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.     

Regulation of transferrin receptor expression at the cell surface by insulin-like growth factors, epidermal growth factor and platelet-derived growth factor.

Addition of platelet-derived growth factor (PDGF), recombinant insulin-like growth factor I (rIGF-I) or epidermal growth factor (EGF) to BALB/c 3T3 fibroblasts causes a marked increase in the binding of [125I]diferric transferrin to cell surface receptors. This effect is very rapid and is complete within 5 min. The effect of EGF is transient, with [125I]diferric transferrin binding returning to control values within 25 min. In contrast, PDGF and rIGF-I cause a prolonged stimulation of [125I]diferric transferrin binding that could be observed for up to 2 h. The increase in the binding of [125I]diferric transferrin caused by growth factors was investigated by analysis of the binding isotherm. Epidermal growth factor, PDGF and rIGF-I were found to increase the cell surface expression of transferrin receptors rather than to alter the affinity of the transferrin receptors. This result was confirmed in human fibroblasts by the demonstration that EGF, PDGF and rIGF-I could stimulate the binding of a monoclonal antibody directed against the transferrin receptor (OKT9) to the cell surface. Furthermore, PDGF and rIGF-I stimulated the sustained uptake of [59Fe]diferric transferrin by BALB/c 3T3 fibroblasts, while EGF transiently increased uptake. Thus the effect of these growth factors to increase the cell surface expression of the transferrin receptor appears to have an important physiological consequence.     

Transforming growth factor alpha production and epidermal growth factor receptor expression in normal and oncogene transformed human mammary epithelial cells

We have characterized the expression of transforming growth factor alpha (TGF alpha) and its receptor, the epidermal growth factor receptor (EGF-R), in normal and malignantly transformed human mammary epithelial cells. Human mammary epithelial cells were derived from a reduction mammoplasty (184), immortalized by benzo-a-pyrene (184A 1N4), and further transformed by the oncogenes simian virus 40 T (SV40 T), v-Ha-ras, and v-mos alone or in combination using retroviral vectors. 184 and 184A 1N4 cells require EGF for anchorage-dependent clonal growth. In mass culture, they secrete TGF alpha at high concentrations and exhibit an attenuated requirement for exogenous EGF/TGF alpha. SV40 T transformed cells have 4-fold increased EGF-R, have acquired the ability to clone in soft agar with EGF/TGF alpha supplementation, but are not tumorigenic. Cells transformed by v-mos or v-Ha-ras are weakly tumorigenic and capable of both anchorage dependent and independent growth in the absence of EGF/TGF alpha. Cells transformed by both SV40 T and v-Ha-ras are highly tumorigenic, are refractory to EGF/TGF alpha, and clone with high efficiency in soft agar. The expression of v-Ha-ras is associated with a loss of the high (but not low) affinity binding component of the EGF-R. Malignant transformation and loss of TGF alpha/EGF responsiveness did not correlate with an increase in TGF alpha production. Thus, TGF alpha production does not appear to be a tumor specific marker for human mammary epithelial cells. Differential growth responses to EGF/TGF alpha, rather than enhanced production of TGF alpha, may determine the transition from normal to malignant human breast epithelium.     

Effects of platelet-derived growth factor on phosphorylation of the epidermal growth factor receptor in human skin fibroblasts

Heterologous regulation of the epidermal growth factor (EGF) receptor by platelet-derived growth factor (PDGF) was studied in FS4 human skin fibroblasts. The addition of PDGF to FS4 cells inhibited high affinity binding of 125I-EGF and stimulated phosphorylation of the EGF receptor. Phosphopeptide analysis by high performance liquid chromatography revealed that PDGF treatment of cells increased phosphorylation at several distinct sites of the EGF receptor. However, PDGF did not stimulate phosphorylation of threonine 654, a residue previously shown to be phosphorylated when protein kinase C is activated. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) also stimulated phosphorylation of the same peptides from the EGF receptor as PDGF, and, in addition, induced phosphorylation of threonine 654. TPA inhibited both high and low affinity 125I-EGF binding by these cells. PDGF treatment of cells had no effect on EGF-dependent, tyrosine-specific autophosphorylation of the receptor, whereas TPA treatment was inhibitory. TPA, but not PDGF, stimulated phosphorylation of a Mr = 80,000 protein, known to be a substrate for protein kinase C, even though PDGF appeared to mediate breakdown of phosphoinositides. These data suggest that regulation of EGF receptor function by PDGF and TPA are distinct in these cells, even though some elements of regulation are shared. The results differ from those previously reported for a human lung fibroblast isolate, indicating that cell type-specific differences may exist in metabolism of the EGF receptor.     

