Controlling Epidermal Growth Factor (EGF)-stimulated Ras Activation in Intact Cells by a Cell-permeable Peptide Mimicking Phosphorylated EGF Receptor

The possible involvement of a stimulatory guanosine triphosphate (GTP)-binding (G) protein in epidermal growth factor (EGF)-induced phosphoinositide hydrolysis has been investigated in permeabilized NIH-3T3 cells expressing the human EGF receptor. The mitogenic phospholipid lysophosphatidate (LPA), a potent inducer of phosphoinositide hydrolysis, was used as a control stimulus. In intact cells, pertussis toxin partially inhibits the LPA-induced formation of inositol phosphates, but has no effect on the response to EGF. In cells permeabilized with streptolysin-O, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) dramatically increases the initial rate of inositol phosphate formation induced by LPA. In contrast, activation of phospholipase C (PLC) by EGF occurs in a GTP-independent manner. Guanine 5'-O-(2-thiodiphosphate) (GDP beta S) which keeps G proteins in their inactive state, blocks the stimulation by LPA and GTP gamma S, but fails to affect the EGF-induced response. Tyrosine-containing substrate peptides, when added to permeabilized cells, inhibit EGF-induced phosphoinositide hydrolysis without interfering with the response to LPA and GTP gamma S. These data suggest that the EGF receptor does not utilize an intermediary G protein to activate PLC and that receptor-mediated activation of effector systems can be inhibited by exogenous substrate peptides.

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Perturbation of epidermal growth factor receptor complex formation and Ras signalling in cells harbouring the hepatitis C virus subgenomic replicon

Journal of General Virology ◽

10.1099/vir.0.80724-0 ◽

2005 ◽

Vol 86 (4) ◽

pp. 1027-1033 ◽

Cited By ~ 16

Author(s):

Andrew Macdonald ◽

Julia Ka Yu Chan ◽

Mark Harris

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Egf Receptor ◽

Adaptor Proteins ◽

Mitogen Activated Protein Kinase ◽

Subgenomic Replicon ◽

Ras Activation ◽

Epidermal Growth

Hepatitis C virus non-structural NS5A protein inhibits epidermal growth factor (EGF)-stimulated activation of the Ras–ERK mitogen-activated protein kinase pathway at a point upstream of Ras activation. To determine the mechanism of this inhibition, the events occurring between the EGF receptor and Ras in Huh-7 cells harbouring the HCV subgenomic replicon were investigated. It was shown that, following EGF stimulation, these cells exhibited decreased EGF receptor tyrosine phosphorylation, aberrant recruitment of the adaptor proteins ShcA and Grb2 to the EGF receptor, reduced phosphorylation of ShcA and reduced Ras activation in comparison with control cells. These data are consistent with effects of NS5A and/or other components of the replicon on multiple events occurring upstream of Ras.

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Role of Phosphoinositide 3-Kinase in Activation of Ras and Mitogen-Activated Protein Kinase by Epidermal Growth Factor

Molecular and Cellular Biology ◽

10.1128/mcb.19.6.4279 ◽

1999 ◽

Vol 19 (6) ◽

pp. 4279-4288 ◽

Cited By ~ 206

Author(s):

Stefan Wennström ◽

Julian Downward

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Map Kinase ◽

Tyrosine Kinases ◽

Kinase Inhibitors ◽

Egf Receptor ◽

Mitogen Activated Protein Kinase ◽

Ras Activation ◽

Mitogen Activated Protein ◽

Epidermal Growth

ABSTRACT The paradigm for activation of Ras and extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinase by extracellular stimuli via tyrosine kinases, Shc, Grb2, and Sos does not encompass an obvious role for phosphoinositide (PI) 3-kinase, and yet inhibitors of this lipid kinase family have been shown to block the ERK/MAP kinase signalling pathway under certain circumstances. Here we show that in COS cells activation of both endogenous ERK2 and Ras by low, but not high, concentrations of epidermal growth factor (EGF) is suppressed by PI 3-kinase inhibitors; since Ras activation is less susceptible than ERK2 activation, PI 3-kinase-sensitive events may occur both upstream of Ras and between Ras and ERK2. However, strong elevation of PI 3-kinase lipid product levels by expression of membrane-targeted p110α is by itself never sufficient to activate Ras or ERK2. PI 3-kinase inhibition does not affect EGF-induced receptor autophosphorylation or adapter protein phosphorylation or complex formation. The concentrations of EGF for which PI 3-kinase inhibitors block Ras activation induce formation of Shc-Grb2 complexes but not detectable EGF receptor phosphorylation and do not activate PI 3-kinase. The activation of Ras by low, but mitogenic, concentrations of EGF is therefore dependent on basal, rather than stimulated, PI 3-kinase activity; the inhibitory effects of LY294002 and wortmannin are due to their ability to reduce the activity of PI 3-kinase to below the level in a quiescent cell and reflect a permissive rather than an upstream regulatory role for PI 3-kinase in Ras activation in this system.

