Cellular progesterone receptor phosphorylation in response to ligands activating protein kinases

Progesterone receptors were immunoprecipitated with monoclonal antibodies KD68 from lysates of human breast carcinoma T47D cells labelled to steady state specific activity with 32Pi. The 120 kDa 32P-labelled progesterone receptor band was resolved by polyacrylamide gel electrophoresis and identified by autoradiography. Phosphoamino acid analysis revealed serine phosphorylation, but no threonine or tyrosine phosphorylation. Treatment of the 32Pi-labelled cells with EGF, TPA or dibutyryl cAMP had no significant quantitative effect on progesterone receptor phosphorylation, though the EGF receptor and the cAMP-dependent protein kinases have been reported to catalyze phosphorylation of purified avian progesterone receptor preparations in cell free systems. Progesterone receptor phosphorylation on serine residues was increased by 2-fold in cells treated with 10 nM progesterone; EGF had no effect on progesterone-mediated progesterone receptor phosphorylation.     

Activation of the epidermal growth factor receptor tyrosine protein kinase in the absence of receptor oligomerization

The epidermal growth factor (EGF) receptor exists in a monomeric (170 kDa) form and in several aggregated states (360 kDa, greater than 500 kDa). The hypothesis that the oligomerization of the receptor is required for the stimulation of the kinase was tested by correlating the oligomeric state of the receptor with the protein kinase activity. EGF and sphingosine stimulate the phosphorylation of an exogenous peptide substrate by the receptor to an equal extent. Chemical cross-linking using disuccinimidyl suberate and the analysis of EGF receptor complexes by Western blotting demonstrated that EGF caused the aggregation of receptors. Similar results were obtained when [32P]phosphate-labeled receptors were cross-linked using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. These results were confirmed by sucrose density gradient sedimentation analysis. In contrast to the effects of EGF, incubation of EGF receptors with sphingosine did not cause the oligomerization of the receptors. These data demonstrate that the EGF receptor kinase can be stimulated independently of the aggregation of the receptors.     

Epidermal growth factor: relationship between receptor down regulation in cultured NRK cells and epidermal growth factor enhancement of phosphorylation of a 170000 molecular weight membrane protein in vitro

Incubation of confluent nondividing NRK cells in serum-free media with unlabeled epidermal growth factor (EGF) leads to a reduction in the specific binding capacity for 125I-labeled EGF. This modulation of the binding capacity for 125I-labeled EGF by unlabeled EGF, termed receptor down regulation, was dependent on EGF concentration and time. Membranes from untreated NRK cells have a phosphorylating system which catalyzed in vitro the phosphorylation of numerous membrane components; this phosphorylating system was stimulated by EGF. Although EGF enhanced the phosphorylation of many membrane proteins, one major component with Mr 170K and a minor band of Mr 150K were primarily affected. A comparison of the membrane phosphoproteins of untreated and down-regulated cells by in vitro phosphorylation and NaDodSO4 gel electrophoresis revealed that down regulation of EGF receptors results in a specific decrease in 32P phosphorylation of the 170K- and 150K-dalton components to subsequent stimulation with EGF in vitro. We further characterized the modulation of phosphorylation of the 170K protein by down regulation with EGF and found it to be dependent on EGF concentration and time. These studies demonstrated a correlation between the loss of 125I-labeled EGF binding activity by the cells and the loss of the vitro EGF-dependent 32P phosphorylation of the 170K-dalton membrane protein. In addition, the results suggest that the major 170K Mr phosphoprotein band is a component of the receptor for EGF which is a substrate of the phosphorylation reaction.     

Evidence that the tyrosine kinase domain of a small fraction of epidermal growth factor receptor molecules is exposed on the outer surface of A431 cells

Intact A431 cells were labeled with [gamma-32P]ATP. The major phosphorylation product of the ecto-kinase activity of A431 cells had the molecular mass of 170 kd and was identified as EGF receptor by specific immunoprecipitation. This phosphorylation was not stimulated by EGF added to the reaction buffer, but replacement of MgCl2 by MnCl2 in the buffer remarkably stimulated phosphorylation. An exogenous protein substrate, alpha-casein, was also phosphorylated by intact A431 cells. The analyses for phospho-amino acids of both EGF receptor and alpha-casein revealed that phosphorylation occurred mainly at phosphotyrosine residues. Tryptic phospho-peptides of the EGF receptor of intact A431 cells labeled with [gamma-32P]ATP were fractionated by HPLC. The elution patterns were essentially the same as that of the autophosphorylated EGF receptor, indicating that the phosphorylation sites of EGF receptor labeled in vivo with [gamma-32P]ATP are located in three tyrosine residues in the carboxyl terminus. These results indicate that the carboxyl-terminal tyrosine kinase domain of a small fraction of the EGF receptor molecules of an A431 cell is exposed on the outer surface of the cells.     

