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1.
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
2.
Similarities and differences between the effects of epidermal growth factor and Rous sarcoma virus 总被引:18,自引:5,他引:13
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《The Journal of cell biology》1981,91(3):878-883
We have derived a line of A431 human tumor cells infected with Rous sarcoma virus (RSV). The infected cells contain the RSV-transforming protein, pp60src, which has characteristic tyrosine specific protein kinase activity. As in other RSV-transformed cells, a 36,000-dalton protein is phosphorylated in RSV-infected A431 cells. Addition of epidermal growth factor (EGF) to the cells induces further phosphorylation of this protein. In contrast, this phosphoprotein is not detected in uninfected A431 cells, except when treated with EGF. Increased phosphorylation of the EGF receptor protein and of an 81,000- dalton cellular protein is dependent upon addition of EGF to the culture fluids, in both control and RSV-infected A431 cells. The results are discussed with reference to the similarities and differences between the tyrosine-specific protein kinases induced by RSV and activated by EGF. 相似文献
3.
Regulation of the epidermal growth factor receptor phosphorylation state by sphingosine in A431 human epidermoid carcinoma cells 总被引:4,自引:0,他引:4
M Faucher N Gironès Y A Hannun R M Bell R J Davis 《The Journal of biological chemistry》1988,263(11):5319-5327
The regulation of protein phosphorylation by sphingosine in A431 human epidermoid carcinoma cells was examined. Sphingosine is a competitive inhibitor of phorbol ester binding to protein kinase C (Ca2+/phospholipid-dependent enzyme) and potently inhibits phosphotransferase activity in vitro. Addition of sphingosine to intact A431 cells caused an inhibition of the phorbol ester-stimulated phosphorylation of two protein kinase C substrates, epidermal growth factor (EGF) receptor threonine 654 and transferrin receptor serine 24. We conclude that sphingosine inhibits the activity of protein kinase C in intact A431 cells. However, further experiments demonstrated that sphingosine-treatment of A431 cells resulted in the regulation of the EGF receptor by a mechanism that was independent of protein kinase C. First, sphingosine caused an increase in the threonine phosphorylation of the EGF receptor on a unique tryptic peptide. Second, sphingosine caused an increase in the affinity of the EGF receptor in A431 and in Chinese hamster ovary cells expressing wild-type (Thr654) and mutated (Ala654) EGF receptors. Sphingosine was also observed to cause an increase in the number of EGF-binding sites expressed at the surface of A431 cells. Examination of the time course of sphingosine action demonstrated that the effects on EGF binding were rapid (maximal at 2 mins) and were observed prior to the stimulation of receptor phosphorylation (maximal at 20 mins). We conclude that sphingosine is a potently bioactive molecule that modulates cellular functions by: 1) inhibiting protein kinase C; 2) stimulating a protein kinase C-independent pathway of protein phosphorylation; and 3) increasing the affinity and number of cell surface EGF receptors. 相似文献
4.
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
5.
Epidermal growth factor (EGF) promotes phosphorylation at threonine-654 of the EGF receptor: possible role of protein kinase C in homologous regulation of the EGF receptor 总被引:16,自引:5,他引:11
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Treatment of cells with tumor-promoting phorbol diesters, which causes activation of protein kinase C, leads to phosphorylation of the epidermal growth factor (EGF) receptor at threonine-654. Addition of phorbol diesters to intact cells causes inhibition of the EGF-induced tyrosine-protein kinase activity of the EGF receptor and it has been suggested that this effect of phorbol diesters is mediated by the phosphorylation of the receptor by protein kinase C. We measured the activity of protein kinase C in A431 cells by determining the incorporation of [32P]phosphate into peptides containing threonine-654 obtained by trypsin digestion of EGF receptors. After 3 h of exposure to serum-free medium, A431 cells had no detectable protein kinase C activity. Addition of EGF to these cells resulted in [32P] incorporation into threonine-654 as well as into tyrosine residues. This indicates that EGF promotes the activation of protein kinase C in A431 cells. The phosphorylation of threonine-654 induced by EGF was maximal after only 5 min of EGF addition and the [32P] incorporation into threonine-654 reached 50% of the [32P] in a tyrosine-containing peptide. This indicates that a significant percentage of the total EGF receptors are phosphorylated by protein kinase C. A variety of external stimuli activate Na+/H+ exchange, including EGF, phorbol diesters, and hypertonicity. To ascertain whether activation of protein kinase C is an intracellular common effector of all of these systems, we measured the activity of protein kinase C after exposure of A431 cells to hyperosmotic conditions and observed no effect on phosphorylation of threonine-654, therefore, activation of Na+/H+ exchange by hypertonic medium is independent of protein kinase C activity. Since stimulation of protein kinase C by phorbol diesters results in a decrease in EGF receptor activity, the stimulation of protein kinase C activity by addition of EGF to A431 cells contributes to a feedback mechanism which results in the attenuation of EGF receptor function. 相似文献
6.
