Suppression of protein tyrosine kinase activity of the epidermal growth factor receptor by epidermal growth factor

Epidermal growth factor (EGF) receptor protein kinase activity, estimated by the use of peptide substrates, was reduced by as much as 70% after the treatment of intact A431 human carcinoma cells with EGF. The apparent decrease in protein kinase activity was observed after immunoprecipitation of the receptor or after purification of the receptor by lectin chromatography. By the use of [35S]methionine, it was determined that the total amount of receptor obtained was the same whether or not cells were treated with EGF. EGF stimulated the purified receptor protein kinase activity in vitro; however, the EGF-stimulated activity of receptor from EGF-treated cells continued to be reduced by as much at 70% compared to the EGF-stimulated activity from untreated cells. The reduction in receptor protein kinase activity induced by EGF may represent a feedback mechanism by which responsiveness to the growth factor is regulated.     

Tyrosine phosphorylation of mitogen-activated protein kinase in cells with tyrosine kinase-negative epidermal growth factor receptors.

Epidermal growth factor (EGF) treatment of cells expressing the human EGF receptor (EGFr) results in rapid tyrosine phosphorylation of several cellular proteins including mitogen-activated protein (MAP) kinase. EGF treatment of cells expressing a tyrosine kinase-inactive EGFr failed to induce the tyrosine phosphorylation of endogenous substrates in response to EGF; however, the tyrosine phosphorylation and activation of MAP kinase did occur. This observation indicates that MAP kinase is activated in response to a signal other than the tyrosine kinase activity of the EGFr. Because EGF does not stimulate cells expressing the inactive EGFr to proliferate, phosphorylation of MAP kinase may not be sufficient for the EGF-dependent mitogenesis.     

Stimulation of epidermal growth factor receptor threonine 654 phosphorylation by platelet-derived growth factor in protein kinase C-deficient human fibroblasts

We have tested the hypothesis that the mechanism of platelet-derived growth factor (PDGF) and phorbol diester action to decrease the apparent affinity of the epidermal growth factor (EGF) receptor is the phosphorylation of the EGF receptor at the Ca2+/phospholipid-dependent protein kinase (protein kinase C) phosphorylation site, threonine 654. Protein kinase C-deficient cells were prepared by prolonged incubation of human fibroblasts with phorbol diester. Addition of phorbol diesters to these cells fails to regulate EGF receptor affinity or threonine 654 phosphorylation. In contrast, PDGF treatment of both control and protein kinase C-deficient fibroblasts causes a decrease in the apparent affinity of the EGF receptor and an increase in threonine 654 phosphorylation. Thus, the ability of PDGF or phorbol diester to modulate EGF receptor affinity occurs only when threonine 654 phosphorylation is increased. The stoichiometry of threonine 654 phosphorylation associated with a 50% decrease in the binding of 125I-EGF to high affinity sites was 0.15 versus 0.3 mol of phosphate per mole of EGF receptor when 32P-labeled fibroblasts are treated with PDGF or phorbol diester, respectively. It is concluded that EGF receptor phosphorylation at threonine 654 can be regulated by PDGF independently of protein kinase C, substoichiometric phosphorylation of the total EGF receptor pool at threonine 654 is caused by maximally effective concentrations of PDGF, and different extents of phosphorylation of EGF receptors at threonine 654 are observed for maximally effective concentrations of PDGF and phorbol diester, respectively. The data are consistent with the hypothesis that a specific subpopulation of EGF receptors that exhibit high affinity for EGF are regulated by threonine 654 phosphorylation.     

Protein kinase A mediates cAMP-induced tyrosine phosphorylation of the epidermal growth factor receptor

An increase in the intracellular cAMP concentration induces tyrosine phosphorylation of the epidermal growth factor receptor (EGFR) followed by activation of extracellular signal-regulated kinases 1/2 (ERK1/2). In this report we demonstrate that these effects of cAMP are mediated via activation of protein kinase A (PKA). Chemical inhibition of PKA suppressed forskolin-induced EGFR tyrosine phosphorylation and ERK1/2 activation in PC12 cells. Furthermore, forskolin failed to induce significant tyrosine phosphorylation of the EGFR and ERK1/2 activation in PKA-defective PC12 cells. Forskolin-induced EGFR tyrosine phosphorylation was also observed in A431 cells and in membranes isolated from these cells. Phosphoamino acid analysis indicated that the recombinant catalytic subunit of PKA elicited phosphorylation of the EGFR on both tyrosine and serine but not threonine residues in A431 membranes. Together, our data indicate that activation of PKA mediates the effects of cAMP on the EGFR and ERK1/2. While PKA may directly phosphorylate the EGFR on serine residues, PKA-induced tyrosine phosphorylation of the EGFR occurs by an indirect mechanism.     

