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1.
Epidermal growth factor receptor metabolism and protein kinase activity in human A431 cells infected with Snyder-Theilen feline sarcoma virus or harvey or Kirsten murine sarcoma virus. 总被引:8,自引:0,他引:8
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When human A431 cells, which carry high numbers of epidermal growth factor (EGF) receptors, are exposed to EGF, the total content of phosphotyrosine in cell protein is increased, the EGF receptor becomes phosphorylated at tyrosine, and new phosphotyrosine-containing 36,000- and 81,000-dalton proteins are detected. We examined the properties of A431 cells infected with Snyder-Theilen feline sarcoma virus, whose transforming protein has associated tyrosine protein kinase activity, and Harvey and Kirsten sarcoma viruses, whose transforming proteins do not. In all cases, the infected cells were more rounded and more capable of anchorage-independent growth than the uninfected cells. EGF receptors were assayed functionally by measuring EGF binding and structurally by metabolic labeling and immunoprecipitation. In no case did infection appear to alter the rate of EGF receptor synthesis, but infection reduced EGF receptor stability by about 50% for cloned Harvey sarcoma virus-infected cells and by 80% for cloned feline sarcoma virus-infected cells. The corresponding reductions in EGF binding were 70 and 90%, respectively. The proteins of feline sarcoma virus-infected A431 cells contained an increased amount of phosphotyrosine, and the 36,000- and 81,000-dalton phosphoproteins were detected. The EGF receptor was not detectably phosphorylated at tyrosine, however, unless the cells were exposed to EGF. The Harvey and Kirsten sarcoma virus-infected cells did not exhibit elevated levels of phosphotyrosine either in the total cell proteins or in the EGF receptor, nor were the 36,000- and 81,000-dalton proteins detectable. However, these phosphoproteins were found in the infected cells after EGF treatment. Thus, all of the infected A431 cells exhibited reduced EGF binding and increased degradation of EGF receptors, yet their patterns of protein phosphorylation were distinct from those of EGF-treated A431 cells. 相似文献
2.
The transferrin receptor is a 180 000-dalton protein which can be dissociated to two 90 000-dalton polypeptides under reducing conditions. It can be labelled by lactoperoxidase-catalysed iodination on the cell surface at 0 degree C. Trypsin digestion of labelled cells at 0 degree C can be used to degrade those receptors on the cell surface; they release a 70 000-dalton soluble fragment which binds to transferrin. When cells are labelled at 0 degree C, then warmed to 37 degrees C, the labelled receptors enter the cells and become trypsin resistant. These receptors enter the cells, probably via coated pits, with a half-life of approximately 5 min. Since there is about three times as much receptor inside cells as on the surface, this means that transit through the cell to the cell surface takes approximately 21 min, if all receptors are on the same cycling pathway. 相似文献
3.
Epidermal growth factor (EGF)-induced increases in cytosolic Ca2+ and inositol polyphosphate production were compared in a human hepatocellular carcinoma-derived cell line, PLC/PRF/5, and in an EGF receptor-overexpressing subline, NPLC/PRF/5. Formation of these second messengers was correlated to EGF receptor display at the cell surface by monitoring ligand-induced EGF receptor down-regulation. Both cell lines exhibited a strikingly similar cytosolic Ca2+ increase upon exposure to EGF. The initial inositol phosphate responses were also similar in the two cell lines; inositol 1,4,5-trisphosphate increased within 10-15 s and returned to prestimulatory values after 2 min in both cell lines, while inositol tetrakisphosphate and inositol 1,3,4-trisphosphate were elevated after a 2-min exposure to EGF. At later times the responses were markedly different; NPLC/PRF/5 cells exhibited prolonged production of inositol 1,3,4-trisphosphate and inositol tetrakisphosphate (maximum at 1-3 h) but PLC/PRF/5 cells showed decreased levels of these isomers after 10 min and a return to basal values by 1 h. Exposure of PLC/PRF/5 cells to EGF caused a progressive decrease in the amount of EGF receptor at the cell surface whereas such treatment did not change the surface receptor levels in NPLC/PRF/5 cells. Kinetic analysis of EGF receptor down-regulation showed that receptor internalization was rapid enough to account for the transient nature of the inositol phosphate response in PLC/PRF/5 cells. Thus, the divergent patterns of signaling exhibited by the two cell lines may reflect differences in the efficiency of EGF-induced down-regulation of surface receptors. 相似文献
4.
