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

1995 ◽  
Vol 305 (2) ◽  
pp. 613-619 ◽  
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.

scholarly journals Epidermal growth factor-induced phosphoinositide hydrolysis in permeabilized 3T3 cells: lack of guanosine triphosphate dependence and inhibition by tyrosine-containing peptides.

1990 ◽  
Vol 1 (9) ◽  
pp. 615-620 ◽  
Author(s):  
G F Verheijden ◽  
I Verlaan ◽  
J Schlessinger ◽  
W H Moolenaar
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
G Protein ◽  
Egf Receptor ◽  
Phosphoinositide Hydrolysis ◽  
3T3 Cells ◽  
Guanosine Triphosphate ◽  
Intact Cells ◽  
Epidermal Growth ◽  
Gamma S

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.

Involvement of Shc in insulin- and epidermal growth factor-induced activation of p21ras

1994 ◽  
Vol 14 (3) ◽  
pp. 1575-1581
Author(s):  
G J Pronk ◽  
A M de Vries-Smits ◽  
L Buday ◽  
J Downward ◽  
J A Maassen ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Egf Receptor ◽  
Growth Factor Receptor ◽  
Nucleotide Exchange ◽  
Similar Time ◽  
Epidermal Growth

Shc proteins are phosphorylated on tyrosine residues and associate with growth factor receptor-bound protein 2 (Grb2) upon treatment of cells with epidermal growth factor (EGF) or insulin. We have studied the role of Shc in insulin- and EGF-induced activation of p21ras in NIH 3T3 cells overexpressing human insulin receptors (A14 cells). A14 cells are equally responsive to insulin and EGF with respect to activation of p21ras. Analysis of Shc immunoprecipitates revealed that (i) both insulin and EGF treatment resulted in Shc tyrosine phosphorylation and (ii) Shc antibodies coimmunoprecipitated both Grb2 and mSOS after insulin and EGF treatment. The induction of tyrosine phosphorylation of Shc and the presence of Grb2 and mSOS in Shc immunoprecipitates followed similar time courses, with somewhat higher levels after EGF treatment. In mSOS immunoprecipitates, Shc could be detected as well. Furthermore, Shc immune complexes contained guanine nucleotide exchange activity toward p21ras in vitro. From these results, we conclude that after insulin and EGF treatment, Shc associates with both Grb2 and mSOS and therefore may mediate, at least in part, insulin- and EGF-induced activation of p21ras. In addition, we investigated whether the Grb2-mSOS complex associates with the insulin receptor or with insulin receptor substrate 1 (IRS1). Although we observed association of Grb2 with IRS1, we did not detect complex formation between mSOS and IRS1 in experiments in which the association of mSOS with Shc was readily detectable. Furthermore, whereas EGF treatment resulted in the association of mSOS with the EGF receptor, insulin treatment did not result in the association of mSOS with the insulin receptor. These results indicate that the association of Grb2-nSOS with Shc may be an important event in insulin-induced, mSOS-mediated activation of p21ras.

Evidence for epidermal growth factor (EGF)-induced intermolecular autophosphorylation of the EGF receptors in living cells

1990 ◽  
Vol 10 (8) ◽  
pp. 4035-4044
Author(s):  
A M Honegger ◽  
A Schmidt ◽  
A Ullrich ◽  
J Schlessinger
Keyword(s):  
Signal Transduction ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tyrosine Phosphorylation ◽  
Egf Receptor ◽  
Living Cells ◽  
Egf Receptors ◽  
Receptor Molecule ◽  
Epidermal Growth ◽  
Negative Mutant

In response to epidermal growth factor (EGF) stimulation, the intrinsic protein tyrosine kinase of EGF receptor is activated, leading to tyrosine phosphorylation of several cellular substrate proteins, including the EGF receptor molecule itself. To test the mechanism of EGF receptor autophosphorylation in living cells, we established transfected cell lines coexpressing a kinase-negative point mutant of EGF receptor (K721A) with an active EGF receptor mutant lacking 63 amino acids from its carboxy terminus. The addition of EGF to these cells caused tyrosine phosphorylation of the kinase-negative mutant by the active receptor molecule, demonstrating EGF receptor cross-phosphorylation in living cells. After internalization the kinase-negative mutant and CD63 have separate trafficking pathways. This limits their association and the extent of cross-phosphorylation of K721A by CD63. The coexpression of the kinase-negative mutant together with active EGF receptors in the same cells suppressed the mitogenic response toward EGF as compared with that in cells that express active receptors alone. The presence of the kinase-negative mutant functions as a negative dominant mutation suppressing the response of active EGF receptors, probably by interfering with EGF-induced signal transduction. It appears, therefore, that crucial events of signal transduction occur before K721A and active EGF receptors are separated by their different endocytic itineraries.