Differential phosphorylation of the progesterone receptor by insulin, epidermal growth factor, and platelet-derived growth factor receptor tyrosine protein kinases

Purified preparations of insulin, epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) receptors were compared for their abilities to phosphorylate purified hen oviduct progesterone receptors. The specific activities of all three peptide hormone-induced receptor kinases were first defined using a synthetic tridecapeptide tyrosine protein kinase substrate. Next, equivalent ligand-activated activities of the three receptor kinases were tested for their abilities to phosphorylate hen oviduct progesterone receptor. Both the insulin and EGF receptors phosphorylated progesterone receptor at high affinity, exclusively at tyrosine residues and with maximal stoichiometries that were near unity. In contrast, the PDGF receptor did not recognize progesterone receptor as a substrate. Insulin decreased the Km of the insulin receptor for progesterone receptor subunits as substrates, but had no significant effect on Vmax values. On the other hand, EGF increased the Vmax of the EGF receptor for progesterone receptor subunits as substrates. Phosphorylation of progesterone receptor by the insulin and EGF receptor kinases differed in two additional ways. 1) EGF-activated receptor phosphorylated the 80- and 105-kDa progesterone receptor subunits to an equal extent, whereas insulin-activated receptor preferentially phosphorylated the 80-kDa subunit. 2) Phosphopeptide fingerprinting analyses revealed that while insulin and EGF receptors phosphorylated one identical major site on both progesterone receptor subunits, they differed in their specificities for other sites.     

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.     

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.     

Activation of p38 mitogen-activated protein kinase promotes epidermal growth factor receptor internalization

Endocytic trafficking plays an important role in the regulation of the epidermal growth factor receptor (EGFR). To address if cellular kinases regulate EGFR internalization, we used anisomycin, a potent activator of kinase cascades in mammalian cells, especially the stress-activated mitogen-activated protein (MAP) kinase subtypes. Here, we report that activation of p38 MAP kinase by anisomycin is sufficient to induce internalization of EGFR. Anisomycin and EGF employ different mechanisms to promote EGFR endocytosis as anisomycin-induced internalization does not require tyrosine kinase activity or ubiquitination of the receptor. In addition, anisomycin treatment did not result in delivery and degradation of EGFR at lysosomes. Incubation with a specific inhibitor of p38, or depletion of endogenous p38 by small interfering RNAs, abolished anisomycin-induced internalization of EGFR while having no effect on transferrin endocytosis, indicating that the effect of p38 activation on EGFR endocytosis is specific. Interestingly, inhibition of p38 activation also abolished endocytosis of EGFR induced by UV radiation. Our results reveal a novel role for p38 in the regulation of EGFR endocytosis and suggest that stimulation of EGFR internalization by p38 might represent a general mechanism to prevent generation of proliferative or anti-apoptotic signals under stress conditions.     

Autophosphorylation and protein kinase C phosphorylation of the epidermal growth factor receptor. Effect on tyrosine kinase activity and ligand binding affinity

The effect of autophosphorylation and protein kinase C-catalyzed phosphorylation on the tyrosine-protein kinase activity and ligand binding affinity of the epidermal growth factor (EGF) receptor has been studied. Kinetic parameters for the phosphorylation by the receptor kinase of synthetic peptide substrates having sequences related to the 3 in vitro receptor autophosphorylation sites (tyrosine residues 1173 (P1), 1148 (P2), and 1068 (P3)) were measured. The Km of peptide P1 (residues 1164-1176) was significantly lower than that for peptides P2 (residues 1141-1151) or P3 (residues 1059-1072). The tyrosine residue 1173 was also the most rapidly autophosphorylated in purified receptor preparations, consistent with previous observations for the receptor in intact cells (Downward, J., Parker, P., and Waterfield, M. D. (1984) Nature 311, 483-485). Variation in the extent of receptor autophosphorylation from 0.1 to 2.8 mol of phosphate/mol of receptor did not influence kinase activity or EGF binding affinity either for purified receptor or receptor in membrane preparations. Phosphorylation of the EGF receptor by protein kinase C was shown to cause a 3-fold decrease in the affinity of purified EGF receptor for EGF and to reduce the receptor kinase activity. In membrane preparations, phosphorylation of the EGF receptor by protein kinase C resulted in conversion of high affinity EGF binding sites to a low affinity state. This suggests that activation of protein kinase C by certain growth promoting agents and tumor promoters is directly responsible for modulation of the affinity of the EGF receptor for its ligand EGF. The regulation of the EGF receptor function by protein kinase C is discussed.     