The effect of epidermal growth factor (EGF) receptor overexpression on ligand-induced EGF receptor downregulation was examined using a hepatoma-derived cell line, PLC/PRF/5, which expresses normal amounts of the EGF receptor, and a subline, NPLC/PRF/5, which expresses 10-fold more receptors at its cell surface. PLC/PRF/5 cells efficiently downregulated surface receptor levels upon exposure to saturating and subsaturating concentrations of EGF; the rate of receptor downregulation corresponded to that of ligand-receptor internalization. Upon internalization, EGF receptors were degraded and receptor biosynthesis remained at basal levels. EGF surface receptor remained downregulated for as long as cells were exposed to EGF. By contrast, surface EGF receptor abundance in NPLC/PRF/5 cells decreased by only 5-15% after 1-4 h incubation with subsaturating doses of EGF and actually increased by 67% within 20 h. Exposure of these cells to saturating concentrations of EGF induced modest decreases in surface receptor abundance during the initial 12 h incubation, followed by a progressive decline to 30% of initial values by 24 h. Relative ligand-receptor internalization rates in NPLC/PRF/5 cells were lower than those in PLC/PRF/5, although their surface receptor population was even higher than that predicted by the decreased internalization rates. EGF receptor degradation in NPLC/PRF/5 cells was also inhibited; exposure to saturating levels of EGF for more than 16 h was necessary before significant degradation occurred. Receptor protein and mRNA biosynthesis in NPLC/PRF/5 were stimulated by 8 h exposure to EGF but when saturating concentrations of EGF were present for 16 h, receptor biosynthesis was inhibited. EGF receptor overexpression circumvents the downregulatory effect of EGF by decreasing the rate of receptor internalization, inhibiting degradation of the internalized receptor pool, and stimulating EGF receptor biosynthesis. Conversely, receptor downregulation becomes pronounced at late times when receptor degradation is high and biosynthesis is inhibited. 相似文献
5.
Epidermal growth factor (EGF) stimulates EGF receptor synthesis 总被引:13,自引:0,他引:13
H S Earp K S Austin J Blaisdell R A Rubin K G Nelson L W Lee J W Grisham 《The Journal of biological chemistry》1986,261(11):4777-4780
Epidermal growth factor (EGF) binds to the extracellular domain of a specific 170,000-dalton transmembrane glycoprotein; this results in rapid removal of both ligand and receptor from the cell surface. In WB cells, a rat hepatic epithelial cell line, ligand-directed receptor internalization leads to receptor degradation. We tested whether the EGF receptor was replenished at a constitutive or enhanced rate following EGF binding by immunoprecipitating biosynthetically labeled EGF receptor from cells cultured with [35S]methionine. EGF stimulated receptor synthesis within 2 h in a dose-dependent manner; this was particularly evident when examining the nascent form of the receptor. To determine the site of EGF action, total WB cell RNA was transferred to nitrocellulose paper after electrophoresis and was hybridized to cDNA probes from both the external and cytoplasmic coding regions of the human EGF receptor. EGF increased receptor mRNA by 3-5-fold. Therefore, at least in some cells, the surface action of EGF that leads to EGF receptor degradation is counterbalanced by a positive effect on receptor synthesis. 相似文献
6.
We have used resonance energy transfer to monitor epidermal growth factor (EGF) receptor micro-aggregation at the surface of intact human epidermoid carcinoma (A431) cells. EGF molecules labeled with fluorescein isothiocyanate and eosin isothiocyanate were demonstrated to bind tightly to cellsurface receptors, to elicit immediate changes in cytosolic free [Ca2+], and to undergo endocytosis. Under conditions which maintain the integrity of the cell, we observed no energy transfer between the donor fluorescein isothiocyanate-labeled EGF molecules and the acceptor eosin isothiocyanate-labeled growth factors bound to receptors. However, after disruption of cells by Dounce homogenization, a significant degree of energy transfer was observed (approximately 10-20%) with membranes, indicative of receptor aggregation. These results suggest that EGF does not cause micro-aggregation of the majority of its receptors on the surface of intact A431 cells within the time period of the early events associated with growth factor action. Moreover, it appears that the A431 cells contain some component which imparts a constraint on the ability of EGF receptors to aggregate, and that some of this component is lost upon the disruption of cells. 相似文献
7.
