scholarly journals Activity of the epidermal-growth-factor receptor and phospholipase C-γ 1 in heat-stressed fibroblasts and A-431 cells

1992 ◽  
Vol 286 (2) ◽  
pp. 541-547 ◽  
Author(s):  
S M Liu ◽  
G Carpenter
Keyword(s):  
Growth Factor ◽  
Heat Shock ◽  
Epidermal Growth Factor ◽  
Phospholipase C ◽  
Egf Receptor ◽  
Inositol Phosphates ◽  
Human Fibroblasts ◽  
Egf Receptors ◽  
Epidermal Growth ◽  
Phospholipase C Gamma

A variety of changes in the functions of specific plasma-membrane components have been reported in cells exposed to a heat shock. In this study, we examined the consequences of heat stress on epidermal-growth-factor (EGF)-induced receptor autophosphorylation and receptor-mediated tyrosine phosphorylation of phospholipase C-gamma 1 (PLC-gamma 1), a cellular substrate. Although the tyrosine kinase activity of the EGF receptor is rapidly inactivated at 45 degrees C in vitro [Carpenter, King & Cohen (1979) J. Biol. Chem. 254, 4884-4891], EGF stimulates autophosphorylation of its receptor in both A-431 cells and human fibroblasts after a prolonged heat shock. Phosphoamino acid analysis of the receptor reveals an EGF-induced increase in phosphotyrosine and phosphoserine at 46 degrees C. EGF also stimulates the phosphorylation of phospholipase C-gamma 1 and induces the formation of inositol phosphates under heat-shock conditions. 125I-EGF binding and internalization in A-431 cells is not decreased during incubations at 46 degrees C for up to 90 min. EGF-induced dimerization of EGF receptors on the cell surface is preserved during heat shock. Though EGF-receptor-mediated endocytosis is not inhibited by elevated temperature, the degradation of internalized 125I-EGF is dramatically decreased. These results indicate that, aside from ligand degradation, the EGF-mediated pathway of signal transduction through phospholipase C-gamma 1 remains remarkably intact during conditions of extreme cellular stress.

scholarly journals Phosphorylation and activation of epidermal growth factor receptors in cells transformed by the src oncogene.

1991 ◽  
Vol 11 (1) ◽  
pp. 309-321 ◽  
Author(s):  
W J Wasilenko ◽  
D M Payne ◽  
D L Fitzgerald ◽  
M J Weber
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Phospholipase C ◽  
Tyrosine Phosphorylation ◽  
Egf Receptor ◽  
Growth Factor Receptors ◽  
Epidermal Growth ◽  
Phospholipase C Gamma ◽  
Signaling Activity ◽  
In Cells

Because functionally significant substrates for the tyrosyl protein kinase activity of pp60v-src are likely to include membrane-associated proteins involved in normal growth control, we have tested the hypothesis that pp60v-src could phosphorylate and alter the signaling activity of transmembrane growth factor receptors. We have found that the epidermal growth factor (EGF) receptor becomes constitutively phosphorylated on tyrosine in cells transformed by the src oncogene and in addition displays elevated levels of phosphoserine and phosphothreonine. High-performance liquid chromatography phosphopeptide mapping revealed two predominant sites of tyrosine phosphorylation, both of which differed from the major sites of receptor autophosphorylation; thus, the src-induced phosphorylation is unlikely to occur via an autocrine mechanism. To determine whether pp60v-src altered the signaling activity of the EGF receptor, we analyzed the tyrosine phosphorylation of phospholipase C-gamma, since phosphorylation of this enzyme occurs in response to activation of the EGF receptor but not in response to pp60v-src alone. We found that in cells coexpressing pp60v-src and the EGF receptor, phospholipase C-gamma was constitutively phosphorylated, a result we interpret as indicating that the signaling activity of the EGF receptor was altered in the src-transformed cells. These findings suggest that pp60v-src-induced alterations in phosphorylation and function of growth regulatory receptors could play an important role in generating the phenotypic changes associated with malignant transformation.