High specific induction of spermidine/spermine N1-acetyltransferase in a human large cell lung carcinoma.

The effects of insulin and anti-(insulin receptor) monoclonal antibodies on tyrosine phosphorylation were investigated in fibroblasts transfected with human insulin receptor cDNA (NIH 3T3HIR3.5 cells) using anti-phosphotyrosine immunoblotting. Insulin increased levels of tyrosine phosphorylation in two major proteins of molecular mass 97 kDa (pp97, assumed to be the insulin receptor beta-subunit) and 185 kDa (pp185). Insulin-mimetic anti-receptor antibodies also stimulated tyrosine phosphorylation of both pp97 and pp185. The observation of antibody-stimulated pp97 phosphorylation, as detected by immunoblotting, is in contrast with previous data which failed to show receptor autophosphorylation in NIH 3T3HIR3.5 cells labelled with [32P]P1. The effect of insulin on pp97 was maximal within 1 min, but the response to antibody was apparent only after a lag of 1-2 min and rose steadily over 20 min. The absolute level of antibody-stimulated phosphorylation of both pp97 and pp185 after 20 min was only about 20% of the maximum level induced by equivalent concentrations of insulin, even at concentrations of antibody sufficient for full occupancy of receptors. Another insulin-mimetic agent, wheat-germ agglutinin, stimulated receptor autophosphorylation with kinetics similar to those produced by the antibody. It is suggested that the relatively slow responses to both agents may be a function of the dependence on receptor cross-linking. These data are consistent with a role for the insulin receptor tyrosine kinase activity in the mechanism of action of insulin-mimetic anti-receptor antibodies.     

Epidermal growth factor dependent phosphorylation of a 35-kilodalton protein in placental membranes

In human placental membranes isolated in the presence of ethylenediaminetetraacetic acid (EDTA), epidermal growth factor (EGF) stimulated the [gamma-32P]ATP-dependent phosphorylation of tyrosine residues on the 170-kilodalton (kDa) EGF receptor and on a 35-kDa protein. The initial rate of phosphorylation of these proteins in the presence of EGF was 5.2 and 3.5 nmol of phosphate min-1 (mg of receptor protein)-1, and this was approximately 10- and 6-fold higher than the basal rate, respectively. Half-maximal phosphorylation of both proteins occurred at about 2.5 nM EGF. In the presence of p-nitrophenyl phosphate, EGF stimulated the phosphorylation of the 35-kDa protein but not the EGF receptor, suggesting that hormone-stimulated autophosphorylation of the receptor/kinase was not required for kinase activation. The 35-kDa protein exists in two forms: (1) 35Keluate, which was associated with the membrane in the presence of Ca2+ but was eluted with EDTA, and (2) 35Kmemb, which was not eluted from membranes with EDTA. Both forms were immunologically related to a 35-kDa protein previously isolated from A431 cells. Antiserum against the 35-kDa protein also reacted with a protein with an apparent size of 66 kDa that was phosphorylated in an EGF-dependent manner. In phosphorylation reactions performed in the presence of Mg2+, Ca2+ was required for phosphorylation of the 35Keluate form, but Ca2+ was not required for phosphorylation of the 35Kmemb form. Phosphorylation appears to change the membrane-binding properties of the 35Kmemb form because 32P-labeled 35Kmemb could be eluted from the membrane by EDTA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epidermal growth factor treatment of A431 cells alters the binding capacity and electrophoretic mobility of the cytoskeletally associated epidermal growth factor receptor