J Castagnola C MacLeod H Sunada J Mendelsohn R Taetle 《Journal of cellular physiology》1987,132(3):492-500
The transferrin (Tf) receptor is a major transmembrane protein which provides iron for normal and malignant cell growth. Epidermal growth factor (EGF) has been reported to rapidly and transiently alter the number of surface Tf receptors in normal and transformed epithelial cells. To investigate mechanisms of EGF-induced changes in surface Tf display, EGF effects on surface Tf receptors were compared in two cell lines which differ in their number of EGF receptors and growth responses to EGF. In cloned A431 cells with high receptor numbers which are growth-inhibited by EGF, EGF caused a 50% decrease in Tf receptor expression after 30 min. In contrast, EGF induced a rapid, transitory increase (within 5 min) in the number of surface Tf receptors on KB carcinoma cells which returned to basal levels by 15 min. The observed changes in Tf receptor display were due to altered receptor distribution and not changes in ligand affinity or total cellular transferrin receptor pools. Anti-EGF receptor monoclonal antibody blocked effects of EGF on transferrin receptor expression. Since the antibody is internalized and causes EGF receptor down-regulation, effects on transferrin receptor expression were independent of these events. EGF-induced alterations in Tf receptor display occurred even when cells were pretreated with colchicine, suggesting that changes in surface Tf binding were not mediated by cytoskeletal components. Na orthovanadate, which mimics some early cellular effects of EGF, duplicated EGF's effects on A431 Tf receptors, but had no effect on KB cells, suggesting these responses occur by differing mechanisms. To determine whether EGF caused changes in Tf receptor phosphorylation, 32P-labelled Tf receptors were immunoprecipitated after EGF treatment. After exposure to EGF, A431 cells showed no change in Tf phosphorylation, but KB cells showed a transient, 6-fold increase in transferrin receptor phosphorylation on serine residues. In both A431 and KB cells, phorbol ester (PMA) also increased phosphorylation on transferrin receptors, but had little effect on surface Tf receptor expression. In malignant cell lines, EGE induces rapid, variable changes in transferrin receptor expression and phosphorylation which differ from the effects of PMA. These early responses to EGF appear to differ with the cell type and correlate poorly with alterations in Tf receptor phosphorylation. These results suggest Tf receptor phosphorylation does not regulate Tf receptor display in all cells. 相似文献
8.
Insulin-EGF receptor chimerae mediate tyrosine transphosphorylation and serine/threonine phosphorylation of kinase-deficient EGF receptors. 总被引:3,自引:0,他引:3
S Tartare R Ballotti R Lammers F Alengrin T Dull J Schlessinger A Ullrich E Van Obberghen 《The Journal of biological chemistry》1991,266(15):9900-9906
To study cross-talk between unoccupied epidermal growth factor (EGF) receptors and activated EGF receptor kinases, we have used double-transfected cells, IHE2 cells, expressing both an enzymatically active insulin-EGF chimeric receptor and an inactive kinase EGF receptor mutant. Using immunoaffinity-purified receptors, we show that insulin increased phosphorylation of the insulin-EGF chimeric beta subunit and of the kinase-deficient EGF receptor. Stimulation of intact IHE2 cells with insulin leads to a rapid tyrosine autophosphorylation of the insulin-EGF chimeric beta subunit and to tyrosine phosphorylation of the unoccupied kinase-deficient EGF receptor. Insulin-stimulated transphosphorylation of the kinase-deficient EGF receptor yields the same pattern of tryptic phosphopeptides as those in EGF-induced autophosphorylation of the wild-type human EGF receptor. We conclude that insulin, through activation of the insulin-EGF chimeric receptor, mediates transphosphorylation of the kinase-deficient EGF receptor, further confirming that EGF receptor autophosphorylation may proceed by an intermolecular mechanism. In addition to receptor tyrosine phosphorylation, we find that exposure of cells to insulin results in enhanced phosphorylation on serine and threonine residues of the unoccupied kinase-deficient EGF receptor. These results suggest that insulin-EGF chimeric receptor activation stimulates at least one serine/threonine kinase, which in turn phosphorylates the kinase-deficient EGF receptor. Finally, we show that transphosphorylation and coexpression of an active kinase cause a decrease in the number of cell surface kinase-deficient EGF receptors without increasing their degradation rate. 相似文献
9.