Internalization of functional epidermal growth factor:receptor/kinase complexes in A-431 cells 总被引:7,自引:0,他引:7
We have isolated and partially purified an intracellular vesicle fraction from A-431 cells that contains both epidermal growth factor (EGF) and enzymatically active EGF:receptor/kinase. Exposure of intact A-431 cells to EGF leads to an accumulation of both EGF and kinase activity in this vesicle fraction. The accumulation is time- and temperature-dependent and is blocked by inhibitors of energy production. The EGF receptor in internalized vesicles is capable of autophosphorylation and, in the presence of Ca2+, of phosphorylation of the previously isolated 35-kDa protein (Fava, R. A., and Cohen, S. (1984) J. Biol. Chem. 259, 2636-2645). The demonstration of an EGF-induced increase in kinase activity of an internalized vesicle fraction lends credence to the hypothesis that EGF-induced endocytosis of the receptor is of physiological significance in the response of cells to this ligand. In addition, these results are consistent with the suggestion that the phosphorylation of the 35-kDa protein is associated with internalization of the EGF:receptor/kinase complex. 相似文献
7.
Comparison of the 34,000-Da pp60src substrate and a 38,000-Da phosphoprotein identified by monoclonal antibodies 总被引:14,自引:0,他引:14
One of the cellular targets of the pp60src tyrosine kinase is a phosphoprotein with a Mr = 34,000 and an isoelectric point of approximately 7.5 (Radke, K., Gilmore, T., and Martin, G. S. (1980) Cell 21, 821-828; Erikson, E., and Erikson, R. L. (1980) Cell 21, 829-836). We report here the preparation of monoclonal antibodies to partially purified 34-kDa protein and to a heretofore unrecognized phosphoprotein that is not a pp60src target. Two antibodies were initially obtained that recognized phosphoproteins in the Mr = 34,000-39,000 range. One of these antibodies immunoprecipitated a 34,000-Da protein which, on the basis of its molecular mass, phosphorylation state, and isoelectric point, was determined to be the 34-kDa pp60src substrate. The second monoclonal antibody bound to a 38,000-Da nucleolar associated protein, which appeared not to be a target of the pp60src kinase and was found by tryptic analysis to be structurally unrelated to the 34-kDa protein. The monoclonal antibody to the 34-kDa protein coupled to Sepharose CL-4B was used to purify the pp60src substrate to homogeneity in milligram quantities. Both the purified 34-kDa protein and the monoclonal antibody are currently being used in studies aimed at elucidating the structure and function of this pp60src target. 相似文献
8.
Epidermal-growth-factor-stimulated phosphorylation of calpactin II in membrane vesicles shed from cultured A-431 cells. 总被引:2,自引:1,他引:1
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Membrane vesicles shed from intact A-431 epidermoid carcinoma cells and harvested in the presence of Ca2+ contained epidermal-growth-factor (EGF) receptor/kinase substrates of apparent molecular masses 185, 85, 70, 55, 38 and 27 kDa. The 38 kDa substrate (p38) was recognized by an antibody that had been raised against the human placental EGF receptor/kinase substrate calpactin II (lipocortin I). The A-431 and placental substrates, isolated by immunoprecipitation after phosphorylation in situ, yielded identical phosphopeptide maps upon limited proteolytic digestion with each of five different enzymes. The A-431-cell vesicular p38 is therefore calpactin II. EGF treatment of the intact A-431 cells before inducing vesiculation was not necessary for the substrate to be present within the vesicles. Our data thus indicate that receptor internalization is not a prerequisite for receptor-mediated phosphorylation of calpactin II. The ability of the protein to function as a substrate for the receptor/kinase depended upon the continued presence of Ca2+ during the vesicle-isolation procedure. EGF-stimulated phosphorylation of calpactin II was much less pronounced in vesicles prepared from A-431 cells in the absence of Ca2+, although comparable amounts of the protein were detectable by immunoblotting. Calpactin II therefore appears to be sequestered in a Ca2+-modulated manner within shed vesicles, along with at least four other major targets for the EGF receptor/kinase. The vesicle preparation may be a useful model system in which to study the phosphorylation and function of potentially important membrane-associated substrates for the receptor. 相似文献
9.