The epidermal growth factor (EGF) receptor interacts with structural elements of A431 cells and remains associated with the cytoskeleton following extraction with nonionic detergents. Extraction of cells with 0.15% Triton X-100 resulted in detection of only approximately 40% of the EGF binding sites on the cytoskeleton. If the cells were exposed to EGF prior to extraction, approximately twofold higher levels of low-affinity EGF binding sites were detected. The difference in number of EGF binding sites was not a consequence of differences in numbers of EGF receptors associated with the cytoskeleton; equal amounts of 35S-labeled receptor were immunoprecipitated from the cytoskeletons of both control and EGF-treated cells. The effect of EGF pretreatment on binding activity was coincident with a change in the mobility of the receptor from a doublet of Mr approximately 160-180 kDa to a single sharp band at 180 kDa. The alteration in receptor mobility was not a simple consequence of receptor phosphorylation in that the alteration was not reversed by alkaline phosphatase treatment, nor was the shift produced by treatment of the cells with phorbol ester. The two EGF receptor species demonstrated differential susceptibility to V8 proteinase digestion. The EGF-induced 180 kDa species was preferentially digested by the proteinase relative to the 160 kDa species, indicating that EGF binding results in a conformational change in the receptor. The EGF-mediated preservation of binding activity and altered conformation may be related to receptor oligomerization.     

Examination of the role of serine phosphorylation in phospholipase C-gamma and its related P47 in cAMP-mediated depression of epidermal growth factor signal transduction.

Forskolin-pretreatment of A431 cells reduced both intrinsic and epidermal growth factor (EGF)-induced EGF receptor phosphorylation, however, phosphorylation of phospholipase C-gamma (PLC-gamma) was stimulated under the same conditions. No significant difference was detected in the amount of phosphotyrosine of PLC-gamma between two cultures with or without forskolin treatment followed by EGF. On the other hand, phosphorylation of a 47 kDa protein (P47) which cross-reacted with an anti-PLC-gamma monoclonal antibody, was stimulated by both forskolin and EGF. Phosphorylation was exclusively on serine residues in this case. These results indicate that both PLC-gamma and P47 are phosphorylated by a cAMP-dependent protein kinase and the EGF-stimulated serine kinase, and suggest that serine phosphorylation of PLC-gamma has no effect on ligand-dependent coupling with the EGF receptor.     

Tumor-promoting phorbol diesters mediate phosphorylation of the epidermal growth factor receptor

4 beta-Phorbol 12 beta-myristate 13 alpha-acetate (PMA) markedly inhibited the binding of low concentrations (less than 10(-9 m) of 125I-epidermal growth factor (EGF) to A431 human epidermoid carcinoma cells. However, very little change in the binding of 125-I-EGF at high concentrations (greater than 10(-8) M) was observed in response to PMA. Affinity labeling of the 170,000-dalton EGF receptor with 125I-EGF and disuccinimidyl suberate was also decreased by the tumor promoter at low, but not high, concentrations of 125I-EGF. In order to examine this action of PMA on the EGF receptor, the receptor phosphorylation state was evaluated in A431 cells that had been incubated with [32P]phosphate for 3 h prior to the addition of PMA. The 32P content of the EGF receptor purified with EGF-Sepharose was increased by 38% compared with the same amount of receptor isolated from control cells. The increase in EGF receptor phosphorylation was dose-dependent with a half-maximal effect between 0.1 and 1 nM PMA and was specific for tumor promoting analogues of phorbol diesters. Phosphoamino acid analysis indicated that the increase in the 32P content of the EGF receptor was mainly due to phosphoserine. These results demonstrate that the EGF receptor is a target for PMA action and suggest that the mechanism of PMA action on the response of cells to epidermal growth factor may be mediated in part by phosphorylation of the EGF receptor.     

Regulation of epidermal growth factor receptor number and phosphorylation by fasting in rat liver