Activation of the purified protein tyrosine kinase domain of the epidermal growth factor receptor

Biological responses to epidermal growth factor (EGF) depend on the ligand-stimulated protein tyrosine kinase activity of its receptor. To further characterize the enzymatic activity of the EGF receptor, the baculovirus expression system was used to express the cytoplasmic protein tyrosine kinase domain of the EGF receptor. Spodoptera frugiperda (Sf9) cells infected with recombinant baculovirus correctly expressed an active tyrosine kinase domain of the EGF receptor as demonstrated by 35S metabolic labeling, immunoblotting with anti-EGF receptor and anti-phosphotyrosine antibodies, and autophosphorylation analysis. The kinase domain (Mr 66,000) was purified to near homogeneity using a monoclonal anti-phosphotyrosine antibody column, providing 0.5 mg of kinase domain/liter of Sf9 cells (23% yield). The purified kinase domain exhibited a strong preference for Mn2+ compared to Mg2+. The specific activity of the kinase domain was low compared to purified, EGF-activated EGF receptor. However, the addition of sphingosine or ammonium sulfate greatly increased the activity of the kinase domain to equal or exceed the activity of ligand-activated holo EGF receptor. These results indicate that the addition of sphingosine or ammonium sulfate to the purified kinase domain can mimic the effect of EGF to induce a conformation of the holo EGF receptor which is optimal for tyrosine kinase activity. Deletion of the ligand binding domain, analogous to that which occurs in erb B, is not sufficient to fully activate the kinase, implying that EGF causes conformational changes additional to removal of an inhibitory constraint.     

Antibody-induced dimerization activates the epidermal growth factor receptor tyrosine kinase

The relationship between epidermal growth factor receptor (EGF-R) protein tyrosine kinase activation and ligand-induced receptor dimerization was investigated using several bivalent anti-EGF-R antibodies directed against various receptor epitopes. In A431 membrane preparations and permeabilized cells, all antibodies were able to activate the EGF-R tyrosine kinase, as measured by EGF-R autophosphorylation and phosphorylation of other substrates on tyrosine residues. EGF-R tyrosine kinase activation correlated strongly with the induction of EGF-R dimerization. (i) Both processes specifically occurred in a narrow antibody concentration range; (ii) both processes required the presence of detergent; and (iii) both processes depended on antibody bivalence since monovalent Fab fragments were inactive yet regained full activity after cross-linking by a second bivalent antibody. These data demonstrate that antibody bivalence is essential and sufficient for EGF-R activation and that activation occurs regardless of the EGF-R epitope recognized. Finally, EGF-R dimerization was shown not to depend on receptor autophosphorylation since it still occurred in the absence of ATP. Also, partial inhibition of the tyrosine kinase activity by the specific EGF-R tyrosine kinase inhibitor tyrphostin AG 213 did not affect formation of EGF-R dimers. Taken together these results demonstrate that induction of EGF-R dimerization is sufficient and in case of antibody action, essential, for activation of the EGF-R tyrosine kinase and thus provide strong support for an intermolecular mechanism of EGF-R tyrosine kinase activation.     

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.     

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+.     

