in a variety of tissues and species. The best characterized form is a protein of relative molecular mass (Mr) approximately 40,000 (40K), which is phosphorylated in vivo by protein tyrosine kinases and by protein serine-threonine kinases. It has been proposed that the phospholipase inhibitory activity of lipocortin can be regulated by its phosphorylation. In the A431 cell line, a protein of approximately 35K is phosphorylated by the protein tyrosine kinase activity of the epidermal growth factor (EGF) receptor. Here we report that human lipocortin is phosphorylated near its amino terminus by the EGF receptor/kinase. By peptide mapping and immunological analyses, we show that lipocortin and the endogenous 35K substrate for the EGF receptor/kinase from A431 cells are the same protein.     

Diminished in vitro tyrosine kinase activity of the EGF receptor of senescent human fibroblasts

Fibroblastic cultures derived from normal human tissues undergo a finite number of population doublings when serially subcultivated in vitro (see refs 1, 2 for reviews). Epidermal growth factor (EGF) serves as a mitogen for early doubling level cultures of the human fetal lung-derived cell strain, WI-38, under serum-free conditions. The ability of cells from late doubling level cultures to respond mitogenically to EGF is lost, however, despite undiminished binding of EGF throughout the replicative lifespan. The ultimate effects of EGF, that is DNA synthesis and mitosis (see ref. 4 for review), occur after a sequence of events initiated by binding of ligand to specific cellular receptors. The receptor for EGF has been characterized as a 145,000-165,000 (145 K-165 K) molecular weight doublet, and, like the receptors for platelet-derived growth factor and insulin, and the transforming proteins of certain of the RNA tumour viruses, is a tyrosine-specific protein kinase with autophosphorylating activity. Moreover, several of the cellular target molecules of tyrosine phosphorylation have been found to be substrates for two or more of these kinases. The hypothesis that tyrosine phosphorylation underlies a common mechanism of growth control prompted us to ask whether the loss of responsiveness to EGF by late doubling level WI-38 cells is accompanied by altered expression of the EGF receptor, and specifically whether changes occur in the ability of receptors from populations of cells of various in vitro ages to catalyse tyrosine autophosphorylation. We show here that autophosphorylating activity is absent from the EGF receptor of cells which have lost their mitogenic responsiveness to EGF.     

表皮生长因子效应评价细胞模型的建立

为了筛选表皮生长因子(EGF)信号通路的未知调控分子,利用人正常乳腺上皮细胞系MCF10A建立了EGF效应评价模型,包括细胞增殖、迁移以及细胞周期的进程等。结果显示,EGF能有效促进MCF10A细胞的增殖。饥饿同步化后,EGF能促进细胞从G1期进入S期,开始DNA的合成。此外,Transwell实验显示EGF能明显促进MCF10A细胞的迁移。     

低功率毫米波辐射对HL60细胞基因表达谱的影响

摘要：研究低功率毫米波辐射对HL60白血病细胞基因表达谱的影响。应用基因芯片检测频率41.32GHz的毫米波辐射HL60白血病细胞和未辐射毫米波HL60白血病细胞组基因表达差异,并进行RT-PCR方法验证IL-7、EGF和LGALS3基因变化。 结果与对照组比较,毫米波辐射60min后,HL60细胞增殖,基因芯片检出基因表达上调18个和下调306个,在下调的基因中,RT-PCR 检出IL-7、EGF和LGALS3基因下调与基因芯片结果一致。表明低功率毫米波可导致HL60细胞基因表达谱发生变化,这些变化的基因与HL60细胞增殖功能相关。提示基因表达变化是低功率毫米波辐射HL60细胞所致生物学反应的重要因素。     

Vaccinia virus encodes a polypeptide homologous to epidermal growth factor and transforming growth factor

Epidermal growth factor (EGF) and transforming growth factor type I (TGF) are polypeptides of 53 and 50 amino acid residues, respectively. Both bind to EGF receptor, a 1,200-residue transmembranous glycoprotein, leading to phosphorylation of the receptor, enhancement of its tyrosine-specific kinase activity and ultimately to stimulation of cell growth. We report here that a 140-residue polypeptide encoded by one of the early genes of vaccinia virus (VV) is related closely to EGF and TGF. The presence of putative signal and transmembranous sequences further suggests that the viral protein might be an integral membrane protein, but that, as in the case of EGF itself, the membrane-associated form may be the precursor of a soluble growth factor. Production of EGF-like growth factors by virally infected cells could account for the proliferative diseases associated with members of the poxvirus family such as Shope fibroma virus, Yaba tumour virus, and molluscum contagiosum virus (MCV).     