The binding of 125I-epidermal growth factor (EGF) to microsomal membrane preparations from the livers of rats fasted for 72 h or fed control or high carbohydrate diets was examined to determine whether alterations in nutrient intake could affect the EGF receptor system. Fasted rats had 40-50% less membrane binding than did control or carbohydrate-fed rats. Scatchard analysis of the binding data indicated that the decrease in EGF binding in fasted rats was due to a decrease in receptor number with no change in receptor affinity. Cross-linking of 125I-EGF to EGF receptors with disuccinimidyl suberate revealed specific binding of a Mr 170,000 protein, which was diminished by approximately 75% in fasting, and a Mr = 150,000 protein, which accounted for 40-50% of the total labeling in the control and carbohydrate-fed rats and which was relatively unchanged by fasting. The sum of the labeling of the 2 bands was reduced by approximately 40% in fasting and is consistent with the reduction in EGF binding detected by Scatchard analysis. EGF stimulated a 1.5-3-fold increase in 32P incorporation into one major protein of 170 kDa in all 3 groups. Basal and EGF-stimulated autophosphorylation of 170 kDa, when normalized for protein, was 75% lower in membranes from fasted animals, compared to those from control or carbohydrate-fed rats. The comparable reduction of 125I-EGF binding to, and 32P incorporation into, the 170-kDa EGF receptor protein suggested that kinase activity/receptor was unaffected by fasting. Moreover, EGF receptor kinase activity in the 3 groups was comparable for an exogenous substrate, as judged by equal basal and EGF-stimulated phosphorylation of Val5-angiotensin II, when normalized for total EGF-binding capacity. These results suggest that fasting regulates EGF receptor kinase activity primarily by regulation of the number of hepatic EGF receptors. The possibility exists that some in vivo effects of fasting may be mediated by a reduction in EGF receptor levels.     

EGF induces tyrosine phosphorylation of phospholipase C-II: a potential mechanism for EGF receptor signaling

Binding of EGF to cells expressing human EGF receptor stimulated rapid tyrosine phosphorylation of phospholipase C-II (PLC-II), as revealed by immunoblotting analysis with phosphotyrosine-specific antibodies. Tyrosine phosphorylation of PLC-II was stimulated by low physiological concentrations of EGF (1 nM), was quantitative, and was already maximal after a 30 sec incubation with 50 nM EGF at 37 degrees C. Interestingly, antibodies specific for PLC-II were able to coimmunoprecipitate the EGF receptor and antibodies against EGF receptor also coimmunoprecipitated PLC-II. According to this analysis, approximately 1% of EGF receptor molecules were associated with PLC-II molecules. The protein tyrosine kinase inhibitor tyrphostin RG50864, which blocks EGF-dependent cell proliferation, blocked EGF-induced tyrosine phosphorylation of PLC-II, its association with EGF receptor, and EGF-induced Ca2+ release. Hence, EGF-induced tyrosine phosphorylation of PLC-II may be a regulatory event linking the tyrosine kinase activity of EGF receptor to the PIP2 hydrolysis signaling pathway.     

Epidermal growth factor stimulates tyrosine phosphorylation of specific proteins in permeabilized human fibroblasts

We have investigated the epidermal growth factor (EGF)-stimulated tyrosine-specific protein kinase activity in quiescent cultures of diploid human fibroblasts that have a well characterized mitogenic response to EGF. We developed a method of permeabilizing cells with digitonin or other agents that permitted the rapid labeling of cellular proteins with exogenously added [gamma-32P]ATP while allowing only about 25% of marker cytosolic enzymes to escape from the cells. When phosphatases were inhibited with zinc and vanadate, EGF induced up to 8-fold stimulation of the incorporation of radioactivity from [gamma-32P]ATP into a 35-kDa band on sodium dodecyl sulfate gels. Alkali treatment of gels showed that EGF stimulated the phosphorylation of bands with apparent molecular masses of 170, 45, 35, 26, 22, and 21 kDa. Phosphoamino acid analysis was performed on the 170- and 35-kDa bands and revealed that the EGF-stimulated phosphorylation was on tyrosyl residues. The 35-kDa band was resolved into four spots by two-dimensional gel electrophoresis. The most acidic form was the most prominent and it was precipitated by an antiserum against a 35-kDa protein from A-431 cells; heretofore, this protein has only been reported to be phosphorylated in an EGF-dependent manner by A-431 membranes in vitro (Fava, R. A., and Cohen, S. (1984) J. Biol. Chem. 259, 2636-2645). This antiserum also precipitated a 35-kDa phospho-protein from extracts of intact [32P]orthophosphate-labeled fibroblasts which was phosphorylated on tyrosine in an EGF-dependent manner. None of the forms of the 35-kDa phosphoproteins labeled in permeabilized cells were immunologically related to the 34-kDa protein that is a substrate for the tyrosyl kinase encoded by Rous sarcoma virus. Other mitogens (serum, insulin, platelet-derived growth factor, and thrombin) did not detectably stimulate phosphorylation in permeabilized cells.     

Ethanol-induced structural and functional alterations of epidermal growth factor receptor in buccal mucosa.