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

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.

scholarly journals Epidermal growth factor stimulates distinct diradylglycerol species generation in Swiss 3T3 fibroblasts: evidence for a potential phosphatidylcholine-specific phospholipase C-catalysed pathway

1994 ◽  
Vol 298 (3) ◽  
pp. 655-660 ◽  
Author(s):  
T R Pettitt ◽  
M Zaqqa ◽  
M J Wakelam
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Phospholipase C ◽  
Phospholipase D ◽  
Kinase Inhibitor ◽  
Inositol Phosphates ◽  
Egf Receptors ◽  
3T3 Fibroblasts ◽  
Epidermal Growth ◽  
Hydrolysis Of

Stimulation of 3T3 fibroblasts with epidermal growth factor (EGF) results in an increase in 1,2-diacylglycerol (DAG) mass which is maximal at 25 s, declining at 1 min and returning to basal levels by 30 min. No changes in alkylacylglycerol or alkenylacylglycerol were detected. Three species account for most of this mass increase: 18:0/20:5,n-3, 18:0/20:4,n-6 and 18:0/20:3,n-9. These species are characteristic of the phosphoinositides; however, previous work failed to detect any EGF-stimulated rise in inositol phosphates in these cells [Cook and Wakelam (1992) Biochem. J. 285, 247-253]. This ruled out phosphoinositide hydrolysis by phospholipase C, but raised the possibility of phospholipase D/phosphatidate phosphohydrolase-catalysed hydrolysis of phosphatidylinositol. The inclusion of butanol in the incubation medium failed to block the diacylglycerol changes, indicating that the phospholipase D pathway is not involved and that DAG must be derived from another source, probably via phospholipase C-catalysed hydrolysis of a phosphatidylcholine pool that is particularly rich in these species. The tyrosine kinase inhibitor ST-271 almost abolished the elevation in 18:0/20:5,n-3, 18:0/20:4, n-6 and 18:0/20:3,n-9 at 25 s, but only reduced the rise in total DAG mass by about 50%. The protein kinase C (PKC) inhibitor Ro-31-8220 increased DAG levels at all time points but had no effect on the species profiles. This provides additional evidence for PKC-mediated regulation of cell-surface EGF receptors, since the inhibition of PKC would increase the availability and/or ligand binding affinity of receptors at the plasma membrane and hence increase and prolong the response to EGF.

Tyrosine kinase activity is essential for the association of phospholipase C-gamma with the epidermal growth factor receptor

1990 ◽  
Vol 10 (2) ◽  
pp. 435-441
Author(s):  
B Margolis ◽  
F Bellot ◽  
A M Honegger ◽  
A Ullrich ◽  
J Schlessinger ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Phospholipase C ◽  
Kinase Activity ◽  
Egf Receptor ◽  
Growth Factor Receptor ◽  
Wild Type ◽  
Epidermal Growth ◽  
Phospholipase C Gamma ◽  
Negative Mutant

Epidermal growth factor (EGF) treatment of NIH 3T3 cells transfected with wild-type EGF receptor induced tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma). The EGF receptor and PLC-gamma were found to be physically associated such that antibodies directed against PLC-gamma or the EGF receptor coimmunoprecipitated both proteins. The association between PLC-gamma and wild-type EGF receptor was dependent on the concentration of EGF, but EGF did not enhance the association between PLC-gamma and a kinase-negative mutant of the EGF receptor. Oligomerization of the EGF receptor was not sufficient to induce association of the EGF receptor with PLC-gamma, since the kinase-negative mutant receptor underwent normal dimerization in response to EGF yet did not associate with PLC-gamma. The form of PLC-gamma associated with the EGF receptor appeared to be primarily the non-tyrosine-phosphorylated form. It is concluded that the kinase activity of the EGF receptor is essential for association of PLC-gamma with the EGF receptor, possibly by stimulating receptor autophosphorylation.

scholarly journals Interactions between the receptors for platelet-derived growth factor and epidermal growth factor.

1983 ◽  
Vol 96 (3) ◽  
pp. 679-683 ◽  
Author(s):  
D F Bowen-Pope ◽  
P E Dicorleto ◽  
R Ross
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Human Fibroblasts ◽  
Insulin Binding ◽  
Platelet Derived Growth Factor ◽  
Egf Receptors ◽  
3T3 Cells ◽  
Low Concentrations ◽  
Epidermal Growth

Preincubation of Swiss 3T3 cells or human fibroblasts with purified platelet-derived growth factor (PDGF) at 4 degrees C or 37 degrees C rapidly inhibits subsequent binding of 125I-epidermal growth factor (125I-EGF). The effect does not result from competition by PDGF for binding to the EGF receptor since (a) very low concentrations of PDGF are effective, (b) cells with EGF receptors but no PDGF receptors are not affected, and (c) the inhibition persists even if the bound PDGF is eluted before incubating the cells with 125I-EGF. PDGF does not affect 125I-insulin binding nor does EGF affect 125I-PDGF binding under these conditions. Endothelial cell-derived growth factor also competes for binding to PDGF receptors and inhibits 125I-EGF binding. The inhibition demonstrated by PDGF seems to result from an increase in the Kd for 125I-EGF binding with no change in the number of EGF receptors.