Abelson murine leukaemia virus transformation involves loss of epidermal growth factor-binding sites

Malignant transformation by mammalian RNA sarcoma viruses has previously been shown to involve a reduction in receptor sites for a well characterized 6,000-molecular weight (MW) growth-promoting substance, designated epidermal growth factor (EGF). Although Abelson murine leukaemia virus (AbLV) resembles sarcoma viruses in its ability to transform embryo fibroblasts in cell culture, AbLV induces a rapid B-cell lymphoid leukaemia rather than fibrosarcomas in vivo. The major translational product of AbLV is a highly phosphorylated polyprotein of MW 120,000 which exhibits an associated tyrosine-specific protein kinase activity and probable transforming function. We show here that AbLV transformation resembles transformation by RNA sarcoma viruses with respect to the abolition of EGF-binding sites. EGF binding is restored to control levels following loss of polyprotein expression in morphological revertants of AbLV-transformed clones and remains uninfluenced in cell lines infected with transformation-defective (td) AbLV mutants encoding polyproteins deficient in protein kinase activity. These findings indicate that AbLV transformation involves a polyprotein-associated, tyrosine-specific protein kinase activity which mediates its effect through a mechanism resulting directly or indirectly in the abolition of EGF-binding sites.     

Induction of a novel epidermal growth factor-secreting cell lineage by mucosal ulceration in human gastrointestinal stem cells

Epidermal growth factor, and its human homologue urogastrone (EGF/URO), are secreted by the gut-associated salivary and Brunner's glands. Recombinant EGF/URO is a powerful stimulator of cell proliferation and differentiation in the rodent and neonatal human intestine. But EGF/URO is not absorbed from the adult gut and has no action when given through the gut lumen; thus the role of secreted EGF/URO is unknown. We now report that ulceration of the epithelium anywhere in the human gastrointestinal tract induces the development of a novel cell lineage from gastrointestinal stem cells. This lineage initially appears as a bud from the base of intestinal crypts, adjacent to the ulcer, and grows locally as a tubule, ramifying to form a new small gland, and ultimately emerges onto the mucosal surface. The lineage produces neutral mucin, shows a unique lectin-binding profile and immunophenotype, is nonproliferative, and contains and secretes abundant immunoreactive EGF/URO. We propose that all gastrointestinal stem cells can produce this cell lineage after mucosal ulceration, secreting EGF/URO to stimulate cell proliferation, regeneration and ulcer healing. This cell lineage is very commonly associated with gastrointestinal mucosal ulceration, and we conclude that a principal in vivo role for EGF/URO is to stimulate ulcer healing throughout the gut through induction of this cell lineage in the adjacent mucosa.     

Amplification, enhanced expression and possible rearrangement of EGF receptor gene in primary human brain tumours of glial origin

Epidermal growth factor (EGF), through interaction with specific cell surface receptors, generates a pleiotropic response that, by a poorly defined mechanism, can induce proliferation of target cells. Subversion of the EGF mitogenic signal through expression of a truncated receptor may be involved in transformation by the avian erythroblastosis virus (AEV) oncogene v-erb-B, suggesting that similar EGF receptor defects may be found in human neoplasias. Overexpression of EGF receptors has been reported on the epidermoid carcinoma cell line A431, in various primary brain tumours and in squamous carcinomas. In A431 cells the receptor gene is amplified. Here we show that 4 of 10 primary brain tumours of glial origin which express levels of EGF receptors that are higher than normal also have amplified EGF receptor genes. Amplified receptor genes were not detected in the other brain tumours examined. Further analysis of EGF receptor defects may show that such altered expression and amplification is a particular feature of certain human tumours.     

Transformation by murine and feline sarcoma viruses specifically blocks binding of epidermal growth factor to cells.

Normal cells in culture have membrane receptors for epidermal growth factor (EGF); EGF stimulates cells to divide by binding to these receptors. Cells transformed by murine and feline sarcoma viruses rapidly lose the ability to bind EGF, whereas cells transformed by the DNA tumour viruses, polyoma and SV40, or infected with non-transforming RNA tumour viruses have normal levels of functional EGF receptors. The results suggest that a product of the sarcoma virus genome specifically changes cell EGF receptors; the sarcoma gene product may, then, be functionally related to EGF.     

Human transforming growth factors induce tyrosine phosphorylation of EGF receptors

Cultured cell lines of human tumour origin as well as cells transformed by various RNA tumour viruses secrete low molecular weight polypeptide transforming growth factors (TGFs). In addition to competing with epidermal growth factor (EGF) for binding to its cellular receptor, TGFs can transform morphologically fibroblast and epithelial cells in culture. In view of accumulating evidence that tyrosine phosphorylation activity is associated with the transforming genes of various tumour viruses, we determined whether phosphotyrosine levels were elevated in these human tumour cells. We show here that TGFs produced by human tumour cells induce phosphorylation of specific tyrosine acceptor sites in the 160,000-molecular weight (160 K) EGF receptor.     