The interaction of fibronectin fragments with fibroblastic cells 总被引:21,自引:0,他引:21
S K Akiyama E Hasegawa T Hasegawa K M Yamada 《The Journal of biological chemistry》1985,260(24):13256-13260
We have examined the interaction of the purified cell-binding domain of fibronectin with fibroblastic baby hamster kidney cells. When the cell-binding region of fibronectin is part of a large 75,000-dalton fragment, the direct binding of the tritium-labeled fragment to cells in suspension can be observed. There is a single class of 10(5) sites/cell with an apparent dissociation constant of 4 X 10(-7) M. When the cell-binding region is part of a smaller 11,500-dalton fragment, an interaction with cells can only be observed indirectly via inhibition assays. The apparent affinity of this fragment for the cell surface fibronectin receptor is low. This 11,500-dalton fragment competitively inhibits both the direct binding of soluble [3H]fibronectin to cells in suspension and the spreading of cells on fibronectin-coated substrates, suggesting that the fragment binds to the same receptor site as intact fibronectin. Possible models describing the mechanism of the interaction of fibronectin with its receptor are proposed. 相似文献
10.
Addition of epidermal growth factor (EGF) to many cell types activates phospholipase C resulting in increased levels of diacylglycerol and intracellular Ca2+ which may lead to activation of protein kinase C. EGF treatment of cells can also lead to phosphorylation of the EGF receptor at threonine 654 (a protein kinase C phosphorylation site) which appears to attenuate some aspects of receptor signaling. Thus, a feedback loop involving the EGF receptor, phospholipase C, and protein kinase C may regulate EGF receptor function. In this report, the role of phosphorylation of threonine 654 of the EGF receptor in regulation of EGF-stimulated activation of phospholipase C was investigated. NIH-3T3 cells expressing the normal human EGF receptor or expressing EGF receptor in which an alanine residue had been substituted at residue 654 of the receptor were used. Addition of EGF to cells expressing wild-type receptor induced a rapid, but transient, increase in phosphorylation of threonine 654. EGF addition also caused the rapid accumulation of inositol phosphates in these cells. EGF-stimulated accumulation of inositol phosphates was significantly higher in cells expressing Ala-654 receptors compared to control cells. Treatment of cells with 12-O-tetradecanoylphorbol 13-acetate (TPA), which stimulated phosphorylation of threonine 654 to a greater degree than EGF, completely inhibited EGF-dependent inositol phosphate accumulation in cells expressing wild-type receptor, but caused only a 20-30% inhibition in Ala-654 expressing cells. EGF stimulated phosphorylation of phospholipase C-gamma on serine and tyrosine residues in cells expressing wild-type of Ala-654 receptors. However, TPA treatment of cells inhibited EGF-induced tyrosine phosphorylation of phospholipase C-gamma only in cells expressing wild-type receptors. Similarly, TPA inhibited tyrosine-specific autophosphorylation of the EGF receptor and tyrosine phosphorylation of several other proteins in wild-type receptor cells, but not in Ala-654 cells. TPA treatment abolished high affinity binding of EGF to cells expressing wild-type receptors, while decreasing the number of high affinity binding sites 20-30% in Ala-654 cells. These data suggest that phosphorylation of threonine 654 can regulate early events in EGF receptor signal transduction such as phosphoinositide turnover, probably through a feedback mechanism involving protein kinase C. Subsequent dephosphorylation of threonine 654 could reactivate the EGF receptor for participation in later signaling events. 相似文献
11.
Biological activities of EGF-receptor mutants with individually altered autophosphorylation sites. 总被引:29,自引:3,他引:26
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A Honegger T J Dull F Bellot E Van Obberghen D Szapary A Schmidt A Ullrich J Schlessinger 《The EMBO journal》1988,7(10):3045-3052
In vitro site-directed mutagenesis was used to replace individually the three known autophosphorylation sites of the epidermal growth factor (EGF)-receptor (i.e. Tyr1173, Tyr1148 and Tyr1068) by phenylalanine, a residue which cannot serve as a phosphate acceptor site. In another mutant, Tyr1173 was substituted by a serine residue. The cDNA constructs encoding either mutant or wild-type EGF-receptors were transfected into NIH-3T3 cells devoid of endogenous EGF-receptors. The mutant receptors were expressed on the cell surface and displayed typical high- and low-affinity binding sites for [125I]EGF. Phorbol ester (PMA) modulated the binding affinity of wild-type and mutant receptors in a similar manner. Mutant EGF-receptors exhibited EGF-dependent tyrosine kinase activity leading to self-phosphorylation and phosphorylation of exogenous substrates both in vitro and in living cells. The internalization and degradation of EGF-receptors were not affected by the mutations. Cells expressing mutant EGF-receptors became mitogenically responsive to EGF, indicating that none of the vital functions of the EGF-receptor were critically impaired by the loss of individual autophosphorylation sites. Maximal mitogenic stimulation correlated with the number of wild-type or mutant receptors per cell, highly expressing cells showing higher maximal stimulation. However, the dose-response curves of cells expressing mutant receptors were slightly shifted to lower concentrations of EGF, rendering the cells mitogenically responsive to lower doses of EGF than cells expressing normal EGF-receptor at similar expression levels. Basal [3H]thymidine incorporation in the presence of 0.5% calf serum was consistently higher for cells expressing mutant receptors, while the response to stimulation with 10% calf serum was not affected. 相似文献
12.
