Role of epidermal growth factor-stimulated protein kinase in control of proliferation of A431 cells

Epidermal growth factor (EGF), which stimulates tyrosine-specific protein kinase activity both in vivo and in vitro, inhibits proliferation of A431 human epidermoid carcinoma cells. After mutagenesis clonal cell lines that were resistant to the growth inhibitory effects of EGF were selected. All six variants examined contained decreased EGF-stimulated protein kinase. The number of EGF receptors in variant cells decreased in parallel with EGF-stimulated protein kinase activity so that the specific activity of EGF-stimulated protein kinase per EGF receptor remained constant in variant cell lines with up to tenfold reductions in both activities. This result suggests that both EGF binding and kinase activities reside in the same or closely coupled molecules. The effect of EGF on growth of two resistant variants was examined in detail. Clone 29 contains approximately 50% and clone 4 contains approximately 20% of the EGF-stimulated protein kinase activity of the parental A431 cell line. In serum-supplemented medium, EGF stimulated proliferation of clone 29 but did not affect growth of clone 4. In a 1:1 mixture of DME and F-12 medium without serum, EGF caused both clone 29 and clone 4 to grow as well as in 10% serum. These variants, which were selected for resistance to the growth inhibitory effects of EGF, thus exhibit a strong mitogenic response to EGF. This result suggests that resistance to the growth inhibitory effect of EGF may involve both a decrease in EGF-stimulated protein kinase and an alteration in the response pathway.     

Mechanism of desensitization of the epidermal growth factor receptor protein-tyrosine kinase.

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.     

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.     

Alteration of epidermal growth factor receptor activity by mutation of its primary carboxyl-terminal site of tyrosine self-phosphorylation

The epidermal growth factor (EGF) receptor, which exhibits intrinsic protein tyrosine kinase activity, undergoes a rapid, intramolecular self-phosphorylation reaction following EGF activation. The primary sites of tyrosine self-phosphorylation in vivo are located in the extreme carboxyl-terminal region of the molecule, principally Tyr-1173. To test the biological and biochemical consequences of this EGF receptor self-phosphorylation, we made the mutation Tyr----Phe-1173. Membranes containing the mutated receptor exhibited an ED50 for EGF activation of tyrosine kinase activity equivalent to control receptor at both high and low substrate levels, but exhibited reduced basal and EGF-stimulated tyrosine kinase activity at low, non-saturating substrate levels. The Tyr----Phe-1173 mutant possessed high affinity EGF binding and could still self-phosphorylate other tyrosine sites in an intramolecular fashion with a low Km for ATP (200 nM), suggesting that this alteration did not grossly change receptor structure. When EGF-dependent growth of Chinese hamster ovary cells expressing comparable levels of control or mutant EGF receptor was measured, the ability of the mutant receptor to mediate cell growth in response to EGF was reduced by approximately 50%, yet both receptors exhibited a similar affinity and ED50 for EGF. These results support the concept that this self-phosphorylation site can act as a competitive/alternate substrate for the EGF receptor, and that this region of the molecule is important in modulating its maximal biological activity.     

Calmodulin inhibits the epidermal growth factor receptor tyrosine kinase.

We demonstrate in this report that the epidermal growth factor (EGF) receptor from rat liver can be isolated by calmodulin affinity chromatography by binding in the presence of Ca2+ and elution with a Ca(2+)-chelating agent. The bulk of the EGF receptor is not eluted by a NaCl gradient in the presence of Ca2+. We ascertained the identity of the isolated receptor by immunoblot and immunoprecipitation using a polyclonal antibody against an EGF receptor from human origin. The purified receptor is autophosphorylated in tyrosine residues in an EGF-stimulated manner, and EGF-dependent phosphorylation of serine residues was also detected. Both the EGF and the transforming growth factor-alpha stimulate the tyrosine-directed protein kinase activity of the isolated receptor with similar affinities. Furthermore, we demonstrate that calmodulin inhibits the EGF-dependent tyrosine-directed protein kinase activity associated to the receptor in a concentration-dependent manner. This inhibition is partially Ca2+ dependent and is not displaced by increasing the concentration of EGF up to an EGF/calmodulin ratio of 10 (mol/mol). In addition, calmodulin was phosphorylated in an EGF-stimulated manner in the presence of a basic protein (histone) as cofactor and in the absence, but not in the presence, of Ca2+.     

