Role of EGF receptor tyrosine kinase activity in antiapoptotic effect of EGF on mouse hepatocytes

The apoptotic Fas pathway is potentially involved in the pathogenesis of liver diseases. Growth factors, such as epidermal growth factor (EGF), can protect cells against apoptosis induced by a variety of stimuli, including Fas receptor stimulation. However, the underlying mechanisms of this protection have yet to be determined. We investigated the involvement of EGF receptor (EGFR) tyrosine kinase (TK) activity in the antiapoptotic effect of EGF on primary mouse hepatocyte cultures. Cells undergoing apoptosis after treatment with anti-Fas antibody were protected by EGF treatment. This protection was significantly but partially decreased when cells were treated with two specific inhibitors of the TK activity of EGFR. Evaluation of the efficacy of these compounds indicated that they were able to abolish EGFR autophosphorylation and postreceptor events such as activation of mitogen-activated protein kinases and the phosphatidylinositol 3′-kinase pathways as well as increases in Bcl-xL mRNA and protein levels. This leads us to postulate that EGF exerts its antiapoptotic action partially through the TK activity of EGFR. In addition, our results suggest that Bcl-xL protein upregulation caused by EGF is linked to the TK activity of its receptor.

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Antiapoptotic effect of EGF on mouse hepatocytes associated with downregulation of proapoptotic Bid protein

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00040.2003 ◽

2003 ◽

Vol 285 (2) ◽

pp. G298-G308 ◽

Cited By ~ 23

Author(s):

Chantal Éthier ◽

Valérie-Ann Raymond ◽

Lina Musallam ◽

Robert Houle ◽

Marc Bilodeau

Keyword(s):

Growth Factors ◽

Egf Receptor ◽

Specific Inhibitor ◽

Pi3 Kinase ◽

Hepatocyte Apoptosis ◽

Antiapoptotic Effect ◽

Primary Mouse ◽

Major Player ◽

Mouse Hepatocytes

Growth factors have been shown to protect cells from a variety of apoptotic stimuli. In the liver, the Fas system is thought to be very important in the genesis of hepatocyte apoptosis. Others have already shown the importance of the phosphatidylinositol 3-kinase (PI3-kinase) pathway and of increased Bcl-xl expression in the antiapoptotic effect of growth factors on hepatocytes. We investigated the effect of EGF on Bid, a BH3-only member of the Bcl-2 family and a major player in the transduction of the Fas apoptotic signal. Hepatocyte apoptosis was induced in vitro with a purified anti-mouse Fas antibody. The effect of EGF on Bid protein expression was studied on those cultures. EGF dose dependently reduced the expression of Bid protein in primary mouse hepatocyte cultures independently of Fas stimulation. This decrease was not the result of the degradation of Bid into its active p15 fragment. Treating cells with a specific inhibitor of the EGF receptor autophosphorylation completely abolished the decrease in Bid expression afforded by EGF. Treatment with LY-294002, a PI3-kinase blocker, partly reverted the effect of EGF. When apoptosis was induced in Bid-deficient hepatocytes, EGF lost its capacity to protect cells against this type of cell death. These results show that EGF decreases the expression of Bid protein and suggest that the effect of EGF on Bid is one of the mechanisms of the antiapoptotic effect of EGF.

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Multiple functions of let-23, a Caenorhabditis elegans receptor tyrosine kinase gene required for vulval induction.

