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.     

cAMP-dependent protein kinase stimulates epidermal growth factor-dependent phosphorylation of epidermal growth factor receptors

Epidermal growth factor (EGF)-dependent transfer of radiolabeled phosphate from [gamma-32P]ATP to 160-kDa EGF receptor solubilized from human epidermoid carcinoma A431 cell surface membranes was stimulated up to 3-fold by addition of 3',5'-cAMP and purified cAMP-dependent protein kinase. Phosphorylation of EGF receptors was stimulated to the same extent when cAMP-dependent protein kinase catalytic subunit was substituted for 3',5'-cAMP and cAMP-dependent protein kinase. Phosphoamino acid analysis revealed that the extent of phosphorylation of EGF receptor at tyrosine residues was the same regardless of whether cAMP-dependent protein kinase catalytic subunit was present in or omitted from the system. Increased EGF receptor phosphorylation occurring in response to cAMP-dependent protein kinase catalytic subunit was accounted for by phosphorylation at serine or threonine residues. In samples phosphorylated in the presence of cAMP-dependent protein kinase catalytic subunit, phosphate was present in tyrosine, serine, and threonine in a ratio of 32:60:8. Two-dimensional mapping of radiolabeled phosphopeptides produced from EGF receptors by digestion with trypsin revealed the generation of one additional major phosphoserine-containing peptide when cAMP-dependent protein kinase was present with EGF in the EGF receptor kinase system. Degradation of 160-kDa EGF receptors to a 145-kDa form by purified Ca2+-activated neutral protease produced a 145-kDa fragment with phosphoserine content increased over that present initially in the 160-kDa precursor.     

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.     

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.     

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.     

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.     

Radiation-induced epidermal growth factor receptor nuclear import is linked to activation of DNA-dependent protein kinase

Ionizing radiation, but not stimulation with epidermal growth factor (EGF), triggers EGF receptor (EGFR) import into the nucleus in a probably karyopherin alpha-linked manner. An increase in nuclear EGFR is also observed after treatment with H2O2, heat, or cisplatin. During, this process, the proteins Ku70/80 and the protein phosphatase 1 are transported into the nucleus. As a consequence, an increase in the nuclear kinase activity of DNA-dependent kinase (DNA-PK) and increased formation of the DNA end-binding protein complexes containing DNA-PK, essential for repair of DNA-strand breaks, occurred. Blockade of EGFR import by the anti-EGFR monoclonal antibody C225 abolished EGFR import into the nucleus and radiation-induced activation of DNA-PK, inhibited DNA repair, and increased radiosensitivity of treated cells. Our data implicate a novel function of the EGFR during DNA repair processes.     

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.     

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.     

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.     

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.     

Stimulation of the mitogen-activated protein kinase cascade and tyrosine phosphorylation of the epidermal growth factor receptor by hepatopoietin

Hepatopoietin (HPO) is a novel human hepatotrophic growth factor, which specifically stimulates proliferation of cultured primary hepatocytes in vitro and liver regeneration after liver partial hepatectomy in vivo. Recently, the identification of the mitogenic effect of HPO on hepatoma cell lines and the existence of HPO-specific receptors indicate that HPO acts via its specific cell surface receptor. However, the molecular mechanism of HPO action is not fully elucidated. In this report, we examined the signal transduction events induced by HPO in hepatoma cell line (HepG2). Our results demonstrated that HPO induces phosphorylation of mitogen-activated protein kinase kinase and mitogen-activated protein kinase (MAPK) in a rapid and transient manner. HPO stimulates tyrosine phosphorylation of epidermal growth factor receptor (EGFR). Furthermore, we observed that both MAPK activation and the mitogenic effect of HPO on HepG2 cells were completely blocked by AG1478, a specific inhibitor of EGFR tyrosine kinase activity. However, the effects of HPO were not antagonized by an EGFR-blocking antibody, mAb528, which blocks the interaction between epidermal growth factor and EGFR, indicating that stimulation of tyrosine phosphorylation of EGFR by HPO was not mediated by epidermal growth factor. In contrast, genistein, a general tyrosine kinase inhibitor, significantly attenuated the tyrosine phosphorylation of EGFR in response to HPO. In conclusion, our results suggest that tyrosine phosphorylation of EGFR may play a critical role in MAPK activation and mitogenic stimulation by HPO.     

Mechanical stretch stimulates protein kinase B/Akt phosphorylation in epidermal cells via angiotensin II type 1 receptor and epidermal growth factor receptor

Mechanical stress is known to modulate fundamental events such as cell life and death. Mechanical stretch in particular has been identified as a positive regulator of proliferation in skin keratinocytes and other cell systems. In the present study it was investigated whether antiapoptotic signaling is also stimulated by mechanical stretch. It was demonstrated that mechanical stretch rapidly induced the phosphorylation of the proto-oncogene protein kinase B (PKB)/Akt at both phosphorylation sites (serine 473/threonine 308) in different epithelial cells (HaCaT, A-431, and human embryonic kidney-293). Blocking of phosphoinositide 3-OH kinase by selective inhibitors (LY-294002 and wortmannin) abrogated the stretch-induced PKB/Akt phosphorylation. Furthermore mechanical stretch stimulated phosphorylation of epidermal growth factor receptor (EGFR) and the formation of EGFR membrane clusters. Functional blocking of EGFR phosphorylation by either selective inhibitors (AG1478 and PD168393) or dominant-negative expression suppressed stretch-induced PKB/Akt phosphorylation. Finally, the angiotensin II type 1 receptor (AT1-R) was shown to induce positive transactivation of EGFR in response to cell stretch. These findings define a novel signaling pathway of mechanical stretch, namely the activation of PKB/Akt by transactivation of EGFR via angiotensin II type 1 receptor. Evidence is provided that stretch-induced activation of PKB/Akt protects cells against induced apoptosis.     

Control of epidermal growth factor receptor endocytosis by receptor dimerization, rather than receptor kinase activation

Given that ligand binding is essential for the rapid internalization of epidermal growth factor receptor (EGFR), the events induced by ligand binding probably contribute to the regulation of EGFR internalization. These events include receptor dimerization, activation of intrinsic tyrosine kinase activity and autophosphorylation. Whereas the initial results are controversial regarding the role of EGFR kinase activity in EGFR internalization, more recent data suggest that EGFR kinase activation is essential for EGFR internalization. However, we have shown here that inhibition of EGFR kinase activation by mutation or by chemical inhibitors did not block EGF-induced EGFR internalization. Instead, proper EGFR dimerization is necessary and sufficient to stimulate EGFR internalization. We conclude that EGFR internalization is controlled by EGFR dimerization, rather than EGFR kinase activation. Our results also define a new role for EGFR dimerization: by itself it can drive EGFR internalization, independent of its role in the activation of EGFR kinase.     

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 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.     

Developments in the mechanism of growth factor action: activation of protein kinase by epidermal growth factor

The interaction between epidermal growth factor (EGF) and its target cells has been used as a model for studying the regulation of cell proliferation. Many of the details of binding and subsequent internalization and degradation of this growth factor have been elucidated by following the fate of [125I]EGF in the presence of responsive cells. To investigate the membrane-localized biochemical consequences of EGF-receptor complex formation, a subcellular membrane system has been developed. In this system, EGF enhances phosphorylation of its receptor as well as other endogenous proteins. This EGF-stimulable protein kinase activity is not separated from the EGF receptor activity either by detergent solubilization or by affinity purification of the solubilized membranes. The data suggest that the EGF-binding activity and EGF-sensitive protein kinase activity reside in a single membrane protein.