1. Ethanol treatment caused a 47% decrease in [125I]EGF binding to the membrane preparations of buccal mucosa resulting from the decrease of binding sites rather than the affinity of high affinity receptor. 2. The immunoblot revealed a protein band of 170 kDa in the control sample, while a barely detectable 200 kDa protein was observed in the ethanol-treated sample. 3. Protein kinase assays using [32P]ATP as probe showed an EGF-stimulated phosphorylation band of 170 kDa in the control but not in ethanol-treated sample. 4. Ethanol did not change the basal incorporation of [3H]thymidine and [35S]methionine, but caused a 38% and 57% decrease of EGF-stimulated thymidine and methionine incorporation, respectively. 5. The data suggest that EtOH decreases EGF receptor binding through modification of the receptor molecule, thereby impairing receptor kinase activity and its physiological function.     

Epidermal growth factor and potent phorbol tumor promoters induce epidermal growth factor receptor phosphorylation in a similar but distinctively different manner in human epidermoid carcinoma A431 cells

When human epidermoid carcinoma A431 cells labeled with 32Pi to steady state specific activity were treated either with epidermal growth factor (EGF) or with active phorbol ester tumor promoters such as 12-O-tetradecanoylphorbol 13-acetate (TPA) or phorbol 12,13-dibutyrate, labeling of 160 kDa EGF receptors isolated by immunoprecipitation with monoclonal anti-EGF receptor IgG was increased 2- to 3-fold. These treatments produced no significant increase in 32Pi labeling of acid-precipitable material present in detergent extracts of the cells. Phosphoamino acid analysis of radiolabeled EGF receptors isolated from these cells revealed several differences: the relative abundance of phosphotyrosine in EGF receptors was increased in cells treated with EGF, but decreased in cells treated with TPA; the overall relative abundance of phosphothreonine in EGF receptors was decreased in cells treated with EGF, but remained constant within the limits of experimental detection in cells treated with TPA. Two-dimensional mapping of the radiolabeled phosphopeptides produced from EGF receptors isolated by immunoprecipitation and treated with trypsin revealed 9 independent labeled regions, 2 of which contained phosphothreonine and were present only in EGF- or TPA-treated cells. These two phosphopeptide regions were more highly labeled in cells treated with TPA than with EGF.     

The epidermal growth factor receptor tyrosine kinase in liver epithelial cells. The effect of ligand-dependent changes in cellular location

Epidermal growth factor (EGF) activates the intrinsic tyrosine-specific protein kinase of its receptor (EGF-R). We studied the effect of EGF-dependent EGF-R internalization on receptor autophosphorylation and on the appearance of tyrosine phosphoproteins in rat liver epithelial cells. Peak receptor autophosphorylation activity (3- to 6-fold over basal) was found in homogenates of EGF-treated cells at times when the majority of receptors (greater than 90%) had been internalized but not yet degraded (15 to 30 min). Stimulated activity persisted for at least 2 h if EGF-R degradation was blocked by methylamine or 18 degrees C incubation. Detection of stimulated autophosphorylation in homogenates of cells treated with EGF in culture required detergent in the assay. Detergent was not necessary to detect stimulated autophosphorylation when EGF was added directly to homogenates of untreated cells. Immunoblots using antibodies against phosphotyrosine (p-Tyr) demonstrated that EGF treatment of intact cells increased the p-Tyr content of at least seven proteins (EGF-R, 115, 100, 75, 66, 57, and 52 kDa) within 5 s. Incubation of intact cells with EGF at 0 degrees C to prevent endocytosis still resulted in tyrosine phosphorylation of these seven proteins. In contrast, several substrates (120, 78, and 38 kDa) showed delayed increases (45-90 s) in tyrosine phosphorylation at 37 degrees C; their phosphorylation was even slower at 18 degrees C and did not occur at 0 degrees C. In cells incubated with EGF at 18 degrees C or in the presence of methylamine, EGF-R p-Tyr in the intact cell was lost by 2 h even though receptor was not degraded and still exhibited enhanced autophosphorylation in the homogenate assay. These findings suggest that tyrosine phosphorylation in response to EGF occurs predominantly during the initial stages of endocytosis and is mediated for the most part by ligand-receptor complexes at the cell surface. A subset of phosphorylations may require intracellular movement.     

Secretion of an epidermal growth factor-like growth factor by epidermal growth factor-independent rat mammary carcinoma cells.