The intrinsic protein-tyrosine kinase activity of the epidermal growth factor (EGF) receptor is required for signal transduction. Increased protein-tyrosine kinase activity is observed following the binding of EGF to the receptor. However, signaling is rapidly desensitized during EGF treatment. We report that EGF receptors isolated from desensitized cells exhibit a lower protein-tyrosine kinase activity than EGF receptors isolated from control cells. The mechanism of desensitization of kinase activity can be accounted for, in part, by the EGF-stimulated phosphorylation of the receptor at Ser1046/7, a substrate for the multifunctional calmodulin-dependent protein kinase II in vitro. Mutation of Ser1046/7 by replacement with Ala residues blocks desensitization of the EGF receptor protein-tyrosine kinase activity. Furthermore, this mutation causes a marked inhibition of the EGF-stimulated endocytosis and down-regulation of cell surface receptors. Thus, the phosphorylation site Ser1046/7 is required for EGF receptor desensitization in EGF-treated cells. This regulatory phosphorylation site is located at the carboxyl terminus of the EGF receptor within the subdomain that binds src homology 2 regions of signaling molecules. 相似文献
13.
Expression of the human EGF receptor with ligand-stimulatable kinase activity in insect cells using a baculovirus vector. 总被引:19,自引:2,他引:17
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The mechanism by which the binding of epidermal growth factor (EGF) to specific cell surface receptors induces a range of biological responses remains poorly understood. An important part of the study of signal transduction in this system involves the production of sufficient native and mutant EGF receptor species for X-ray crystallographic and spectroscopic analysis. Baculovirus vectors containing the cDNA encoding the human EGF receptor protein have here been utilized to infect insect cells. This results in expression of a 155-kb transmembrane protein which is recognized by four antibodies against different regions of the human EGF receptor. Studies with tunicamycin, monensen and endoglycosidase H show the difference in size between the recombinant and the native receptor is due to alterations in glycocsylation. Studies of [125I] EGF binding shows a Kd of 2 X 10(-9) M in intact infected insect cells which falls to 2 X 10(-7) M upon detergent solubilization. The recombinant protein exhibits an EGF-stimulated tyrosine protein kinase activity and an analysis of tryptic peptides shows that the phosphate acceptor sites are similar to those of the EGF receptor isolated from A431 cells. These observations indicate that functional EGF receptor can be expressed in insect cells, and furthermore, this system can be used for large-scale production. 相似文献
14.
Point mutation at the ATP binding site of EGF receptor abolishes protein-tyrosine kinase activity and alters cellular routing 总被引:100,自引:0,他引:100
A M Honegger T J Dull S Felder E Van Obberghen F Bellot D Szapary A Schmidt A Ullrich J Schlessinger 《Cell》1987,51(2):199-209
Cultured NIH 3T3 cells devoid of endogenous EGF receptors were transfected with cDNA constructs encoding either the human EGF receptor or an EGF receptor mutant in which Lys721, a key residue in the ATP binding site, was replaced with an alanine residue. The mutant receptor was properly processed, and it displayed both high- and low-affinity surface binding sites. Unlike the wild-type receptor, the mutant receptor did not possess intrinsic protein-tyrosine kinase activity. The initial rate of EGF internalization was similar for wild-type and mutant EGF receptors. Surprisingly, the mutant receptors were not down regulated, but appeared to recycle in transfected cells. These data suggest that degradation of normal EGF receptors after endocytosis is due to the kinase activity endogenous to this receptor. A single amino acid substitution rendered a "down-regulated" receptor into a receptor that can recycle from cytoplasmic compartment back to the cell surface. 相似文献
15.