Structural analysis of the transmembrane domain of the epidermal growth factor receptor

The ligand-binding domain of the epidermal growth factor (EGF) receptor is separated from the cytoplasmic protein tyrosine kinase domain by a predicted single transmembrane segment. Antipeptide antibodies prepared against the outer portion of the predicted transmembrane segment confirmed this area was exposed only when cells were treated with permeabilizing agents. To investigate structural requirements for signal transduction by the transmembrane domain, three types of mutant EGF receptor were prepared. The first type was designed to shorten the transmembrane domain, the second to place proline substitutions within this domain, and the third to make amino acid substitutions analogous to those present in the transforming c-erbB2/neu oncoprotein. Mutant human receptors were expressed in null recipient mouse B82L and Chinese hamster ovary cells. All receptors bound EGF and exhibited EGF-stimulated protein tyrosine kinase activity in vivo as assayed using a 125I-labeled monoclonal anti-phosphotyrosine antibody. EGF stimulated growth of cells expressing each mutant receptor with similar dose-response characteristics. In contrast to other growth factor receptors, the transmembrane domain of the EGF receptor is tolerant to a variety of changes which neither mimic EGF action by constitutive activation nor interfere with ligand-induced signal transduction.     

Analysis of morphology and receptor metabolism in clonal variant A431 cells with differing growth responses to epidermal growth factor

Human epidermoid carcinoma A431 cell clones have been obtained whose growth is inhibited, stimulated, or unaffected by epidermal growth factor (EGF). In clones exhibiting each type of growth response, EGF induced similar morphologic changes consisting of aggregation of cells into dense clusters with baring of large areas of the culture dish. The similarity of the clones' morphologic responses, despite their differing growth responses, indicates that the effects of EGF on morphology are distinct from effects on growth. Cells whose growth was inhibited by EGF contained high numbers of EGF receptors, whereas the concentration of EGF receptors was reduced in cells whose growth was stimulated or unaffected by EGF. There were, however, no consistent differences in EGF receptor concentrations between stimulated or null clones. Cells that exhibited each type of growth response displayed similar rates of EGF binding to receptors, rates of internalization of EGF, and rates and extent of EGF-induced receptor down-regulation. Changes in EGF-stimulated tyrosine-specific protein kinase activity paralleled changes in EGF receptors, both between clones and upon down-regulation. These studies indicate that a reduction in the concentration of EGF receptors in A431 cells allows escape from the growth inhibitory effects of EGF, but suggest that the pattern of growth response depends on biochemical events subsequent to EGF-receptor metabolism and activation of tyrosine-specific protein kinase.     

Tannic acid, a potent inhibitor of epidermal growth factor receptor tyrosine kinase

Increasing evidence supports the hypothesis that tannic acid, a plant polyphenol, exerts anticarcinogenic activity in chemically induced cancers. In the present study, tannic acid was found to strongly inhibit tyrosine kinase activity of epidermal growth factor receptor (EGFr) in vitro (IC50 = 323 nM). In contrast, the inhibition by tannic acid of p60(c-src) tyrosine kinase (IC50 = 14 microM) and insulin receptor tyrosine kinase (IC50 = 5 microM) was much weaker. The inhibition of EGFr tyrosine kinase by tannic acid was competitive with respect to ATP and non-competitive with respect to peptide substrate. In cultured cells, growth factor-induced tyrosine phosphorylation of growth factor receptors, including EGFr, platelet-derived growth factor receptor, and basic fibroblast growth factor receptor, was inhibited by tannic acid. No inhibition of insulin-induced tyrosine phosphorylation of insulin receptor and insulin-receptor substrate-1 was observed. EGF-stimulated growth of HepG2 cells was inhibited in the presence of tannic acid. The inhibition of serine/threonine-specific protein kinases, including cAMP-dependent protein kinase, protein kinase C and mitogen-activated protein kinase, by tannic acid was only detected at relatively high concentration, IC50 being 3, 325 and 142 microM respectively. The molecular modeling study suggested that tannic acid could be docked into the ATP binding pockets of either EGFr or insulin receptor. These results demonstrate that tannic acid is an in vitro potent inhibitor of EGFr tyrosine kinase.     

C-kinase phosphorylates the epidermal growth factor receptor and reduces its epidermal growth factor-stimulated tyrosine protein kinase activity

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.     

Effects of substitution of threonine 654 of the epidermal growth factor receptor on epidermal growth factor-mediated activation of phospholipase C

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.     

Monoclonal antibodies against the human epidermal growth factor receptor from A431 cells. Isolation, characterization, and use in the purification of active epidermal growth factor receptor

A431 cells have been used as an immunogen for generating monoclonal antibodies against the epidermal growth factor (EGF) receptor. Two immunoglobulin M and eight immunoglobulin G3 anti-EGF receptor antibodies were cloned. All ten antibodies immunoprecipitated biosynthetically labeled mature A431 cell EGF receptor and were able to recognize the receptor in Western blotting. However, none of the antibodies immunoprecipitated precursor polypeptides of the A431 cell EGF receptor, neither did they recognize EGF receptors from human foreskin fibroblasts, human placenta, nor a human-mouse hybrid cell expressing EGF receptor. The antibodies were found to bind to glycolipids from A431 cells and it was shown that the determinant involved was the blood group A antigen. It appears that this determinant is present on both the EGF receptor and glycolipids of A431 cells but is not expressed on EGF receptors from other human cells tested. One of the monoclonal antibodies raised was used for immunoaffinity purification of the EGF receptor. The procedure took advantage of the carbohydrate nature of the antigenic determinant by employing sugar-specific elution. The mild conditions permitted the purification of A431 cell EGF receptor (70-80% pure) that possessed an intrinsic EGF-stimulated tyrosine kinase activity with a specific activity of about 20 nmol/min/mg.     