Tyrosine phosphorylation by the epidermal growth factor receptor kinase induces functional alterations in microtubule-associated protein 2

We have examined the effect of tyrosine phosphorylation of microtubule-associated protein 2 (MAP2) by the epidermal growth factor (EGF) receptor kinase on its functions. Incubation of MAP2 with the EGF receptor in the presence of ATP resulted in a great decrease in the ability of MAP2 to promote tubulin polymerization. Under a variety of conditions, the decrease in the ability correlated with the extent of phosphorylation of MAP2. Furthermore, another function of MAP2, the actin filament cross-linking activity, was also inactivated by the incubation of MAP2 with the EGF receptor and ATP. The loss of this activity also correlated well with the extent of phosphorylation. These data indicate that tyrosine phosphorylation of MAP2 by the EGF receptor kinase inactivates both the tubulin polymerizing activity and actin filament cross-linking activity of MAP2. Thus, this study has clearly shown that tyrosine phosphorylation could modify the function of a cytoskeletal protein.     

Abl tyrosine kinase regulates endocytosis of the epidermal growth factor receptor

Signal attenuation from ligand-activated epidermal growth factor receptor (EGFR) is mediated in part by receptor endocytosis and trafficking to the lysosomal degradative compartment. Uncoupling the activated EGFR from endocytosis and degradation has emerged as a mechanism for oncogenic activation of the EGFR. The Abl nonreceptor tyrosine kinase is activated by ligand-stimulated EGFR, but the role of Abl in EGFR signaling has not been defined. Here we uncovered a novel role for the activated Abl kinase in the regulation of EGFR endocytosis. We show that activated Abl impairs EGFR internalization. Moreover, we show that activated Abl phosphorylates the EGFR primarily on tyrosine 1173, and that mutation of this site to phenylalanine restores ligand-dependent endocytosis of the EGFR in the presence of activated Abl. Furthermore, we show that activated Abl allows the ligand-activated EGFR to escape Cbl-dependent down-regulation by inhibiting the accumulation of Cbl at the plasma membrane in response to epidermal growth factor stimulation and disrupting the formation of the EGFR.Cbl complex without affecting Cbl protein stability. These findings reveal a novel role for Abl in promoting increased cell-surface expression of the EGFR and suggest that Abl/EGFR signaling may cooperate in human tumors.     

Inhibition of epidermal growth factor receptor tyrosine kinase by chalcone derivatives.

In our previous study, butein, a chalcone derivative, was found to be an inhibitor of tyrosine kinases and the inhibition was ATP-competitive. In this work, chalcone and seven chalcone derivatives were used to analyse the relationship between the structure of these compounds and their inhibition of tyrosine kinase activity. Three of chalcone derivatives, including butein, marein and phloretin, were found to have an ability to inhibit the tyrosine kinase activity of epidermal growth factor receptor (EGFR) in vitro. IC(50) was 8 microM for butein, 19 microM for marein and 25 microM for phloretin. The structural characterisations of these inhibitors suggest that the hydroxylations at C4 and C4' of these molecules may be required for them to act as EGFR tyrosine kinase inhibitors. The inhibition of EGF-induced EGFR tyrosine phosphorylation by butein was also observed in human hepatocellular carcinoma HepG2 cells, while marein and phloretin were inactive at the doses tested. Molecular modelling suggests that butein, marein and phloretin can be docked into the ATP binding pocket of EGFR. Hydrogen bonds and hydrophobic interaction appear to be important in the binding of these inhibitors to EGFR.     

Ratiometric assay of epidermal growth factor receptor tyrosine kinase activation

Activation of cells is frequently followed by tyrosine phosphorylation of proteins. To quantify this process, we developed a ratiometric enzyme-linked immunosorbent assay (ELISA) using epidermal growth factor receptors (EGFR) as a model. Microtiter dishes were coated with anti-EGFR monoclonal antibodies to capture the receptor followed by parallel detection of receptor and phosphotyrosine content with secondary antibodies. The ratio of these two parameters was found to directly reflect EGFR activation and was insensitive to the effect of receptor downregulation. Our assay could resolve differences in EGFR activation due to small changes (less than 1 ng/ml) in ligand. We found that phosphotyrosine detection by ELISA was 8- to 32-fold more sensitive than Western blot detection and could be reliably detected using as little as 4 ng of cellular lysate. Detection of EGFR levels by ELISA was 30 times more sensitive than Western blot analysis and was reliable for as low as 8 ng of cellular lysate per well. Because of the wide linear range of the ELISA, we could directly compare receptor activation in cell types with different EGFR expression levels. Our assay provides a rapid and sensitive method of determining EGFR activation status and could be easily modified to evaluate any tyrosine-phosphorylated protein.