Rat mammary carcinoma (RMC) cells derived from serially transplantable mammary tumors are independent of epidermal growth factor (EGF) for long-term growth in serum-free medium. This phenotype is in contrast to that of normal mammary epithelial cells or cells derived from nontransplantable tumors that express an absolute requirement for EGF for growth in culture. The results of the experiments reported here indicate that EGF-independent RMC cells secrete a growth factor with potent EGF-like mitogenic activity. Conditioned media obtained from these cells can substitute for EGF for the growth of the EGF-dependent cell line MCF-10. This growth factor is neither EGF nor transforming growth factor alpha and does not compete with 125I-EGF for binding to EGF receptors. Phosphotyrosine Western blot analysis of lysates obtained from EGF-independent RMC cells revealed the presence of a 190 kilodalton (kDa) protein that was distinct from the EGF receptor. Similarly, growth of MCF-10 cells to confluence in serum-free medium supplemented with conditioned medium growth factor in place of EGF resulted in the disappearance of the EGF receptor band and appearance of the 190 kDa band in phosphotyrosine Western blots. The 190 kDa tyrosine-phosphorylated protein detected in cells stimulated by the conditioned medium factor is unlikely to be the c-erbB-2 protein, as indicated by negative results in immunoprecipitation experiments and in vitro kinase assays. In summary, EGF-independent RMC cells secrete a factor with potent EGF-like mitogenic activity. This suggests that an autocrine loop involving this growth factor mediates EGF independence in these cells.     

Effect of glucocorticoid on epidermal growth factor receptor in human salivary gland adenocarcinoma cell line HSG

Human salivary gland adenocarcinoma (HSG) cells treated with 10(-6) M triamcinolone acetonide for 48 h exhibited a 1.7- to 2.0-fold increase in [125I]human epidermal growth factor (hEGF) binding capacity as compared with untreated HSG cells. Scatchard analysis of [125I]EGF binding data revealed that the number of binding sites was 83,700 (+/- 29,200) receptors/cell in untreated cells and 160,500 (+/- 35,500) receptors/cell in treated cells. No substantial change in receptor affinity was detected. The dissociation constant of the EGF receptor was 0.78 (+/- 0.26).10(-9) M for untreated cells, whereas it was 0.93 (+/- 0.31).10(-9)M for treated cells. The triamcinolone acetonide-induced increase in [125I]EGF binding capacity was dose-dependent between 10(-9) and 10(-6)M, and maximal binding was observed at 10(-6)M. EGF receptors on HSG cells were affinity-labeled with [125I]EGF by use of the cross-linking reagent disuccinimidyl suberate (DSS). The cross-linked [125I]EGF was 3-4% of the total [125I]EGF bound to HSG cells. The affinity-labeled EGF receptor was detected as a specific 170 kDa band in the autoradiograph after SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis revealed that triamcinolone acetonide amplified the intensity of this band 2.0-fold over that of the band of untreated cells. EGF receptor synthesis was also measured by immunoprecipitation of [3H]leucine-labeled EGF receptor protein with anti-hEGF receptor monoclonal antibody. Receptor synthesis was increased 1.7- to 1.8-fold when HSG cells were treated with 10(-8)-10(-6)M triamcinolone acetonide for 48 h. When the immunoprecipitated, [35S]methionine-pulse-labeled EGF receptor was analyzed by SDS-PAGE and fluorography, the newly synthesized EGF receptor was detected at the position of 170 kDa; and treatment of HSG cells with triamcinolone acetonide resulted in a 2.0-fold amplification of this 170 kDa band. There was no significant difference in turnover rate of EGF receptor between treated and untreated HSG cells. These results demonstrate that the triamcinolone acetonide-induced increase in [125I]EGF binding capacity is due to the increased synthesis of EGF receptor protein in HSG cells.     

Regulation of the phosphorylation state and function of the epidermal growth factor receptor by vitamin K-3