Genetics ◽

10.1093/genetics/128.2.251 ◽

1991 ◽

Vol 128 (2) ◽

pp. 251-267 ◽

Cited By ~ 4

Author(s):

R V Aroian ◽

P W Sternberg

Keyword(s):

Caenorhabditis Elegans ◽

Tyrosine Kinase ◽

Receptor Tyrosine Kinase ◽

Egf Receptor ◽

Kinase Gene ◽

Multiple Functions ◽

Tyrosine Kinase Gene ◽

Receptor Tyrosine Kinase Gene ◽

Epidermal Growth ◽

Mutant Phenotypes

Abstract The let-23 gene, which encodes a putative tyrosine kinase of the epidermal growth factor (EGF) receptor subfamily, has multiple functions during Caenorhabditis elegans development. We show that let-23 function is required for vulval precursor cells (VPCs) to respond to the signal that induces vulval differentiation: a complete loss of let-23 function results in no induction. However, some let-23 mutations that genetically reduce but do not eliminate let-23 function result in VPCs apparently hypersensitive to inductive signal: as many as five of six VPCs can adopt vulval fates, in contrast to the three that normally do. These results suggest that the let-23 receptor tyrosine kinase controls two opposing pathways, one that stimulates vulval differentiation and another that negatively regulates vulval differentiation. Furthermore, analysis of 16 new let-23 mutations indicates that the let-23 kinase functions in at least five tissues. Since various let-23 mutant phenotypes can be obtained independently, the let-23 gene is likely to have tissue-specific functions.

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Ontogeny of epidermal growth factor receptor tyrosine kinase in rat liver

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1991.260.2.g290 ◽

1991 ◽

Vol 260 (2) ◽

pp. G290-G298 ◽

Cited By ~ 1

Author(s):

B. K. De ◽

T. L. Brown ◽

F. J. Suchy

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Tyrosine Kinase ◽

Rat Liver ◽

Plasma Membranes ◽

Egf Receptor ◽

Specific Binding ◽

Liver Plasma Membranes ◽

Epidermal Growth ◽

Autokinase Activity

The binding of epidermal growth factor (EGF) to its receptor and the activity of the receptor intrinsic protein-tyrosine kinase were studied during the ontogeny of rat liver. The number of EGF receptors during pre- and postnatal development was first compared in crude liver plasma membranes using 1) specific binding of 125I-labeled EGF and 2) immunoblot analysis using any antireceptor polyclonal rabbit antibody. Both methods detected the expression of the EGF receptor in fetal rat liver on day 17 of gestation, but in an amount markedly less than the adult. Within 24 h, there was a more than twofold increase in EGF binding to plasma membranes as well as a marked increase in receptor immunoreactivity. However, after birth, there was a precipitous drop in receptor number to less than 20% of the adult level by the end of the first postnatal day (P less than 0.001). Next, the presence of EGF-stimulated tyrosine kinase activity (autophosphorylation) was determined during the same stages of development. Electrophoresis of membranes phosphorylated in the presence or absence of EGF followed by autoradiography demonstrated autokinase activity stimulated by EGF in day 18 and 19 fetal liver plasma membranes, but not in membranes on day 17 of gestation. Similar to the pattern observed with EGF binding, there was a decrease in autokinase activity in early neonatal plasma membranes followed by an increase to near adult levels by 7 days postnatally. Quantitation of the amount of 32P radioactivity associated with the EGF receptor bands in each age group, correlated with the degree of autophosphorylation assessed by autoradiography.(ABSTRACT TRUNCATED AT 250 WORDS)

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Rapid uptake of tyrphostin into A431 human epidermoid cells is followed by delayed inhibition of epidermal growth factor (EGF)-stimulated EGF receptor tyrosine kinase activity.

Molecular and Cellular Biology ◽

10.1128/mcb.11.5.2697 ◽

1991 ◽

Vol 11 (5) ◽

pp. 2697-2703 ◽

Cited By ~ 28

Author(s):

C A Faaland ◽

F H Mermelstein ◽

J Hayashi ◽

J D Laskin

Keyword(s):

Cell Cycle ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Tyrosine Kinase ◽