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Nitric oxide stimulates tyrosine phosphorylation in murine fibroblasts in the absence and presence of epidermal growth factor

Biochemical Journal ◽

10.1042/bj3050613 ◽

1995 ◽

Vol 305 (2) ◽

pp. 613-619 ◽

Cited By ~ 45

Author(s):

T M S Peranovich ◽

A M da Silva ◽

D M Fries ◽

A Stern ◽

H P Monteiro

Keyword(s):

Methylene Blue ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Tyrosine Phosphorylation ◽

Cyclic Gmp ◽

Egf Receptor ◽

Tyrosine Phosphatase ◽

Intact Cells ◽

Murine Fibroblasts ◽

Epidermal Growth

In the present study, utilizing anti-phosphotyrosine monoclonal antibodies, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) as sources of NO and murine fibroblasts expressing the human epidermal growth factor (EGF) receptor (HER14 cells), we showed that tyrosine phosphorylation of a set of proteins (126, 56 and 43 kDa) was stimulated when cells were incubated with either SNP or SNAP and abolished by Methylene Blue and oxyhaemoglobin. Inhibition by Methylene Blue suggested an involvement of cyclic GMP in the process, which was evidenced by the effects of 8-bromo cyclic GMP. This analogue of cyclic GMP stimulated tyrosine phosphorylation of the same set of proteins phosphorylated after incubation with the NO source. Tyrosine phosphorylation of the same set of proteins was stimulated when cells were incubated simultaneously with SNP and EGF, showing that NO also potentiates EGF-evoked tyrosine kinase activity in HER14 cells. However, stimulation of the autophosphorylation of the EGF receptor, above the levels obtained for EGF alone, was not observed under those conditions. Additionally, we investigated the effects of NO on EGF-receptor tyrosine phosphatase activities in HER14 cells. Increasing concentrations of NO correlate with a gradual inhibition of these activities in HER14 cells, either in intact cells or in cell lysates. Taken together, these observations suggest that NO modulates tyrosine phosphorylation in HER14 cells.

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

Molecular and Cellular Biology ◽

10.1128/mcb.8.3.1345 ◽

1988 ◽

Vol 8 (3) ◽

pp. 1345-1351 ◽

Cited By ~ 37

Author(s):

E Sturani ◽

R Zippel ◽

L Toschi ◽

L Morello ◽

P M Comoglio ◽

...

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Egf Receptor ◽

Human Fibroblasts ◽

Atp Depletion ◽

Egf Receptors ◽

A431 Cells ◽

Intact Cells ◽

Receptor Phosphorylation ◽

Epidermal Growth

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

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Epidermal growth factor receptor cytoplasmic domain mutations trigger ligand-independent transformation

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.3048-3055.1990 ◽

1990 ◽

Vol 10 (6) ◽

pp. 3048-3055

Author(s):

S Massoglia ◽

A Gray ◽

T J Dull ◽

S Munemitsu ◽

H J Kun ◽

...

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Egf Receptor ◽

Transforming Growth Factor ◽

Growth Factor Receptor ◽

Cytoplasmic Domain ◽

Control Mechanisms ◽

Egf Receptors ◽

Intact Cells ◽

Epidermal Growth

The transforming gene product of avian erythroblastosis virus, v-erbB, is derived from the epidermal growth factor (EGF) receptor but has lost its extracellular ligand-binding domain and was mutated in its cytoplasmic portion, which is thought to be responsible for biological signal generation. We have repaired the deletion of extracellular EGF-binding sequences and investigated the functional consequences of cytoplasmic erbB mutations. Within the resulting EGF receptors, the autophosphorylation activities of the cytoplasmic domains of v-erbB-H and v-erbB-ES4 were fully ligand dependent in intact cells. However, the mitogenic and transforming signaling activities of an EGF receptor carrying v-erbB-ES4 (but not v-erbB-H) cytoplasmic sequences remained ligand independent, whereas those of a receptor with a v-erbB-H cytoplasmic domain were regulated by EGF or transforming growth factor alpha. Thus, structural alterations in the cytoplasmic domain of growth factor receptor tyrosine kinases may induce constitutive signaling activity without autophosphorylation. These findings provide new insight into the mechanism of receptor-mediated signal transduction and suggest a novel alternative for subversion of cellular control mechanisms and proto-oncogene activation.

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

Molecular and Cellular Biology ◽

10.1128/mcb.8.3.1345-1351.1988 ◽

1988 ◽

Vol 8 (3) ◽

pp. 1345-1351

Author(s):

E Sturani ◽

R Zippel ◽

L Toschi ◽

L Morello ◽

P M Comoglio ◽

...

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Egf Receptor ◽

Human Fibroblasts ◽

Atp Depletion ◽

Egf Receptors ◽

A431 Cells ◽

Intact Cells ◽

Receptor Phosphorylation ◽

Epidermal Growth

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

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Human growth hormone augmentation of epidermal growth factor binding sites on rat granulosa cells

Journal of Endocrinology ◽

10.1677/joe.0.1420069 ◽

1994 ◽

Vol 142 (1) ◽

pp. 69-75 ◽

Cited By ~ 6

Author(s):

M-A Hattori ◽

Y Shinohara ◽

E Yoshino ◽

M Kanzaki ◽

I Kojima ◽

...