Stimulation of a MR 80,000 protein phosphorylation by EGF in EGF receptor-hyperproducing human tumor cells 总被引:2,自引:0,他引:2
NA and Ca9-22 cells derived from squamous cell carcinomas of the tongue possess a large number of epidermal growth factor (EGF) receptors (2.0 X 10(6) and 1.3 X 10(6) receptors/cell, respectively). In these cell lines, EGF stimulated receptor autophosphorylation and phosphatidylinositol (PI) turnover. Furthermore, EGF enhanced the phosphorylation of an acidic protein of Mr 80,000. Phosphorylation of this protein was also stimulated by 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a phorbol ester tumor promoter, and was mainly at serine residues. Phosphopeptide mapping using protease V8 or trypsin indicated that Mr 80,000 proteins isolated from the EGF- and TPA-treated cells were identical. The Mr 80,000 protein was present mainly in the cytosol, but it became closely associated with the membrane as a phosphorylated form upon EGF or TPA stimulation. These results suggest that the EGF-stimulated phosphorylation of the Mr 80,000 acidic phosphoprotein in EGF receptor-hyperproducing tumor cells is mediated through the activation of PI turnover and protein kinase C. 相似文献
10.
Exposure of A-431 cells to epidermal growth factor (EGF) results in a rapid enhancement (approximately 10-fold) of cytosolic serine protein kinase activity. The increase in serine kinase activity may be detected using a number of peptide and protein substrates. Enhancement of kinase activity occurs within 1 min of exposure of the cells to EGF and reaches a maximum in 5 min. Similar results were obtained with a variety of cell lines. We have partially purified the EGF-activated kinase from A-431 cells. It has an apparent molecular mass of approximately 100 kDa by gel filtration. One distinguishing property of the enzyme is its sensitivity to inhibition by micromolar quantities of polyarginine; polylysine has no effect. The EGF-activated kinase is unaffected by cyclic nucleotides, Ca2+/calmodulin, Ca2+/diolein/phosphatidylserine, or heparin. The enhancement of cytosolic serine kinase activity in A-431 cells appears to be an early event in cell "activation" by a number of biological response modifiers including EGF, bradykinin, 12-O-tetradecanoylphorbol-13-acetate, and histamine. 相似文献
11.
Affinity labeling of the protein kinase associated with the epidermal growth factor receptor in membrane vesicles from A431 cells 总被引:25,自引:0,他引:25
Epidermal growth factor (EGF), a mitogenic polypeptide hormone, stimulates the phosphorylation of certain endogenous proteins in membrane preparations derived from A431 cells, a human tumor cell line. Membrane vesicles prepared from A431 cells were reacted with 5'-p-fluorosulfonylbenzoyl adenosine (5'-p-FSO2BzAdo). Reaction of the vesicles with 5'-p-FSO2BzAdo results in a time-dependent inhibition of EGF-stimulable protein kinase activity which parallels an increase in incorporation into the vesicles of the 5'-p-sulfonylbenzoyl-[8-14C]adenosine moiety from 5'-p-FSO2Bz[14C]Ado. The primary bands labeled have Mr = 170,000 and 150,000. Labeling of these bands by 5'-p-FSO2Bz[14C]Ado is inhibited by incubation of the membrane vesicles with adenyl-5'-yl imidodiphosphate, an ATP analog. Inactivation of the kinase with N-ethylmaleimide or by heating results in a sharply decreased labeling of the proteins with Mr = 170,000 and 150,000. Proteins of these molecular weights have previously been identified in these cells as the EGF receptor and a degradation product of the receptor. These experiments provide chemical evidence that the EGF receptor and the EGF-stimulable kinase are the same protein. 相似文献
12.