Receptor Tyrosine Kinase ◽

Kinase Activity ◽

Egf Receptor ◽

Tyrosine Kinase Activity ◽

A431 Cells ◽

Epidermal Growth

Treatment of A431 human epidermoid cells with epidermal growth factor (EGF; 20 nM) results in decreased proliferation. This is associated with blockage of the cells in the S and/or G2 phases of the cell cycle. We found that tyrphostin, a putative tyrosine kinase inhibitor, in the range of 50 to 100 microM, partially reversed the growth-inhibitory and cell cycle changes induced by EGF. By using high-pressure liquid chromatography with electrochemical detection, we found that tyrphostin was readily incorporated into A431 cells, reaching maximal levels within 1 h. Although tyrphostin (50 to 100 microM) had no effect on high-affinity binding of EGF to its receptor in A431 cells for up to 24 h, the compound partially inhibited EGF-stimulated EGF receptor tyrosine kinase activity. However, this effect was evident only after prolonged treatment of the cells (4 to 24 h) with the drug. When the peak intracellular concentration of tyrphostin occurred (1 h), no inhibition of tyrosine kinase activity was observed. After both 1 and 24 h, tyrphostin was a less effective inhibitor of tyrosine kinase activity than the potent tumor promoter 12-O-tetradecanoyl phorbol-13-acetate, which almost completely blocked EGF receptor autophosphorylation. On the basis of our data, we hypothesize that tyrphostin is not a competitive inhibitor of the EGF receptor tyrosine kinase in intact cells and that it functions by an indirect mechanism.

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Praeruptorin B Mitigates the Metastatic Ability of Human Renal Carcinoma Cells through Targeting CTSC and CTSV Expression

International Journal of Molecular Sciences ◽

10.3390/ijms21082919 ◽

2020 ◽

Vol 21 (8) ◽

pp. 2919

Author(s):

Chia-Liang Lin ◽

Tung-Wei Hung ◽

Tsung-Ho Ying ◽

Chi-Jui Lin ◽

Yi-Hsien Hsieh ◽

...

Keyword(s):

Growth Factor Receptor ◽

Cellular Migration ◽

Mitogen Activated Protein Kinase ◽

Migration And Invasion ◽

Antitumor Effects ◽

Protein Levels ◽

Extracellular Signal ◽

Adult Kidney ◽

Mitogen Activated Protein ◽

Underlying Mechanisms

Renal cell carcinoma (RCC) is the most common adult kidney cancer, and accounts for 85% of all cases of kidney cancers worldwide. Praeruptorin B (Pra-B) is a bioactive constituent of Peucedanum praeruptorum Dunn and exhibits several pharmacological activities, including potent antitumor effects. However, the anti-RCC effects of Pra-B and their underlying mechanisms are unclear; therefore, we explored the effects of Pra-B on RCC cells in this study. We found that Pra-B nonsignificantly influenced the cell viability of human RCC cell lines 786-O and ACHN at a dose of less than 30 μM for 24 h treatment. Further study revealed that Pra-B potently inhibited the migration and invasion of 786-O and ACHN cells, as well as downregulated the mRNA and protein expression of cathepsin C (CTSC) and cathepsin V (CTSV) of 786-O and ACHN cells. Mechanistically, Pra-B also reduced the protein levels of phospho (p)-epidermal growth factor receptor (EGFR), p-mitogen-activated protein kinase kinase (MEK), and p-extracellular signal-regulated kinases (ERK) in RCC cells. In addition, Pra-B treatment inhibited the effect of EGF on the upregulation of EGFR–MEK–ERK, CTSC and CTSV expression, cellular migration, and invasion of 786-O cells. Our findings are the first to demonstrate that Pra-B can reduce the migration and invasion ability of human RCC cells through suppressing the EGFR-MEK-ERK signaling pathway and subsequently downregulating CTSC and CTSV. This evidence suggests that Pra-B can be developed as an effective antimetastatic agent for the treatment of RCC.