Vitamin K-3 or 12-O-tetradecanoylphorbol 13-acetate (TPA) reduced the binding of epidermal growth factor (EGF) to its receptor by more than 90% in human foreskin fibroblasts. After the equilibration of fibroblasts with [32P]orthophosphate, vitamin K-3 or TPA markedly increased the amount of 32P found in the receptor; the increase was principally due to serine and threonine phosphorylation. By the use of two-dimensional tryptic phosphopeptide mapping, using a synthetic phosphopeptide as a standard, threonine-654 was identified as one of the residues whose phosphorylation state was elevated by vitamin K-3 or TPA. Because of the large amounts of EGF receptor present on A431 human carcinoma cells, these cells were used to study further the relationship between the phosphorylation state of threonine-654, the tyrosine phosphorylation state of the receptor, and the receptor's protein tyrosine kinase activity toward exogenous substrates. Vitamin K-3 and TPA both increased the amount of phosphate on threonine-654 in A431 cells. However, whereas receptor from TPA-treated cells lacked phosphotyrosine, vitamin K-3-treated cells contained receptor with markedly elevated levels of phosphotyrosine. The addition of vitamin K-3, TPA or EGF to intact A431 cells followed by homogenization of the cells and the assay of EGF receptor protein tyrosine kinase activity by the use of a synthetic peptide substrate resulted in marked decreases in apparent receptor kinase activity. Therefore, assuming that the activity measured in the peptide assay reflects the protein tyrosine kinase activity of the receptor in the intact cell, the activity of the EGF receptor kinase cannot be deduced from the amount of phosphotyrosine associated with the receptor.     

Kinetics and regulation of the tyrosine phosphorylation of epidermal growth factor receptor in intact A431 cells.

We have previously reported that antibodies to phosphotyrosine recognize the phosphorylated forms of platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors (Zippel et al., Biochim. Biophys. Acta 881:54-61, 1986, and Sturani et al., Biochem. Biophys. Res. Commun. 137:343-350, 1986). In this report, the time course of receptor phosphorylation is investigated. In normal human fibroblasts, ligand-induced phosphorylation of PDGF and EGF receptors is followed by rapid dephosphorylation. However, in A431 cells the tyrosine-phosphorylated form of EGF receptor persists for many hours after EGF stimulation, allowing a detailed analysis of the conditions affecting receptor phosphorylation and dephosphorylation. In A431 cells, the number of receptor molecules phosphorylated on tyrosine was quantitated and found to be about 10% of total EGF receptors. The phosphorylated receptor molecules are localized on the cell surface, and they are rapidly dephosphorylated upon removal of EGF from binding sites by a short acid wash of intact cells and upon a mild treatment with trypsin. ATP depletion also results in rapid dephosphorylation, indicating that continuous phosphorylation-dephosphorylation reactions occur in the ligand-receptor complex at steady state. Phorbol 12-myristate 13-acetate added shortly before EGF reduces the rate and the final extent of receptor phosphorylation. Moreover, it also reduces the amount of phosphorylated receptors if it is added after EGF. Down-regulation of protein kinase C by chronic treatment with phorbol dibutyrate increases the receptor phosphorylation induced by EGF, suggesting a homologous feedback regulation of EGF receptor functions.     

Angiotensin II stimulates rat astrocyte mitogen-activated protein kinase activity and growth through EGF and PDGF receptor transactivation

We showed that the intracellular tyrosine kinases src and pyk2 mediate angiotensin II (Ang II) stimulation of growth and ERK1/2 mitogen-activated protein (MAP) kinase phosphorylation in astrocytes. In this study, we investigated whether the membrane-bound receptor tyrosine kinases platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors mediate Ang II stimulation of ERK1/2 and astrocyte growth. Ang II significantly stimulated PDGF and EGF receptors in a dose- and time-dependent manner. The PDGF receptor and the EGF receptor were maximally stimulated with 100 nM Ang II (0.98+/-0.18- and 4.4+/-1.4-fold above basal, respectively). This stimulation occurred as early as 5 min, and was sustained for at least 15 min for both receptor tyrosine kinases. Moreover, 1 microM AG1478 and 0.25 microM PDGFRInhib attenuated Ang II stimulation of the EGF and PDGF receptors, respectively. Ang II-induced phosphorylation of ERK1/2 and astrocyte growth was mediated by both PDGF and EGF receptors. This report also provides novel findings that co-inhibiting EGF and PDGF receptors had a greater effect to decrease Ang II-induced ERK1/2 (90% versus 49% and 71% with PDGF receptor and EGF receptor inhibition, respectively), and astrocyte growth (60% versus 10% and 32% with PDGF receptor and EGF receptor inhibition, respectively). In conclusion we showed in astrocytes that the PDGF and the EGF receptors mediate Ang II-induced ERK1/2 phosphorylation and astrocyte growth and that these two receptors may exhibit synergism to regulate effects of the peptide in these cells.