In contrast to the intact EGF, a cyanogen bromide derivative of EGF (EGF-CNBr) does not induce an increase in uridine phosphorylation rate in 3T3 cells, the ability of the EGF-CNBr to stimulate autophosphorylation of the EGF-receptor in A-431 cells being reserved. EGF and EGF-CNBr were used in concentrations promoting their equivalent binding with EGF receptor in both the series of experiments, which was necessary because of a decreased affinity to binding EGF-CNBr. Thus, the EGF-induced receptor autophosphorylation was not enough for uridine kinase activation. The differences between EGF and EGF-CNBr cellular processing made it possible to discuss potential ways of uridine-kinase activity regulation during the early period of stimulation of quiescent cell cultures. 相似文献
13.
A Sahai K B Smith M Panneerselvam D S Salomon 《Biochemical and biophysical research communications》1982,109(4):1206-1214
A calcium and phospholipid-dependent protein kinase (protein kinase C) was detected in the crude soluble extracts of A431 human epidermoid carcinoma cells. The enzyme required calcium, phosphatidylserine or phosphatidylinositol, and diacylglycerol (DG) for maximal activation. Protein kinase C phosphorylated both endogenous cytosolic proteins and various histones. Addition of epidermal growth factor (EGF) to A431 cultures resulted in a 2 to 3-fold stimulation of protein kinase activity. 12-0-tetradecanoylphorbol-13-acetate (TPA) in concert with EGF attenuated the EGF-induced enhanced phosphorylation of endogenous proteins. It is conceivable that DG, derived from phosphatidylinositol turnover, acts as a natural activator of protein kinase C activity. 相似文献
14.
Epidermal growth factor stimulates the phosphorylation of the calcium-dependent 35,000-dalton substrate in intact A-431 cells 总被引:18,自引:0,他引:18
We have previously reported the isolation of a 35-kDa protein from A-431 cells that, in the presence of Ca2+, is an excellent in vitro substrate for the epidermal growth factor (EGF) receptor/kinase present in membrane preparations (Fava, R. A., and Cohen, S. (1984) J. Biol. Chem. 259, 2636-2645). In this communication we demonstrate that the phosphorylation of the 35-kDa protein is markedly enhanced in intact, 32P-labeled, A-431 cells following exposure of the cells to EGF. The 35-kDa protein immunoprecipitated from cells treated with EGF is phosphorylated to a 20-120-fold greater extent than comparable preparations from control cells. Both phosphotyrosine and phosphoserine residues are detected in the protein after treatment of the cells with EGF. EGF-dependent phosphorylation of the 35-kDa protein is barely detected unless the intact cells are exposed to EGF for periods greater than 5 min. We suggest that endosomes containing internalized EGF X receptor/kinase complexes are primarily responsible for the observed phosphorylation of the 35-kDa protein in intact cells. 相似文献
15.
C-kinase phosphorylates the epidermal growth factor receptor and reduces its epidermal growth factor-stimulated tyrosine protein kinase activity 总被引:84,自引:0,他引:84
C Cochet G N Gill J Meisenhelder J A Cooper T Hunter 《The Journal of biological chemistry》1984,259(4):2553-2558
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. 相似文献
16.