Mechanism of phosphorylation of the epidermal growth factor receptor at threonine 669

The major site of phosphorylation of the epidermal growth factor (EGF) receptor after treatment of cells with EGF is threonine 669. Phosphorylation of this site is also associated with the transmodulation of the EGF receptor caused by platelet-derived growth factor and phorbol ester. A distinctive feature of the primary sequence surrounding threonine 669 is the proximity of 2 proline residues (-Pro-Leu-Thr669-Pro-). This site is not a substrate for phosphorylation by protein kinase C. To investigate the mechanism of the increased phosphorylation of the EGF receptor at threonine 669, in vitro assays were used to measure protein kinase and protein phosphatase activities present in homogenates prepared from cells treated with and without EGF. No evidence for the regulation of protein phosphatase activity was obtained in experiments using the [32P]phosphate-labeled EGF receptor as a substrate. A synthetic peptide corresponding to residues 663-681 of the EGF receptor was used as a substrate for protein kinase assays. Incubation of murine 3T3 L1 pre-adipocytes and human WI-38 fibroblasts with EGF caused a rapid increase (3-10-fold) in the level of threonine protein kinase activity detected in cell homogenates. Similar results were obtained after EGF treatment of Chinese hamster ovary cells expressing wild-type (Thr669) and mutated (Ala669) human EGF receptors. Activation of the threonine protein kinase activity was also observed in cells treated with platelet-derived growth factor, serum, and phorbol ester. Insulin-like growth factor-1 caused no significant change in protein kinase activity. Together these data indicate a role for the regulation of the activity of a threonine protein kinase in the control of the phosphorylation state of the EGF receptor at threonine 669. The significance of the identification of a growth factor-stimulated threonine protein kinase to the mechanism of signal transduction is discussed.     

High-yield covalent attachment of epidermal growth factor to its receptor by kinetically controlled, stepwise affinity cross-linking.

We report here the use of a stepwise affinity cross-linking technique in the specific covalent attachment of epidermal growth factor (EGF) to its receptor. A heterobifunctional cross-linking reagent, sulfo-N-succinimidyl 4-(fluorosulfonyl)benzoate (SSFSB), which contains a rapidly reacting sulfo-N-succinimidyl active ester and a much more slowly reacting aromatic fluorosulfonyl moiety, was synthesized and characterized. Murine EGF (mEGF) was modified by the cross-linker to yield as the major product a derivative of mEGF having the (fluorosulfonyl)benzoyl moiety attached covalently at the amino terminus. SSFSB-modified, 125I-labeled mEGF was separated from unreacted SSFSB by size-exclusion chromatography and applied to shed membrane vesicles from A431 human carcinoma cells. Binding of derivatized 125I-mEGF to vesicles led to high yields (greater than 60%) of covalent linkage of 125I-mEGF to the EGF receptor, as determined by measurement of the fraction of specifically bound radiolabel which comigrated with the EGF receptor in NaDodSO4-polyacrylamide gels. The specificity of affinity cross-linking was evident in the negligible degree of labeling of species other than the EGF receptor and in the retention of EGF-stimulated receptor kinase activity after cross-linking.     

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.     

Effects of protein kinase C activation after epidermal growth factor binding on epidermal growth factor receptor phosphorylation

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.     

Treatment of A431 cells with epidermal growth factor (EGF) induces desensitization of EGF-stimulated phosphatidylinositol turnover

Epidermal growth factor (EGF) stimulates the turnover of phosphoinositides in A431 cells. In cells that were pretreated with EGF for 30 min at 37 degrees C and then washed to remove surface-bound hormone, a 70-100% decrease in the EGF-stimulated production of inositol monophosphate, inositol bisphosphate, and inositol triphosphate was noted when the cells were exposed to the agonist a second time. Since only a 15% decrease in receptor number was observed in these pretreated cells, the loss of responsiveness to EGF for the production of inositol phosphates could not be attributed to a down-regulation of the EGF receptors. These data suggest that pretreatment of A431 cells with high concentrations of EGF leads to a desensitization of the EGF receptor. This desensitization of the receptor by EGF is apparent within 10-15 min of the addition of EGF and is maximal by 30 min. The desensitization appears to be homologous in nature since pretreatment of cells with EGF did not diminish their responsiveness to bradykinin; and conversely, pretreatment with bradykinin did not diminish the subsequent responsiveness of the cells to EGF. Desensitization to EGF was observed in cells in which protein kinase C had been down-regulated by prolonged treatment with 12-O-tetradecanoylphorbol-13-acetate, implying that EGF receptor desensitization is independent of protein kinase C. The desensitizing effects of EGF on growth factor-induced phosphatidylinositol turnover could be prevented by pretreatment of the cells with the calmodulin antagonist trifluoperazine, suggesting that calmodulin may be involved in the regulation of EGF receptor sensitivity.     