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Granulosa Cell ◽

Granulosa Cells ◽

Binding Sites ◽

Egf Receptor ◽

Scatchard Analysis ◽

Maximal Effect ◽

Intact Cells ◽

Epidermal Growth

Abstract The effect of human GH (hGH) on the regulation of epidermal growth factor (EGF) receptor was investigated during differentiation of FSH-treated rat granulosa cells, which has been reported to be mediated by a cAMP-dependent mechanism. By measuring the binding of [125I]iodo-EGF to the intact cells, FSH was shown to cause increases in the number of EGF binding sites after culture for 72 h. When granulosa cells were cultured with hGH, the number of FSH-induced EGF binding sites was augmented, with a half-maximal effect at about 10 μg hGH/l and a maximal stimulatory concentration of 100 μg/l. The stimulatory effect of hGH was absolutely dependent on insulin which by itself showed stimulatory effects on EGF binding sites. Scatchard analysis of EGF binding sites indicated that treatment with hGH increased the number of EGF binding sites (17 200 sites/cell after treatment with FSH; 31 700 sites/cell after FSH plus hGH), but did not alter the binding affinity. The augmentation was observed after culturing for 48 h and increased progressively with time, reaching 280% of the level after FSH treatment by 120 h. Although progesterone synthesis was increased by hGH, the markers of cell differentiation such as cAMP synthesis and LH binding sites were suppressed, indicating hGH inhibition of the cAMP-mediated signal. The action of hGH on the EGF binding sites was not accompanied by cell proliferation. These findings indicate that hGH has a novel action on the regulation of rat granulosa cell EGF binding sites and that the granulosa cell may possess both cAMP-dependent and -independent mechanisms for expression of EGF binding sites. Journal of Endocrinology (1994) 142, 69–75

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Expression of epidermal-growth-factor receptor in the K562 cell line by transfection. Altered receptor biochemistry

Biochemical Journal ◽

10.1042/bj2710785 ◽

1990 ◽

Vol 271 (3) ◽

pp. 785-790 ◽

Cited By ~ 4

Author(s):

H Allen ◽

J Hsuan ◽

S Clark ◽

R Maziarz ◽

M D Waterfield ◽

...

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Cell Line ◽

Egf Receptor ◽

Transforming Growth Factor ◽

Growth Factor Receptor ◽

K562 Cells ◽

Intact Cells ◽

Autocrine Stimulation ◽

Epidermal Growth

The epidermal-growth-factor (EGF) receptor was expressed in the human erythroleukaemic cell line K562 by transfection of the receptor cDNA. EGF-receptor biochemistry appears altered in the K562 transfectants. Autophosphorylation of the K562 receptor is not stimulated substantially by EGF. Tyrosine kinase activity of the receptor is high in the absence of EGF, whereas receptor affinity for EGF is low. K562 cells are shown to lack mRNA for transforming growth factor alpha (TGF alpha). Therefore autocrine stimulation of the K562 receptor, at least by TGF alpha, does not explain the observed receptor biochemistry. The K562 receptor is phosphorylated at a single major site in intact cells, a threonine residue that may be Thr-669. Possible mechanisms of regulation of the EGF receptor in the K562 transfectants are discussed.

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Epidermal growth factor receptor cytoplasmic domain mutations trigger ligand-independent transformation.

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.3048 ◽

1990 ◽

Vol 10 (6) ◽

pp. 3048-3055 ◽

Cited By ~ 19

Author(s):

S Massoglia ◽

A Gray ◽

T J Dull ◽

S Munemitsu ◽

H J Kun ◽

...

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Egf Receptor ◽

Transforming Growth Factor ◽

Growth Factor Receptor ◽

Cytoplasmic Domain ◽

Control Mechanisms ◽

Egf Receptors ◽

Intact Cells ◽

Epidermal Growth

The transforming gene product of avian erythroblastosis virus, v-erbB, is derived from the epidermal growth factor (EGF) receptor but has lost its extracellular ligand-binding domain and was mutated in its cytoplasmic portion, which is thought to be responsible for biological signal generation. We have repaired the deletion of extracellular EGF-binding sequences and investigated the functional consequences of cytoplasmic erbB mutations. Within the resulting EGF receptors, the autophosphorylation activities of the cytoplasmic domains of v-erbB-H and v-erbB-ES4 were fully ligand dependent in intact cells. However, the mitogenic and transforming signaling activities of an EGF receptor carrying v-erbB-ES4 (but not v-erbB-H) cytoplasmic sequences remained ligand independent, whereas those of a receptor with a v-erbB-H cytoplasmic domain were regulated by EGF or transforming growth factor alpha. Thus, structural alterations in the cytoplasmic domain of growth factor receptor tyrosine kinases may induce constitutive signaling activity without autophosphorylation. These findings provide new insight into the mechanism of receptor-mediated signal transduction and suggest a novel alternative for subversion of cellular control mechanisms and proto-oncogene activation.

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