The receptor for epidermal growth factor (EGF) is a glycosylated transmembrane phosphoprotein that exhibits EGF-stimulable protein tyrosine kinase activity. On EGF stimulation, the receptor undergoes a self-phosphorylation reaction at tyrosine residues located primarily in the extreme carboxyl-terminal region of the protein. Using enzymatically active EGF receptor purified by immunoaffinity chromatography from A431 human epidermoid carcinoma cells, the self-phosphorylation reaction has been characterized as a rapid, intramolecular process which is maximal at 30-37 degrees C and exhibits a very low Km for ATP (0.2 microM). When phosphorylation of exogenous peptide substrates was measured as a function of receptor self-phosphorylation, tyrosine kinase activity was found to be enhanced two to threefold at 1-2 mol of phosphate per mol of receptor. Analysis of the dependence of the tyrosine kinase activity on ATP concentration yielded hyperbolic kinetics when plotted in double-reciprocal fashion, indicating that ATP can serve as an activator of the enzyme. Higher concentrations of peptide substrates were found to inhibit both the self- and peptide phosphorylation, but this inhibition could be overcome by first self-phosphorylating the enzyme. These results suggest that self-phosphorylation can remove a competitive/inhibitory constraint so that certain exogenous substrates can have greater access to the enzyme active site. In addition to self-phosphorylation, the EGF receptor can be phosphorylated on threonine residues by the calcium- and phospholipid-dependent protein kinase C. The sites on the EGF receptor phosphorylated in vitro by protein kinase C are identical to the sites phosphorylated on the receptor isolated from A431 cells exposed to the tumor promoters 12-O-tetradecanoylphorbol 13-acetate or teleocidin. This phosphorylation of the EGF receptor results in a suppression of its tyrosine kinase and EGF binding activities both in vivo and in vitro. The EGF receptor can thus be variably regulated by phosphorylation: self-phosphorylation can enhance tyrosine kinase activity whereas protein kinase C-catalyzed phosphorylation can depress enzyme activity. Because these two phosphorylations account for only a fraction of the phosphate present in the EGF receptor in vivo, other protein kinases can apparently phosphorylate the receptor and these may exert additional controls on EGF receptor/kinase function. 相似文献
17.
BACKGROUND/AIMS: Phosphatases are involved in regulation of MAP kinase (MAPK). A431 cells migrate on collagen after EGF stimulation using MAPK. To clarify the involvement of PP2A in this MAPK-dependent migration, the expression of an isoform of the B regulatory subunit was inhibited. METHODS: An antisense sequence corresponding to Bbeta cDNA was transfected into A431 cells. Their migratory activity on collagen was examined using Transwell, and MAPK phosphorylation and phosphatase activity were measured, and the results were compared with those obtained with mock-transfected cells. RESULTS: Antisense-transfected cells showed less Bbeta protein and phosphatase activity than mock-transfected controls. Migration of antisense-transfected cells showed a low response to EGF. The response of MAPK phosphorylation of antisense-transfected cells to EGF stimulation and adhesion to collagen in the presence or absence of EGF were markedly decreased. Phosphatase activity of PP2A-Bbeta also did not respond to EGF, collagen or EGF plus collagen, and remained at low levels. CONCLUSION: These results suggested that PP2A-Bbeta promotes cell migration through the MAPK cascade. 相似文献
18.
Regulation of casein kinase II activity by epidermal growth factor in human A-431 carcinoma cells 总被引:15,自引:0,他引:15
In order to characterize more fully the mechanism by which casein kinase II is regulated in mammalian cells, the effect of epidermal growth factor (EGF) on the activity of the kinase in human A-431 carcinoma cells was examined. Treatment of cells with EGF prior to lysis consistently resulted in a transient 4-fold increase in the activity of cytosolic casein kinase II. Activity rose sharply between 20 and 30 min, peaked at approximately 50 min, and returned to basal levels by approximately 120 min. Similar results were obtained using the casein kinase II specific peptide substrate, Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu, or DNA topoisomerase II (which is specifically modified by the kinase in vivo and serves as a high affinity substrate in vitro) as the phosphate acceptor in assays. Identification of casein kinase II as the stimulated activity was confirmed by partial proteolytic mapping and phosphoamino acid analysis of modified topoisomerase II, by inhibition at nanomolar levels of heparin or micromolar levels of nonradioactive GTP, and by the ability to employ radioactive GTP as a direct phosphate donor. The EGF stimulation of casein kinase II was dependent on the availability of intracellular (but not extracellular) calcium. In addition, hormonal action was modulated by calcium/phospholipid-dependent protein kinase (protein kinase C). Casein kinase II stimulation did not require an increase in the concentration of the kinase, protein synthesis, the continual presence of a small effector molecule, or a direct interaction with the EGF receptor/tyrosine kinase. In contrast, hormonal activation of the kinase was dependent on the phosphorylation of casein kinase II or a terminal stimulatory factor. 相似文献
19.