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Insulin Growth Factor-I and Epidermal Growth Factor Receptors Recruit Distinct Upstream Signaling Molecules to Enhance AKT Activation in Mammary Epithelial Cells

Endocrinology ◽

10.1210/en.2006-0349 ◽

2006 ◽

Vol 147 (12) ◽

pp. 6027-6035 ◽

Cited By ~ 11

Author(s):

Jodie M. Fleming ◽

Gwenaëlle Desury ◽

Tiffany A. Polanco ◽

Wendie S. Cohick

Keyword(s):

Growth Factors ◽

Growth Factor ◽

Dna Synthesis ◽

Egf Receptor ◽

Mammary Epithelial ◽

Insulin Receptor Substrate ◽

Phosphatidylinositol 3 Kinase ◽

Mapk Activation ◽

Igf I ◽

Epidermal Growth

IGF-I and epidermal growth factor (EGF) stimulate both normal mammary epithelial cell (MEC) growth and tumorigenesis. Whereas both growth factors increase DNA synthesis in MECs, how they evoke a greater response in combination when they activate similar signaling pathways remains unknown. In the present study, we investigated the signaling pathways by which these mitogens act in concert to increase DNA synthesis. Only EGF activated the MAPK pathway, and no further increase in MAPK activation was observed when both mitogens were added together. Both growth factors activated the phosphatidylinositol-3 kinase pathway, and simultaneous treatment enhanced phosphorylation of both AKT and its downstream target, p70S6K. The enhanced activation of AKT was observed at multiple time points (5 and 15 min) and growth factor concentrations (2.5–100 ng/ml). IGF-I activated AKT via insulin receptor substrate-1 and p85, the regulatory subunit of phosphatidylinositol-3 kinase. Treatment with EGF had no effect on insulin receptor substrate-1; however, it activated the EGF receptor, SHC, and c-Src. EGF treatment caused the association of SHC with Grb2 and Gab2 with phospho-SHC, phospho-Gab1, Grb2, and p85. Interestingly, inhibition of Src activation blocked the ability of EGF, but not IGF-I, to activate AKT. This corresponded with a decrease in phosphorylation of the EGF receptor and its association with phospho-SHC as well as downstream signaling. Unexpectedly, inhibition of Src increased basal MAPK activation. This is the first study to show that EGF and IGF-I use separate upstream components within a given MEC line to enhance AKT phosphorylation, contributing to increased DNA synthesis.

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Signal transduction by epidermal growth factor and heregulin via the kinase-deficient ErbB3 protein

Biochemical Journal ◽

10.1042/bj3340189 ◽

1998 ◽

Vol 334 (1) ◽

pp. 189-195 ◽

Cited By ~ 71

Author(s):

Hong-Hee KIM ◽

Ulka VIJAPURKAR ◽

Nathan J. HELLYER ◽

Dolores BRAVO ◽

John G. KOLAND

Keyword(s):

Signal Transduction ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Tyrosine Kinase ◽

Protein Tyrosine Kinase ◽

Kinase Activity ◽

Egf Receptor ◽

Tyrosine Kinase Activity ◽

Epidermal Growth ◽

Protein Tyrosine Kinase Activity

The role of protein tyrosine kinase activity in ErbB3-mediated signal transduction was investigated. ErbB3 was phosphorylated in vivo in response to either heregulin (HRG) in cells expressing both ErbB3 and ErbB2, or epidermal growth factor (EGF) in cells expressing both ErbB3 and EGF receptor. A recombinant receptor protein (ErbB3-K/M, in which K/M stands for Lys → Met amino acid substitution) containing an inactivating mutation in the putative ATP-binding site was also phosphorylated in response to HRG and EGF. Both the wild-type ErbB3 and mutant ErbB3-K/M proteins transduced signals to phosphatidylinositol 3-kinase, Shc and mitogen-activated protein kinases. Separate kinase-inactivating mutations in the EGF receptor and ErbB2 proteins abolished ErbB3 phosphorylation and signal transduction activated by EGF and HRG respectively. Hence the protein tyrosine kinase activity necessary for growth factor signalling via the ErbB3 protein seems to be provided by coexpressed EGF and ErbB2 receptor proteins.