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 Epidermal Growth Factor-Induced Heterologous Desensitization of the Luteinizing Hormone/Choriogonadotopin Receptor in a Cell-Free Membrane Preparation Is Associated with the Tyrosine Phosphorylation of the Epidermal Growth Factor Receptor**This work was supported by USDA Grant NRICGP-9401432 (to M.H.D.).

Endocrinology ◽  
1999 ◽  
Vol 140 (1) ◽  
pp. 29-36 ◽  
Author(s):  
Marilyn L. G. Lamm ◽  
Rajsree M. Rajagopalan-Gupta ◽  
Mary Hunzicker-Dunn
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Adenylyl Cyclase ◽  
Tyrosine Phosphorylation ◽  
Egf Receptor ◽  
Cyclase Activity ◽  
Adenylyl Cyclase Activity ◽  
Heterologous Desensitization ◽  
Dependent Protein ◽  
Epidermal Growth

Abstract Epidermal growth factor (EGF) attenuated hCG-stimulated adenylyl cyclase activity in rat luteal and follicular membranes. H7, an equipotent serine/threonine protein kinase inhibitor of cAMP-dependent protein kinases, cGMP-dependent protein kinases, and lipid-dependent protein kinase C, did not effect the ability of EGF to decrease hCG-responsive adenylyl cyclase activity, suggesting that a serine/threonine phosphorylation event catalyzed by these kinases was not critically involved in EGF-induced desensitization. Likewise, pertussis toxin-catalyzed ADP-ribosylation of a 40-kDa luteal membrane protein, which exhibited immunoreactivity with an antibody against Giα, did not hinder the ability of EGF to attenuate hCG-stimulated adenylyl cyclase activity, indicating that Gi did not mediate EGF-induced desensitization. Rather, EGF-induced heterologous desensitization of LH/CG receptor in ovarian membranes was closely associated with the specific and prominent tyrosine phosphorylation of the 170-kDa EGF receptor. Both EGF-stimulated autophosphorylation of EGF receptor and EGF-induced LH/CG receptor desensitization were attenuated by genistein, a tyrosine kinase inhibitor. These results suggest that tyrosine phosphorylation of the 170-kDa EGF receptor is a necessary component of the signaling pathway in EGF-induced heterologous desensitization of the LH/CG receptor.

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

1991 ◽  
Vol 2 (8) ◽  
pp. 663-673 ◽  
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.

Processing Temporal Growth Factor Patterns by an Epidermal Growth Factor Receptor Network Dynamically Established in Space

2020 ◽  
Vol 36 (1) ◽  
pp. 359-383 ◽  
Author(s):  
Aneta Koseska ◽  
Philippe I.H. Bastiaens
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Tyrosine Phosphatase ◽  
Operation Mode ◽  
Growth Factor Receptor ◽  
Couple Growth ◽  
Kinetic Mechanisms ◽  
Cellular Context ◽  
Epidermal Growth

The proto-oncogenic epidermal growth factor (EGF) receptor (EGFR) is a tyrosine kinase whose sensitivity and response to growth factor signals that vary over time and space determine cellular behavior within a developing tissue. The molecular reorganization of the receptors on the plasma membrane and the enzyme-kinetic mechanisms of phosphorylation are key determinants that couple growth factor binding to EGFR signaling. To enable signal initiation and termination while simultaneously accounting for suppression of aberrant signaling, a coordinated coupling of EGFR kinase and protein tyrosine phosphatase activity is established through space by vesicular dynamics. The dynamical operation mode of this network enables not only time-varying growth factor sensing but also adaptation of the response depending on cellular context. By connecting spatially coupled enzymatic kinase/phosphatase processes and the corresponding dynamical systems description of the EGFR network, we elaborate on the general principles necessary for processing complex growth factor signals.

Malignant transformation of murine fibroblasts by a human c-Ha-ras-1 oncogene does not require a functional epidermal growth factor receptor

1986 ◽  
Vol 6 (10) ◽  
pp. 3382-3387
Author(s):  
I A McKay ◽  
P Malone ◽  
C J Marshall ◽  
A Hall
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Transforming Growth Factor ◽  
Cellular Transformation ◽  
Egf Receptors ◽  
Ras Gene ◽  
Ras Genes ◽  
Murine Fibroblasts ◽  
Epidermal Growth

Although mutations in ras genes are thought to be important for the development of about 20% of human tumors, almost nothing is known about the way in which these mutations lead to cellular transformation. The known biochemical properties of the 21-kilodalton ras proteins suggest that they may behave as G proteins, regulating the proliferation of cells in response to growth factor stimulation of a receptor. Although the putative receptor(s) has not been identified, several lines of evidence, in particular the fact that rodent cell lines containing ras oncogenes produce transforming growth factor alpha, have suggested that the epidermal growth factor (EGF) receptor is involved in ras transformation. Here we show that murine fibroblasts with no EGF receptors can be transformed to a completely malignant phenotype with a mutated ras gene. It appears, therefore, that the EGF receptor is not required for ras-mediated transformation of these cells.

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