The hydrolysis of cell surface glycosphingolipids by endoglycoceramidase reduces epidermal growth factor receptor phosphorylation in A431 cells

This paper presents a new method to evaluate the biologicalsignificance of glycosphingolipids (GSLs) using a GSL-spedficenzyme, endoglycoceramidase (EGCase), by which GSL-sugar chainsare removed from the cell surface of living cells. In this report,the effects of EGCase on epidermal growth factor (EGF)-dependenttyrosine-specific EGF receptor (EGFR) phosphorylation of A431cells are described. After treatment of A431 cells with EGCaseII (20 mU/ml) in the presence of the activator for 12 h, allacidic GSLs tested were reduced to     

Regulation of the epidermal growth factor receptor by phosphorylation

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.     

Imaging epidermal growth factor receptor phosphorylation in human colorectal cancer cells and human tissues

In tumor cells, high phosphorylation levels of receptor tyrosine kinases may occur in the absence of exogenous ligands due to autocrine signaling or enhanced tyrosine kinase activity. Here we show that the phosphorylation state of the endogenous epidermal growth factor receptor (EGFR) can be quantitatively imaged in tumor cells and tissues by detecting fluorescence resonance energy transfer between fluorophores conjugated to antibodies against the receptor and phosphotyrosine, respectively. Five different human colorectal cell lines were analyzed for activity and expression of EGFR. All cell lines exhibited basal EGFR phosphorylation under serum starvation conditions. Phosphorylation levels increased after stimulation with EGF or pervanadate, dependent on the level of basal EGFR phosphorylation in the respective cell lines. This basal activity correlated inversely with receptor expression. Using the acceptor photobleaching fluorescence resonance energy transfer imaging approach, a significantly higher phosphorylation state of EGFR was also found in resected human colorectal tumor samples as compared with adjacent healthy tissue. Imaging of EGFR phosphorylation may thus serve as a valuable tool to investigate the role of receptor tyrosine kinase activity in malignant cell growth.     

Imaging phosphorylation dynamics of the epidermal growth factor receptor

Epidermal growth factor receptor (EGFR) signaling is initiated by ligand binding followed by homodimerization and rapid receptor autophosphorylation. Monitoring EGFR phosphorylation was achieved by measuring translocation and binding of an enhanced yellow fluorescent protein (EYFP)-labeled phosphotyrosine-binding domain (PTB) to enhanced cyan fluorescent protein (ECFP)-tagged EGFR using fluorescence lifetime imaging microscopy or sensitized emission measurements. To simplify dynamic phosphorylation pattern measurements in cells, FLAME, a ratiometric sensor containing both EGFR-ECFP and PTB-EYFP in one molecule, was designed and examined in COS7 cells. Epidermal growth factor (EGF) treatment demonstrated rapid and reversible changes in the EYFP/ECFP fluorescence emission ratios, due to binding of the PTB domain to its consensus binding sites upon phosphorylation at the cell periphery, whereas perinuclear regions failed to respond to EGF but were responsive to tyrosine kinase inhibition. Long-term EGF treatment resulted in accumulation of dephosphorylated receptor in the perinuclear region due to active dephosphorylation occurring at intracellular sites. This indicates that the sensor closely approaches the true dynamics of tyrosine kinase autophosphorylation and dephosphorylation. Phosphatase inhibition by pervanadate resulted in an irreversible response in all cellular compartments. These data show that EGFR is under tonic phosphatase suppression maintaining the receptor in an unphosphorylated (silent) state and is dephosphorylated at endomembranes after ligand-mediated endocytosis.     

Activation of epidermal growth factor receptor via CCR3 in bronchial epithelial cells

We have previously found that bronchial epithelial cells express CCR3 whose signaling elicits mitogen-activated protein (MAP) kinase activation and cytokine production. Several investigators have focused on the signaling crosstalk between G protein-coupled receptors (GPCRs) and epidermal growth factor receptor (EGFR) in cancer cells. In this study, we investigated the role of EGFR in CCR3 signaling in the bronchial epithelial cell line NCI-H292. Eotaxin (1-100 nM) induced dose-dependent tyrosine phosphorylation of EGFR in NCI-H292 cells. Pretreatment of the cells with the EGFR inhibitor (AG1478) significantly inhibited the MAP kinase phosphorylation induced by eotaxin. Eotaxin stimulated IL-8 production, which was inhibited by AG1478. The transactivation of EGFR through CCR3 is a critical pathway that elicits MAP kinase activation and cytokine production in bronchial epithelial cells. The delineation of the signaling pathway of chemokines will help to develop a new therapeutic strategy to allergic diseases including bronchial asthma.     