Mutation of proline-1003 to glycine in the epidermal growth factor (EGF) receptor enhances responsiveness to EGF.

We have shown previously that the epidermal growth factor (EGF) receptor is phosphorylated at Ser-1002 and that this phosphorylation is associated with desensitization of the EGF receptor. Ser-1002 is followed immediately by Pro-1003, a residue that may promote the adoption of a specific conformation at this site or severe as a recognition element for the interaction of the EGF receptor with other proteins. To examine these possibilities, we have mutated Pro-1003 of the EGF receptor to a Gly residue and have analyzed the effect of this mutation on EGF-stimulated signaling. Cells expressing the P1003G EGF receptors exhibited higher EGF-stimulated autophosphorylation and synthetic peptide phosphorylation compared to cells expressing wild-type EGF receptors. In addition, the ability of EGF to stimulate PI 3-kinase activity and mitogen-activated protein kinase activity was enhanced in cells expressing the P1003G EGF receptor. Cells expressing P1003G receptors also demonstrated an increased ability to form colonies in soft agar in response to EGF. These results indicate that mutation of Pro-1003 leads to a potentiation of the biological effects of EGF. The findings are consistent with the hypothesis that Pro-1003 plays a role in a form of regulation that normally suppresses EGF receptor function.     

Reconstitution of epidermal growth factor receptor transmodulation by platelet-derived growth factor in Chinese hamster ovary cells

Platelet-derived growth factor (PDGF) causes an acute decrease in the high affinity binding of epidermal growth factor (EGF) to cell surface receptors and an increase in the phosphorylation state of the EGF receptor at threonine654. The hypothesis that PDGF action to regulate the EGF receptor is mediated by the activation of protein kinase C and the subsequent phosphorylation of EGF receptor threonine654 was tested. The human receptors for PDGF and EGF were expressed in Chinese hamster ovary cells that lack expression of endogenous receptors for these growth factors. The heterologous regulation of the EGF receptor by PDGF was reconstituted in cells expressing [Thr654]EGF receptors or [Ala654]EGF receptors. PDGF action was also observed in phorbol ester down-regulated cells that lack detectable protein kinase C activity. Together these data indicate that neither protein kinase C nor the phosphorylation of EGF receptor threonine654 is required for the regulation of the apparent affinity of the EGF receptor by PDGF.     

Constitutive phosphorylation of the epidermal growth factor receptor blocks mitogenic signal transduction

The epidermal growth factor (EGF) receptor is phosphorylated by protein kinase C at Thr654. It has been proposed that the phosphorylation of this site is an important regulatory mechanism for the control of EGF receptor function. However, the physiological significance of the phosphorylation of EGF receptor Thr654 in intact cells is not understood. To address this question, the design of an experimental strategy is required that can be used to distinguish between the pleiotropic effects of kinase C activation and the specific effects of kinase C that are mediated by the phosphorylation of the EGF receptor at Thr654. The approach that we used was to examine the function of EGF receptors that are constitutively phosphorylated at residue 654. It was observed that the constitutive phosphorylation of the EGF receptor blocked mitogenic signal transduction by the receptor. These data are consistent with the hypothesis that the phosphorylation of the EGF receptor at residue 654 in intact cells inhibits EGF-stimulated cellular proliferation.     

Inhibition of stimulus-dependent epidermal growth factor receptor and transforming growth factor-alpha mRNA accumulation by the protein kinase C inhibitor staurosporine

The ability of staurosporine, a potent inhibitor of protein kinase C, to block certain cellular events initiated by 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF) was examined. Treatment of MDA468 breast cancer cells with TPA decreases EGF binding to the cell surface and this effect is blocked by pretreatment with staurosporine with an IC50 of 30 nM. Either 10(-9) M EGF or 100 ng/ml TPA stimulated the accumulation of both EGF receptor and TGF-alpha mRNA and staurosporine (50 nM) completely abolished these mRNA accumulations. Staurosporine did not block EGF-stimulated tyrosine phosphorylation of its receptor as measured by immunoblotting with anti-phosphotyrosine antibodies. The ability of staurosporine to block the mRNA responses of either EGF or TPA suggests that these two agents have common signaling pathways and it implies a role for protein kinase C in the control of EGF receptor and TGF-alpha expression.