scholarly journals Rap2B-Dependent Stimulation of Phospholipase C-ε by Epidermal Growth Factor Receptor Mediated by c-Src Phosphorylation of RasGRP3

2004 ◽  
Vol 24 (11) ◽  
pp. 4664-4676 ◽  
Author(s):  
Matthias B. Stope ◽  
Frank vom Dorp ◽  
Daniel Szatkowski ◽  
Anja Böhm ◽  
Melanie Keiper ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Phospholipase C ◽  
Tyrosine Phosphorylation ◽  
Egf Receptor ◽  
Dominant Negative ◽  
Nucleotide Exchange ◽  
Egf Receptors ◽  
Epidermal Growth ◽  
Stimulation Of

ABSTRACT Receptor tyrosine kinase regulation of phospholipase C-ε (PLC-ε), which is under the control of Ras-like and Rho GTPases, was studied with HEK-293 cells endogenously expressing PLC-coupled epidermal growth factor (EGF) receptors. PLC and Ca2+ signaling by the EGF receptor, which activated both PLC-γ1 and PLC-ε, was specifically suppressed by inactivation of Ras-related GTPases with clostridial toxins and expression of dominant-negative Rap2B. EGF induced rapid and sustained GTP loading of Rap2B, binding of Rap2B to PLC-ε, and Rap2B-dependent translocation of PLC-ε to the plasma membrane. GTP loading of Rap2B by EGF was inhibited by chelation of intracellular Ca2+ and expression of lipase-inactive PLC-γ1 but not of PLC-ε. Expression of RasGRP3, a Ca2+/diacylglycerol-regulated guanine nucleotide exchange factor for Ras-like GTPases, but not expression of various other exchange factors enhanced GTP loading of Rap2B and PLC/Ca2+ signaling by the EGF receptor. EGF induced tyrosine phosphorylation of RasGRP3, but not RasGRP1, apparently caused by c-Src; inhibition of c-Src interfered with EGF-induced Rap2B activation and PLC stimulation. Collectively, these data suggest that the EGF receptor triggers activation of Rap2B via PLC-γ1 activation and tyrosine phosphorylation of RasGRP3 by c-Src, finally resulting in stimulation of PLC-ε.

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

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.

scholarly journals Tyrosine kinase activity is essential for the association of phospholipase C-gamma with the epidermal growth factor receptor.

1990 ◽  
Vol 10 (2) ◽  
pp. 435-441 ◽  
Author(s):  
B Margolis ◽  
F Bellot ◽  
A M Honegger ◽  
A Ullrich ◽  
J Schlessinger ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Phospholipase C ◽  
Kinase Activity ◽  
Egf Receptor ◽  
Growth Factor Receptor ◽  
Wild Type ◽  
Epidermal Growth ◽  
Phospholipase C Gamma ◽  
Negative Mutant

Epidermal growth factor (EGF) treatment of NIH 3T3 cells transfected with wild-type EGF receptor induced tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma). The EGF receptor and PLC-gamma were found to be physically associated such that antibodies directed against PLC-gamma or the EGF receptor coimmunoprecipitated both proteins. The association between PLC-gamma and wild-type EGF receptor was dependent on the concentration of EGF, but EGF did not enhance the association between PLC-gamma and a kinase-negative mutant of the EGF receptor. Oligomerization of the EGF receptor was not sufficient to induce association of the EGF receptor with PLC-gamma, since the kinase-negative mutant receptor underwent normal dimerization in response to EGF yet did not associate with PLC-gamma. The form of PLC-gamma associated with the EGF receptor appeared to be primarily the non-tyrosine-phosphorylated form. It is concluded that the kinase activity of the EGF receptor is essential for association of PLC-gamma with the EGF receptor, possibly by stimulating receptor autophosphorylation.

scholarly journals Transmembrane signalling at the epidermal growth factor receptor. Positive regulation by the C-terminal phosphotyrosine residues