Differential response to keratinocyte growth factor receptor and epidermal growth factor receptor ligands of proliferating and differentiating intestinal epithelial cells

The expression of the keratinocyte growth factor receptor (KGFR) has been analyzed on intestinal epithelial Caco-2 cells upon confluence-induced spontaneous differentiation. Western blot and immunofluorescence analysis showed that the expression of functional KGFRs, differently from that of epidermal growth factor receptor (EGFR), was up-modulated in post-confluent differentiated cultures compared with the pre-confluent cells. Confocal microscopy and immunoelectron microscopy revealed that the up-regulated KGFRs displayed a basolateral polarized distribution on the cell surfaces in the monolayer. In vivo immunohistochemical analysis on normal human colon tissue sections showed that KGFRs, differently from EGFRs, were mostly distributed on the more differentiated cells located on the upper portion of the intestinal crypt. Bromodeoxyuridine incorporation assay and Ki67 labeling indicated that the differentiated cells were able to proliferate in response to the two ligands of KGFR, KGF and FGF-10, whereas they were not stimulated by the EGFR ligands TGFalpha and EGF. Western blot and quantitative immunofluorescence analysis of the expression of carcinoembryonic antigen (CEA) in post-confluent cells revealed that incubation with KGF induced an increase of cell differentiation. Taken together these results indicate that up-modulation of KGFR may be required to promote proliferation and differentiation in differentiating cells and that, among the cells componing the intestinal epithelial monolayer, the target cells for KGFR ligands appear to be different during differentiation from those responsive to EGFR ligands.     

Insulin-like growth factor I and epidermal growth factor regulate the expression of transferrin receptors at the cell surface by distinct mechanisms

The transferrin receptor cycles rapidly between cell surface and endosomal membrane compartments. Treatment of cultured cells with epidermal growth factor (EGF) or insulin-like growth factor I (IGF-I) at 37 degrees C causes a rapid redistribution of transferrin receptors from an intracellular compartment to the cell surface. The effects of EGF and IGF-I on the kinetics of the cycling of the transferrin receptor in A431 human epidermoid carcinoma cells were compared. The primary site of EGF action was found to be an increase in the rate of transferrin receptor exocytosis. The exocytotic rate constant was measured to be 0.11 min-1 in control cells and 0.33 min-1 in EGF-treated cells. In contrast, IGF-I was found to increase the cell surface expression of transferrin receptors by causing a small increase in the rate of exocytosis (from 0.11 to 0.17 min-1) and a decrease in the rate of endocytosis (from 0.33 to 0.24 min-1). It is concluded that the mechanisms for EGF and IGF-I action to increase the cell surface expression of the transferrin receptor are distinct. A kinetic model of the cycling of the transferrin receptor based on experimentally determined rate constants is presented. The model predicts that a consequence of IGF-I action on transferrin receptor cycling is to decrease the apparent Km for the uptake of diferric transferrin by cells. This prediction is confirmed by direct measurement of the accumulation of 59Fe-labeled diferric transferrin by A431 cells. These data demonstrate that the accumulation of iron by cultured cells is a complex function of the rate of cycling of the transferrin receptor and that this process is under acute regulation by growth factors.     

Aldosterone stimulates epidermal growth factor receptor expression

The steroid hormone aldosterone plays an important role during pathological tissue modifications, similar to cardiovascular or renal fibrosis. The underlying mechanisms for the pathological actions are not understood. Interaction of aldosterone with the epidermal growth factor (EGF) receptor is an attractive hypothesis to explain pathological tissue remodeling elicited by aldosterone, because (i) mineralocorticoids can sensitize cells for EGF, (ii) mineralocorticoid receptor (MR)-antagonists reduce EGFR-mRNA expression, (iii) EGFR itself supports the development of cardiovascular or renal fibrosis, and (iv) signaling elements involved in the pathological action of aldosterone (similar to ERK1/2 or NFkB) are typical downstream modules during EGF signaling. In addition, an interaction of aldosterone and EGF with respect to ERK1/2 activation has been described. Here we show that aldosterone stimulates EGFR expression in renal tissue of adrenalectomized rats and in human renal primary cell cultures. Furthermore, Chinese hamster ovary (CHO) cells normally devoid of EGFR or MR express EGFR after transfection with human MR (CHO-MR cells) but not after transfection with human glucocorticoid receptor (CHO-GR cells). In CHO-MR cells, EGFR-expression is up-regulated by aldosterone and inhibited by spironolactone. CHO-MR cells but not CHO-GR cells respond with ERK1/2 phosphorylation to EGF exposure. The responsiveness to other peptide hormones was virtually not affected. These data suggest that EGFR is an aldosterone-induced protein and is involved in the manifold (patho)biological actions of aldosterone.     

Transforming growth factor beta modulates phosphorylation of the epidermal growth factor receptor and proliferation of A431 cells.