Effects of protein kinase C activation after epidermal growth factor binding on epidermal growth factor receptor phosphorylation 总被引:8,自引:0,他引:8
The possible role of epidermal growth factor (EGF) receptor phosphorylation at threonine 654 in modulating the protein-tyrosine kinase activity of EGF-treated A431 cells has been studied. It has been suggested that EGF could indirectly activate a protein-serine/threonine kinase, protein kinase C, that can phosphorylate the EGF receptor at threonine 654. Protein kinase C is known to be activated, and threonine 654 is phosphorylated, when A431 cells are exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA). The protein-tyrosine kinase activity of EGF receptors is normally evidenced in EGF-treated cells by phosphorylation of the receptor at tyrosine. This is inhibited when TPA-treated cells are exposed to EGF. We now show that receptor phosphorylation at threonine 654 can also be detected in EGF-treated A431 cells, presumably due to indirect stimulation of protein kinase C or a similar kinase. Some receptor molecules are phosphorylated both at threonine 654 and at tyrosine. Since prior phosphorylation at threonine 654 inhibits autophosphorylation, we propose that protein kinase C can phosphorylate the threonine 654 of autophosphorylated receptors. This provides evidence for models in which protein kinase C activation, consequent upon EGF binding, could reduce the protein-tyrosine kinase activity of the EGF receptor. Indeed, we find that 12-O-tetradecanoylphorbol-13-acetate, added 10 min after EGF, further increases threonine 654 phosphorylation and induces the loss of tyrosine phosphate from A431 cell EGF receptors. 相似文献
16.
Glycosylation of the epidermal growth factor receptor in A-431 cells. The contribution of carbohydrate to receptor function 总被引:21,自引:0,他引:21
A-431 cells were treated with inhibitors of either N-linked glycosylation (tunicamycin or glucosamine) or of N-linked oligosaccharide processing (swainsonine or monensin) to examine the glycosylation of epidermal growth factor (EGF) receptors and to determine the effect of glycosylation modification on receptor function. The receptor was found to be an Mr = 130,000 polypeptide to which a relatively large amount of carbohydrate is added co-translationally in the form of N-linked oligosaccharides. Processing of these oligosaccharides accounts for the 10,000-dalton difference in electrophoretic migration between the Mr = 160,000 precursor and Mr = 170,000 mature forms of the receptor. No evidence was found for O-linked oligosaccharides on the receptor. Mr = 160,000 receptors resulting from swainsonine or monensin treatment were present on the cell surface and retained full function, as judged by 125I-EGF binding to intact cells and detergent-solubilized extracts and by in vitro phosphorylation in the absence or presence of EGF. On the other hand, when cells were treated with tunicamycin or glucosamine, ligand binding was reduced by more than 50% in either intact cells or solubilized cell extracts. The Mr = 130,000 receptors synthesized in the presence of these inhibitors were not found on the cell surface. In addition, no Mr = 130,000 phosphoprotein was detected in the in vitro phosphorylation of tunicamycin or glucosamine-treated cells. It appears, therefore, that although terminal processing of N-linked oligosaccharides is not necessary for proper translocation or function of the EGF receptor, the addition of N-linked oligosaccharides is required. 相似文献
17.
Epidermal growth factor and epidermal growth factor receptor-dependent phosphorylation of a Mr = 34,000 protein substrate for pp60src 总被引:6,自引:0,他引:6
A Mr = 34,000 protein present in the 100,000 X g supernatant fraction from A431 human epidermoid carcinoma cells is the major radiolabeled phosphate acceptor from [gamma-32P]ATP in a cell-free system requiring epidermal growth factor (EGF) and EGF receptor kinase. This protein is immunoprecipitated by IgG directed against avian Mr = 34,000 cellular substrate for pp60src. Phosphoamino acid analysis of the Mr = 34,000 protein labeled with 32Pi from [gamma-32P]ATP in a cell-free system requiring EGF and EGF receptor kinase yielded radiolabeled phosphotyrosine with no detectable radioactivity in phosphoserine or phosphothreonine. 相似文献
18.
cAMP-dependent protein kinase stimulates epidermal growth factor-dependent phosphorylation of epidermal growth factor receptors 总被引:6,自引:0,他引:6
Epidermal growth factor (EGF)-dependent transfer of radiolabeled phosphate from [gamma-32P]ATP to 160-kDa EGF receptor solubilized from human epidermoid carcinoma A431 cell surface membranes was stimulated up to 3-fold by addition of 3',5'-cAMP and purified cAMP-dependent protein kinase. Phosphorylation of EGF receptors was stimulated to the same extent when cAMP-dependent protein kinase catalytic subunit was substituted for 3',5'-cAMP and cAMP-dependent protein kinase. Phosphoamino acid analysis revealed that the extent of phosphorylation of EGF receptor at tyrosine residues was the same regardless of whether cAMP-dependent protein kinase catalytic subunit was present in or omitted from the system. Increased EGF receptor phosphorylation occurring in response to cAMP-dependent protein kinase catalytic subunit was accounted for by phosphorylation at serine or threonine residues. In samples phosphorylated in the presence of cAMP-dependent protein kinase catalytic subunit, phosphate was present in tyrosine, serine, and threonine in a ratio of 32:60:8. Two-dimensional mapping of radiolabeled phosphopeptides produced from EGF receptors by digestion with trypsin revealed the generation of one additional major phosphoserine-containing peptide when cAMP-dependent protein kinase was present with EGF in the EGF receptor kinase system. Degradation of 160-kDa EGF receptors to a 145-kDa form by purified Ca2+-activated neutral protease produced a 145-kDa fragment with phosphoserine content increased over that present initially in the 160-kDa precursor. 相似文献
19.
Activation of the EGF receptor tyrosine kinase by ligand indirectly activates a series of other cellular enzymes, including protein kinase C. To test the hypothesis that phosphorylation of the EGF receptor by protein kinase C provides an intracellular negative feedback loop to attenuate EGF receptor signaling, we used scanning EM to follow the characteristic EGF-induced retraction of lamellipodia and concomitant cell shape changes. Wild type and mutant EGF receptors were expressed in receptor-deficient NR6 cells. The mutant receptors were prepared by truncation at C' terminal residue 973 (c'973) to provide resistance to ligand-induced down regulation that strongly attenuates receptor signaling and by replacement of threonine 654 (T654) with alanine (A654) to remove the site of phosphorylation by protein kinase C. Cells expressing WT and c'973 EGF receptors demonstrated characteristic lamellipodial retraction after exposure to EGF, with the non-down regulating c'973 EGF receptors responding more rapidly. Exposure of cells to TPA blocked this response. Replacement of T654 by alanine resulted in EGF receptors that were resistant to TPA. Cells expressing the A654 mutation underwent more rapid and more extensive morphologic changes than cells with the corresponding T654 EGF receptor. In cells expressing T654 EGF receptors, down regulation of protein kinase C resulted in more rapid and extensive EGF-induced changes similar to those seen in cells expressing A654 EGF receptors. These data indicate that activation of protein kinase C and subsequent phosphorylation of the EGF receptor at T654 lead to rapid physiological attenuation of EGF receptor signaling. 相似文献
20.
Receptor-mediated vectorial transcytosis of epidermal growth factor by Madin-Darby canine kidney cells 总被引:15,自引:4,他引:11
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Transcellular transport of a variety of ligands may be an important mechanism by which regulatory substances reach their site of action. We have studied the transcellular transport of two 6,000-mol-wt proteins, epidermal growth factor (EGF) and insulin, across polarized Madin-Darby canine kidney (MDCK) cells grown on dual-sided chambers on a nitrocellulose filter substrate. When grown on these chambers, MDCK cells are polarized and express distinct basal and apical surfaces. MDCK cells are capable of unidirectional transport of EGF from the basal-to-apical direction, 50% of bound EGF transported in 2 h. Transport was inhibited by the addition of unlabeled EGF in a dose-dependent manner. Anti-EGF receptor Ab, which inhibited binding, also inhibited transport. No transport in the apical-to-basal direction is noted. Insulin transport is not observed in either direction. Transport correlates with the presence of ligand-specific receptors on the cell surface. Hence, EGF receptors (Ro = 48,000, Kd = 3.5 X 10(-10) M) are found only on the basal surface of the MDCK cells and neither surface expresses insulin receptors. Characterization of the EGF receptors on MDCK cells, as assessed by affinity, molecular mass, and anti-receptor antibody binding reveals that this receptor has similar characteristics to EGF receptors previously described on a variety of cells. Hence, the EGF receptor can function as a transporter of EGF across an epithelial cell barrier. 相似文献