Regulation of epidermal growth factor-stimulated formation of inositol phosphates in A-431 cells by calcium and protein kinase C 总被引:15,自引:0,他引:15
Epidermal growth factor (EGF) treatment of A-431 cells induces a biphasic increase in the levels of inositol phosphates. The growth factor produces an initial, rapid increase in the level of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) due to hydrolysis of phosphatidyl-inositol-4,5-bisphosphate (Wahl, M., Sweatt, J. D., and Carpenter, G. (1987) Biochem. Biophys. Res. Commun. 142, 688-695). The level of inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4) also rises rapidly in response to treatment with EGF. The initial formation (less than 1 min) of Ins-1,4,5-P3 and Ins-1,3,4,5-P4 does not require Ca2+ present in the culture medium. However, the addition of Ca2+ to the medium at levels of 100 microM or greater potentiates the growth factor-stimulated increases in the levels of all inositol phosphates at later times after EGF addition (1-60 min). The data suggest that EGF-receptor complexes initially stimulate the enzyme phospholipase C in a manner that is independent of an influx of extracellular Ca2+. The presence of Ca2+ in the medium allows prolonged growth factor activation of phospholipase C. Treatment of A-431 cells with Ca2+ ionophores (A23187 and ionomycin) did not mimic the activity of EGF in producing a rapid increase in the formation of the Dowex column fraction containing Ins-1,4,5-P3, Ins-1,3,4,5-P4, and inositol 1,3,4-trisphosphate (InsP3). However, the initial EGF-stimulated formation of inositol phosphates was substantially diminished in cells loaded with the Ca2+ chelator Quin 2/AM. EGF receptor occupancy studies indicated that maximal stimulation of InsP3 accumulation by EGF requires nearly full (75%) occupancy of available EGF binding sites, while half-maximal stimulation requires 25% occupancy. 12-O-Tetradecanoylphorbol-13-acetate (TPA), an exogenous activator of Ca2+/phospholipid-dependent protein kinase (protein kinase C), causes a dramatic, but transient, inhibition of the EGF-stimulated formation of inositol phosphates. Tamoxifen and sphingosine, reported pharmacologic inhibitors of protein kinase C activity, potentiate the capacity of EGF to induce formation of inositol phosphates. Neither TPA nor tamoxifen significantly affects the 125I-EGF binding capacity of A-431 cells; however, TPA appeared to enhance internalization of the ligand. Ligand occupation of the EGF receptor on the A-431 cell appears to initiate a complex signaling mechanism involving production of intracellular messengers for Ca2+ mobilization and activation of protein kinase C.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
20.
Addition of amiloride to A431 human epidermoid carcinoma cell membranes inhibited autophosphorylation of the epidermal growth factor (EGF) receptor. The tyrosine phosphorylation of histone H2B catalyzed by an affinity-purified preparation of EGF receptor was also inhibited by amiloride. The inhibition was noncompetitive with respect to histone but competitive with ATP, suggesting that amiloride may act as an ATP analogue which causes the formation of nonproductive enzyme-substrate complexes. The tyrosine phosphorylation of histone H2B catalyzed by the purified EGF receptor was inhibited by amiloride at concentrations identical to those previously reported to block EGF action on cell proliferation (Ki = 350 microM). Amiloride similarly inhibited the tyrosine phosphorylation of the human placental insulin receptor and the platelet-derived growth factor receptor of Swiss 3T3 cells. Immunoprecipitation of the EGF receptor from A431 cells labeled for 24 h with [32P]phosphate demonstrated that amiloride decreased the phosphorylation of the EGF receptor on serine and threonine residues and blocked the effect of EGF to cause phosphorylation of the receptor on tyrosine residues. Phosphoamino acid analysis of total cell proteins indicated that amiloride inhibited the increase in phosphotyrosine levels caused by EGF. We conclude that amiloride directly inhibits the tyrosine kinase activity of the receptors for EGF, insulin, and platelet-derived growth factor in in vitro and can mediate such actions in vivo. This effect of amiloride demonstrates that it is unsuitable as a drug to test the hypothesis that the stimulation of the Na+/H+ antiporter is essential for mitogenic signaling by growth factor receptors. 相似文献