Transforming growth factor beta (TGF-beta) increased the phosphorylation of the epidermal growth factor (EGF) receptor and inhibited the growth of A431 cells. Incubation with TGF-beta induced maximal EGF receptor phosphorylation to levels 1.5-fold higher than controls. Phosphorylation increased more prominently (4-5-fold) on tyrosine residues as determined by phosphoamino acid analysis and antiphosphotyrosine antibody immunoblotting. The kinase activity of EGF receptor was also elevated 2.5-fold when cells were cultured in the presence of TGF-beta. The antiproliferative effect of TGF-beta on A431 cells was accompanied by prolongation of G0-G1 phase and by morphological changes. TGF-beta augmented the growth inhibition of A431 cells which could be induced by EGF. In parallel, the specific EGF-induced increase in total phosphorylation of the EGF receptor was also augmented in the presence of TGF-beta. In cells cultured with TGF-beta, the phosphorylation of EGF receptor tyrosines induced by 20-min exposure to EGF was further increased 2-3-fold, suggesting additive effects upon receptor phosphorylation. EGF receptor activation by TGF-beta is characterized by kinetics quite distinct from that induced by EGF and therefore appears to take place through an independent mechanism. The TGF-beta-induced elevation in the phosphorylation of the EGF receptor may have a role in the augmented growth inhibition of A431 cells observed in the presence of EGF and TGF-beta.     

Diacylglycerol modulates binding and phosphorylation of the epidermal growth factor receptor

Tumor promoters cause a variety of effects in cultured cells, at least some of which are thought to result from activation of the Ca2+-phospholipid-stimulated protein kinase C. One action of tumor promoters is the modulation of the binding and phosphorylation of the epidermal growth factor (EGF) receptor in A431 cells. To determine if these compounds act on the EGF receptor by substituting for the endogenous activator of C kinase, diacylglycerol, we compared the effects of the potent tumor promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA) with those of the synthetic diacylglycerol analog 1-oleyl 2-acetyl diglycerol (OADG). When A431 cells were treated with TPA, the subcellular distribution of C kinase activity shifted from a predominantly cytosolic location to a membrane-associated state; OADG also caused the disappearance of cytosolic C kinase activity. The shift in the subcellular distribution of C kinase, caused by TPA or OADG, correlated with changes in binding and phosphorylation of the EGF receptor. OADG, like TPA, caused loss of binding to an apparent high affinity class of receptors, blocked EGF-induced tyrosine phosphorylation of the EGF receptor, and stimulated phosphorylation of the EGF receptor at both serine and threonine residues. No difference between the phosphopeptide maps of receptors from cells treated with OADG or TPA was observed. Thus, it appears that tumor promoters can exert their effects on the EGF receptors by substituting for diacylglycerol, presumably by activating protein kinase C. Further, these results suggest that endogenously produced diacylglycerol may have a role in normal growth regulatory pathways.     

Direct protective action of epidermal growth factor on isolated gastric mucosal surface epithelial cells.

Epidermal growth factor (EGF) protects gastric mucosa against acute injury produced by a variety of damaging agents, but the mechanism of its protective action is not clear. Since the surface epithelial cells (SEC) are important component of gastric mucosal defence, we studied whether EGF may directly protect isolated gastric SEC against ethanol injury in vitro, in condition independent of systemic factors and whether endogenous prostaglandins may play a role in EGF's protective action. The isolated SEC from rat gastric mucosa were preincubated in medium only, or medium containing 0.0001-10.0 micrograms/ml of h-rEGF for 15 minutes, and incubated with 8% ethanol for 1 hour. In another study the above experiment was repeated but cells were pretreated with 10(-4) or 10(-5) M indomethacin before EGF treatment. The cell viability was assessed by fast green exclusion test. Incubation of SEC with 8% ethanol significantly reduced SEC cell viability to 50 +/- 2%: EGF 0.1 or 1.0 microgram/ml significantly reduced ethanol induced damage (cell viability 59 +/- 3 and 62 +/- 3% respectively). Pretreatment with 10(-4) M indomethacin (the dose which does not affect SEC viability but inhibit PGE2 and PGI2 generation), significantly reduced protective action of EGF against 8% ethanol injury. EGF 1.0 and 10.0 micrograms/ml alone without ethanol increased PGE2 and 6 keto PGF1 alpha generation by SEC. These studies demonstrated: 1) EGF is able to protect gastric surface epithelial cells directly without mediation by systemic factors. 2) EGF induced protection of SEC may in part be mediated by prostaglandins.     

Erratum: The hydrolysis of cell surface glycosphingolipids by endoglycoceramidase reduces epidermal growth factor receptor phosphorylation in A431 cells

Figure 3 was inadvertently printed incorrectly. The correctfigure is given below: