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

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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Activation of the Epidermal Growth Factor (EGF) Receptor Induces Formation of EGF Receptor- and Grb2-Containing Clathrin-Coated Pits

Molecular and Cellular Biology ◽

10.1128/mcb.26.2.389-401.2006 ◽

2006 ◽

Vol 26 (2) ◽

pp. 389-401 ◽

Cited By ~ 81

Author(s):

Lene E. Johannessen ◽

Nina Marie Pedersen ◽

Ketil Winther Pedersen ◽

Inger Helene Madshus ◽

Espen Stang

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Egf Receptor ◽

Point Mutations ◽

Adaptor Protein ◽

Mitogen Activated Protein Kinase ◽

Coated Pits ◽

Α Subunit ◽

Sh3 Domains ◽

Epidermal Growth

ABSTRACT In HeLa cells depleted of adaptor protein 2 complex (AP2) by small interfering RNA (siRNA) to the μ2 or α subunit or by transient overexpression of an AP2 sequestering mutant of Eps15, endocytosis of the transferrin receptor (TfR) was strongly inhibited. However, epidermal growth factor (EGF)-induced endocytosis of the EGF receptor (EGFR) was inhibited only in cells where the α subunit had been knocked down. By immunoelectron microscopy, we found that in AP2-depleted cells, the number of clathrin-coated pits was strongly reduced. When such cells were incubated with EGF, new coated pits were formed. These contained EGF, EGFR, clathrin, and Grb2 but not the TfR. The induced coated pits contained the α subunit, but labeling density was reduced compared to control cells. Induction of clathrin-coated pits required EGFR kinase activity. Overexpression of Grb2 with inactivating point mutations in N- or C-terminal SH3 domains or in both SH3 domains inhibited EGF-induced formation of coated pits efficiently, even though Grb2 SH3 mutations did not block activation of mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3-kinase (PI3K). Our data demonstrate that EGFR-induced signaling and Grb2 are essential for formation of clathrin-coated pits accommodating the EGFR, while activation of MAPK and PI3K is not required.

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Apical Epidermal Growth Factor Receptor Signaling: Regulation of Stretch-dependent Exocytosis in Bladder Umbrella Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e06-09-0842 ◽

2007 ◽

Vol 18 (4) ◽

pp. 1312-1323 ◽

Cited By ~ 36

Author(s):

Elena M. Balestreire ◽

Gerard Apodaca

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Egf Receptor ◽

Physiological Role ◽

Receptor Activation ◽

Mitogen Activated Protein Kinase ◽

Apical Surface ◽

Mechanical Stimuli ◽

Epidermal Growth ◽

Umbrella Cells

The apical surface of polarized epithelial cells receives input from mediators, growth factors, and mechanical stimuli. How these stimuli are coordinated to regulate complex cellular functions such as polarized membrane traffic is not understood. We analyzed the requirement for growth factor signaling and mechanical stimuli in umbrella cells, which line the mucosal surface of the bladder and dynamically insert and remove apical membrane in response to stretch. We observed that stretch-stimulated exocytosis required apical epidermal growth factor (EGF) receptor activation and that activation occurred in an autocrine manner downstream of heparin-binding EGF-like growth factor precursor cleavage. Long-term changes in apical exocytosis depended on protein synthesis, which occurred upon EGF receptor-dependent activation of mitogen-activated protein kinase signaling. Our results indicate a novel physiological role for the EGF receptor that couples upstream mechanical stimuli to downstream apical EGF receptor activation that may regulate apical surface area changes during bladder filling.

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P129 HEPATITIS C VIRUS MEK1- AND TC-PTP-DEPENDENTLY CONVERTS EPIDERMAL GROWTH FACTOR (EGF) INTO A POTENT INDUCTOR OF CXCR2 LIGAND EXPRESSION

Journal of Hepatology ◽

10.1016/s0168-8278(14)60291-9 ◽

2014 ◽

Vol 60 (1) ◽

pp. S108-S109

Author(s):

C. Gröpper ◽

N. Triller ◽

S. Eisenbürger ◽

R. Bartenschlager ◽

D. Häussinger ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Ligand Expression ◽

Epidermal Growth ◽

Cxcr2 Ligand

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Enhanced Wound Healing by Recombinant Escherichia coli Nissle 1917 via Human Epidermal Growth Factor Receptor in Human Intestinal Epithelial Cells: Therapeutic Implication Using Recombinant Probiotics

Infection and Immunity ◽

10.1128/iai.05820-11 ◽

2011 ◽

Vol 80 (3) ◽

pp. 1079-1087 ◽

Cited By ~ 17

Author(s):

Hye Jin Choi ◽

Jung Hoon Ahn ◽

Seong-Hwan Park ◽

Kee Hun Do ◽

Juil Kim ◽

...