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Temperature-dependent tyrosine phosphorylation of microtubule-associated protein kinase in epidermal growth factor-stimulated human fibroblasts.

Cell Regulation ◽

10.1091/mbc.2.8.663 ◽

1991 ◽

Vol 2 (8) ◽

pp. 663-673 ◽

Cited By ~ 23

Author(s):

R Campos-González ◽

J R Glenney

Keyword(s):

Growth Factor ◽

Protein Kinase ◽

Epidermal Growth Factor ◽

Tyrosine Kinase ◽

Map Kinase ◽

Tyrosine Phosphorylation ◽

Egf Receptor ◽

Human Fibroblasts ◽

C Terminus ◽

Epidermal Growth

Treatment of normal human fibroblasts with epidermal growth factor (EGF) results in the rapid (0.5 min) and simultaneous tyrosine phosphorylation of the EGF receptor (EGFr) and several other proteins. An exception to this tyrosine phosphorylation wave was a protein (42 kDa) that became phosphorylated on tyrosine only after a short lag time (5 min). We identified this p42 kDa substrate as the microtubule-associated protein (MAP) kinase using a monoclonal antibody to a peptide corresponding to the C-terminus of the predicted protein (Science 249, 64-67, 1990). EGF treatment of human fibroblasts at 37 degrees C for 5 min resulted in the tyrosine phosphorylation of 60-70% of MAP kinase as determined by the percent that was immunoprecipitated with antiphosphotyrosine antibodies. Like other tyrosine kinase growth factor receptors, the EGFr is activated and phosphorylated at 4 degrees C but is not internalized. Whereas most other substrates were readily tyrosine phosphorylated at 4 degrees C, MAP kinase was not. When cells were first stimulated with EGF at 4 degrees C and then warmed to 37 degrees C without EGF, tyrosine phosphorylation of MAP kinase was again observed. Treatment of cells with the protein kinase C activator phorbol myristate acetate (PMA) also resulted in the tyrosine phosphorylation of MAP kinase, and again only at 37 degrees C. Tryptic phosphopeptide maps demonstrated that EGF and PMA both induced the phosphorylation of the same peptide on tyrosine and threonine. This temperature and PMA sensitivity distinguishes MAP kinase from most other tyrosine kinase substrates in activated human fibroblasts.

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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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Inhibition of tyrosine kinase activity of the epidermal growth factor (EGF) receptor by a truncated receptor form that binds to EGF: role for interreceptor interaction in kinase regulation.

Molecular and Cellular Biology ◽

10.1128/mcb.9.2.671 ◽

1989 ◽

Vol 9 (2) ◽

pp. 671-677 ◽

Cited By ~ 77

Author(s):

A Basu ◽

M Raghunath ◽

S Bishayee ◽

M Das

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Tyrosine Kinase ◽

Binding Site ◽

Kinase Activity ◽

Egf Receptor ◽

Kinase Domain ◽

Tyrosine Kinase Activity ◽

Truncated Receptor ◽

Epidermal Growth

The tyrosine kinase activity of the epidermal growth factor (EGF) receptor is regulated by a truncated receptor of 100 kilodaltons (kDa) that contains the EGF-binding site but not the kinase domain. The inhibition of kinase is not due to competition for available EGF or for the kinase substrate-binding site. Chemical cross-linking studies suggest that the 100-kDa receptor may form a heterodimer with the intact EGF receptor. Structurally related receptor kinases, such as the platelet-derived growth factor receptor, the insulin receptor, and the Neu receptor, were not inhibited by the 100-kDa receptor. The results indicate that (i) the inhibition was specific for the EGF receptor, (ii) the kinase domain had little or no role in determining target specificity, and (iii) the regulation of kinase may be due to a specific interaction of the 100-kDa receptor with the ligand-binding domain of the EGF receptor kinase.

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