1991 ◽  
Vol 277 (2) ◽  
pp. 305-311 ◽  
Author(s):  
M Magni ◽  
A Pandiella ◽  
K Helin ◽  
J Meldolesi ◽  
L Beguinot
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Phospholipase C ◽  
Tyrosine Phosphorylation ◽  
Egf Receptor ◽  
Membrane Hyperpolarization ◽  
Transmembrane Signalling ◽  
Tyrosine Residues ◽  
Epidermal Growth ◽  
Phospholipase C Gamma

Mutant epidermal growth factor (EGF) receptors (obtained by substitution of one, two or three C-terminal autophosphorylable tyrosine residues with phenylalanine residues or by deletion of the C-terminal 19 amino acids, including the distal tyrosine) were expressed in mouse NIH-3T3 fibroblast clones at densities comparable (less than 25% difference) with those in control clones expressing the wild-type receptor. Total EGF-induced phosphorylation of the mutated receptors was not appreciably changed with respect to controls, whereas autophosphorylation at tyrosine residues was decreased, especially in the double and the triple mutants. In the latter mutant, expression of the EGF-receptor-activated lipolytic enzyme phospholipase C gamma was unchanged, whereas its tyrosine phosphorylation induced by the growth factor was lowered to approx. 25% of that in the controls. In all of the cell clones employed, the accumulation of inositol phosphates induced by treatment with fetal calf serum varied only slightly, whereas the same effect induced by EGF was consistently lowered in those lines expressing mutated receptors. This decrease was moderate for those receptors missing only the distal tyrosine (point and deletion mutants), intermediate in the dual mutants and almost complete in the triple mutants. Likewise, increases in intracellular Ca2+ concentrations [(Ca2+]i) induced by fibroblast growth factor were approximately the same in all of the clones, whereas those induced by EGF were decreased in the mutants, again in proportion to the loss of the phosphorylable C-terminal tyrosine residues. The same trend occurred with membrane hyperpolarization, an effect secondary to the increase in [Ca2+]i via the activation of Ca2(+)-dependent K+ channels. We conclude that C-terminal autophosphorylable tyrosine residues play a positive role in the regulation of transmembrane signalling at the EGF receptor. The stepwise decrease in signal generation observed in single, double and triple point mutants suggest that the role of phosphotyrosine residues is not in the participation in specific amino acid sequences, but rather in the introduction of strong negative charges at strategic sites of the receptor tail. As a consequence of autophosphorylation, the receptor could become competent for specific association with phospholipase C gamma, with ensuing activation by tyrosine phosphorylation followed by the chains of intracellular responses ultimately leading to DNA synthesis and cell duplication.

Phosphorylation and activation of epidermal growth factor receptors in cells transformed by the src oncogene

1991 ◽  
Vol 11 (1) ◽  
pp. 309-321
Author(s):  
W J Wasilenko ◽  
D M Payne ◽  
D L Fitzgerald ◽  
M J Weber
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Phospholipase C ◽  
Tyrosine Phosphorylation ◽  
Egf Receptor ◽  
Growth Factor Receptors ◽  
Epidermal Growth ◽  
Phospholipase C Gamma ◽  
Signaling Activity ◽  
In Cells

Because functionally significant substrates for the tyrosyl protein kinase activity of pp60v-src are likely to include membrane-associated proteins involved in normal growth control, we have tested the hypothesis that pp60v-src could phosphorylate and alter the signaling activity of transmembrane growth factor receptors. We have found that the epidermal growth factor (EGF) receptor becomes constitutively phosphorylated on tyrosine in cells transformed by the src oncogene and in addition displays elevated levels of phosphoserine and phosphothreonine. High-performance liquid chromatography phosphopeptide mapping revealed two predominant sites of tyrosine phosphorylation, both of which differed from the major sites of receptor autophosphorylation; thus, the src-induced phosphorylation is unlikely to occur via an autocrine mechanism. To determine whether pp60v-src altered the signaling activity of the EGF receptor, we analyzed the tyrosine phosphorylation of phospholipase C-gamma, since phosphorylation of this enzyme occurs in response to activation of the EGF receptor but not in response to pp60v-src alone. We found that in cells coexpressing pp60v-src and the EGF receptor, phospholipase C-gamma was constitutively phosphorylated, a result we interpret as indicating that the signaling activity of the EGF receptor was altered in the src-transformed cells. These findings suggest that pp60v-src-induced alterations in phosphorylation and function of growth regulatory receptors could play an important role in generating the phenotypic changes associated with malignant transformation.

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