The neu oncogene encodes an epidermal growth factor receptor-related protein

The neu oncogene is repeatedly activated in neuro- and glioblastomas derived by transplacental mutagenesis of the BDIX strain of rat with ethylnitrosourea. Foci induced by the DNAs from such tumours on NIH 3T3 cells contain the neu oncogene and an associated phosphoprotein of relative molecular mass 185,000 (p185). Previous work has shown that the neu gene is related to, but distinct from, the gene encoding the EGF receptor (c-erb-B). Here we describe a neu complementary DNA clone isolated from a cell line transformed by this oncogene; the clone has biological activity in a focus-forming assay. The nucleotide sequence of this clone predicts a 1,260-amino-acid transmembrane protein product similar in overall structure to the EGF receptor. We found that 50% of the predicted amino acids of neu and the EGF receptor are identical; greater than 80% of the amino acids in the tyrosine kinase domain are identical. Our results suggest strongly that the neu gene encodes the receptor for an as yet unidentified growth factor.     

TGF-β1与EGF对肺泡Ⅱ型上皮细胞表型及功能的影响

目的:探讨TGF-β1与EGF对肺泡Ⅱ型上皮细胞(RLE-6TN)的影响。方法:将细胞分成对照(C)、TGF-β1(T)、EGF(E)及TGF-β1+EGF(TE)组。采用相差显微镜观察细胞形态,免疫荧光检测α-SMA表达,W.B检测上皮、间质细胞标记物、MMPs及信号蛋白的表达;流式细胞术检测凋亡情况。结果:T及TE组RLE-6TN发生EMT转变并见间质标记物及MT1-MMP、MMP2表达上调,E组Vimentin、MT1-MMP、MMP2及CD147表达上调;T与E组p-ERK、p-38MAPK及p-Akt表达上调,T组p-Smad2表达上调;T与E组均见凋亡,TE组凋亡加剧。结论:单用EGF不能诱导RLE-6TN发生EMT,合用TGF-β1无协同诱导EMT,却能协同诱导凋亡。TGF-β1诱导EMT时,主要通过EGFR信号通路诱导MMPs表达。     

Retinoids specifically enhance the number of epidermal growth factor receptors

Retinoids elicit many biological and biochemical responses from cells in vitro. One widely used criterion for the responsiveness of cells to retinoids is inhibition of growth; retinoids reduce the saturation density and/or growth rate of many normal and tumorigenic cell lines. Propagation of eukaryotic cells has been demonstrated to be dependent on the presence of macromolecular growth factors such as epidermal growth factor (EGF), which can stimulate proliferation of epithelial and fibroblastic cell lines. We now describe the effect of retinoids on the binding of EGF to its receptor. Retinoic acid enhances binding of 125I-labelled EGF to various fibroblastic and epidermal cell lines. It has no marked effect on the affinity of this growth factor for its receptor, but increases the number of EGF receptor sites. Retinoic acid has little effect on the binding of concanavalin A (Con A) and insulin, indicating the specific nature of the action of retinoids on cell-surface glycoproteins. Treatment of cells with the phorbol ester 12-o-tetradecanoyl phorbol-13-acetate (TPA) and retinoic acid shows poor antagonism between these compounds on EGF binding. It has been previously shown that retinoids induce or stimulate differentiation of embryonal carcinoma cells. EGF binding can be used as a marker to monitor differentiation of these cells.     

Autophosphorylation sites on the epidermal growth factor receptor

The epidermal growth factor (EGF) receptor is a tyrosine-specific protein kinase with autophosphorylating activity. A 300 amino acid-long region of the receptor's cytoplasmic domain matches (35-90% homology) sequences of transforming proteins from the src family and includes a putative nucleotide binding site. Several of the src transforming proteins have tyrosine kinase activity, but v-erb-B, which appears to be a truncated EGF receptor, is virtually identical to the receptor over this region and yet lacks detectable kinase activity. To locate possible acceptor sites in the v-erb-B protein, we have mapped these sites in the human EGF receptor. We report here that three tyrosine sites near the C-terminus are phosphorylated in vitro. In intact cells, we find that EGF stimulates phosphorylation of several sites, the tyrosine 14 residues from the C-terminus being modified the most extensively. The equivalent site is absent in the v-erb-B protein of avian erythroblastosis virus (AEV) and may influence tyrosine kinase activity.