Keyword(s):

Wound Healing ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Abc Transporter ◽

Egf Receptor ◽

Mitogen Activated Protein Kinase ◽

Content Type ◽

E Coli ◽

Epithelial Migration ◽

Epidermal Growth

The gastrointestinal mucosa has a remarkable ability to repair damage with the support of epidermal growth factor (EGF), which stimulates epithelial migration and proliferative reepithelialization. For the treatment of mucosal injuries, it is important to develop efficient methods for the localized delivery of mucoactive biotherapeutics. The basic idea in the present study came from the assumption that an intestinal probiotic vehicle can carry and deliver key recombinant medicinal proteins to the injured epithelial target in patients with intestinal ulcerative diseases, including inflammatory bowel disease. The study was focused on the use of the safe probioticE. coliNissle 1917, which was constructed to secrete human EGF in conjunction with the lipase ABC transporter recognition domain (LARD). Using thein vitrophysically wounded monolayer model, ABC transporter-mediated EGF secretion by probioticE. coliNissle 1917 was demonstrated to enhance the wound-healing migration of human enterocytes. Moreover, the epithelial wound closure was dependent on EGF receptor-linked activation, which exclusively involved the subsequent signaling pathway of the mitogen-activated protein kinase kinase (MEK) extracellular-related kinases 1 and 2 (ERK1/2). In particular, the migrating frontier of the wounded edge displayed the strongest EGF receptor-linked signaling activation in the presence of the recombinant probiotic. The present study provides a basis for the clinical application of human recombinant biotherapeutics via an efficient, safe probiotic vehicle.

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Alternative Pathways of Epidermal Growth Factor Receptor Mediated Contractile Force in NR6 Fibroblasts

10.1115/imece1999-0403 ◽

1999 ◽

Author(s):

Fred D. Allen ◽

Clara F. Asnes ◽

Alan Wells ◽

Elliot L. Elson ◽

Douglas A. Lauffenburger

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Egf Receptor ◽

Mapk Pathway ◽

Contractile Force ◽

Mitogen Activated Protein Kinase ◽

Egfr Signaling ◽

Signaling Mechanism ◽

Force Increase ◽

Epidermal Growth

Abstract We investigated the contractile force response to epidermal growth factor (EGF) stimulation in 3T3-derived NR6 fibroblast cells in order to determine significant pathways of biochemical signaling that mediate the response. We examined the force generating specificity of the EGF receptor (EGFR) signaling mechanism by using mutant NR6 fibroblasts expressing variations of the EGFR construct. The wild-type (WT) cell presented the complete internalizing EGFR signaling construct while the c’973 cell presented an internalization-defective EGFR construct, and the M721 cell presented a kinase-defective EGFR construct making it signaling inert. Additionally we examined the roles of the phospholipasc C-γ (PLCγ) pathway by using the PLC inhibitor U73122 (1 μM) and the mitogen activated protein kinase (MAPK) pathway using the inhibitor PD98059 (10 μM) in the observed contractile force responses. We found that the WT cells showed a rapid but transient force increase within the first hour post-stimulation and the c’973 showed a more gradual increase in force which it sustained for several hours post-stimulation. Blocking the PLCγ activation in the WT cells reduced the peak force increase by 50% while blocking MAPK did not affect the force development in either WT or c’973 cells.

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Hepatitis C virus NS5A protein blocks epidermal growth factor receptor degradation via a proline motif- dependent interaction

Journal of General Virology ◽

10.1099/vir.0.000145 ◽

2015 ◽

Vol 96 (8) ◽

pp. 2133-2144 ◽

Cited By ~ 9

Author(s):

Zsofia Igloi ◽

Arunas Kazlauskas ◽

Kalle Saksela ◽

Andrew Macdonald ◽

Jamel Mankouri ◽

...

Keyword(s):

Epidermal Growth Factor Receptor ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Growth Factor Receptor ◽

Ns5a Protein ◽

Epidermal Growth ◽

Protein Blocks ◽

Dependent Interaction

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

Molecular Biology of the Cell ◽

10.1091/mbc.5.7.739 ◽

1994 ◽

Vol 5 (7) ◽

pp. 739-746 ◽

Cited By ~ 6

Author(s):

S M Schuh ◽

E P Newberry ◽

M A Dalton ◽

L J Pike

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Kinase Activity ◽

Egf Receptor ◽

Biological Effects ◽

Soft Agar ◽

Mitogen Activated Protein Kinase ◽

Protein Kinase Activity ◽

Egf Receptors ◽

Epidermal Growth

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

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