scholarly journals Carboxy-terminal truncations of epidermal growth factor (EGF) receptor affect diverse EGF-induced cellular responses.

1991 ◽  
Vol 2 (8) ◽  
pp. 641-649 ◽  
Author(s):  
W Li ◽  
N Hack ◽  
B Margolis ◽  
A Ullrich ◽  
K Skorecki ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tyrosine Phosphorylation ◽  
Egf Receptor ◽  
Phosphatidyl Inositol ◽  
Cellular Targets ◽  
Epidermal Growth ◽  
Carboxy Terminal ◽  
Pi Hydrolysis ◽  
Nih 3T3

The binding of epidermal growth factor (EGF) to its receptor induces tyrosine phosphorylation of phospholipase C gamma (PLC gamma), which appears to be necessary for its activation leading to phosphatidyl inositol (PI) hydrolysis. Moreover, EGF-receptor (EGF-R) activation and autophosphorylation results in binding of PLC gamma to the tyrosine phosphorylated carboxy-terminus of the receptor. To gain further insights into the mechanisms and interactions regulating these processes, we have analyzed transfected NIH-3T3 cells expressing two EGF-R carboxy-terminal deletion mutants (CD63 and CD126) with reduced capacity to stimulate PI hydrolysis, Ca2+ rises, and DNA synthesis. In fact, the CD126 mutant lacking 126 carboxy-terminal amino acids, including four tyrosine autophosphorylation sites, was unable to stimulate PI hydrolysis or Ca2+ rise in response to EGF. Surprisingly, EGF binding to the cell lines expressing CD63 or CD126 mutants was followed by similar stimulation of tyrosine phosphorylation of PLC gamma. Our results suggest that although necessary, tyrosine phosphorylation of PLC gamma may not be sufficient for stimulation and PI hydrolysis. It is clear, however, that the carboxy-terminal region of EGF-R is involved in regulation of interactions with cellular targets and therefore plays a crucial role in postreceptor signaling pathways.

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.

scholarly journals Chicken epidermal growth factor (EGF) receptor: cDNA cloning, expression in mouse cells, and differential binding of EGF and transforming growth factor alpha.

1988 ◽  
Vol 8 (5) ◽  
pp. 1970-1978 ◽  
Author(s):  
I Lax ◽  
A Johnson ◽  
R Howk ◽  
J Sap ◽  
F Bellot ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Alpha ◽  
3T3 Cells ◽  
Epidermal Growth ◽  
Factor Alpha ◽  
Differential Binding ◽  
Nih 3T3

The primary structure of the chicken epidermal growth factor (EGF) receptor was deduced from the sequence of a cDNA clone containing the complete coding sequence and shown to be highly homologous to the human EGF receptor. NIH-3T3 cells devoid of endogenous EGF receptor were transfected with the appropriate cDNA constructs and shown to express either chicken or human EGF receptors. Like the human EGF receptor, the chicken EGF receptor is a glycoprotein with an apparent molecular weight of 170,000. Murine EGF bound to the chicken receptor with approximately 100-fold lower affinity than to the human receptor molecule. Surprisingly, human transforming growth factor alpha (TGF-alpha) bound equally well or even better to the chicken EGF receptor than to the human EGF receptor. Moreover, TGF-alpha stimulated DNA synthesis 100-fold better than did EGF in NIH 3T3 cells that expressed the chicken EGF receptor. The differential binding and potency of mammalian EGF and TGF-alpha by the avian EGF receptor contrasts with the similar affinities of the mammalian receptor for the two growth factors.

scholarly journals Tyrosine Phosphorylation of the c-cblProto-oncogene Protein Product and Association with Epidermal Growth Factor (EGF) Receptor upon EGF Stimulation

1995 ◽  
Vol 270 (35) ◽  
pp. 20242-20245 ◽  
Author(s):  
Maria L. Galisteo ◽  
Ivan Dikic ◽  
Andreas G. Batzer ◽  
Wallace Y. Langdon ◽  
Joseph Schlessinger
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tyrosine Phosphorylation ◽  
Egf Receptor ◽  
Protein Product ◽  
Epidermal Growth ◽  
Oncogene Protein

Release of a phorbol ester-induced mitogenic block by mutation at Thr-654 of the epidermal growth factor receptor

1988 ◽  
Vol 8 (6) ◽  
pp. 2302-2308
Author(s):  
E Livneh ◽  
T J Dull ◽  
E Berent ◽  
R Prywes ◽  
A Ullrich ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Ligand Binding ◽  
Binding Affinity ◽  
Egf Receptor ◽  
Wild Type ◽  
3T3 Cells ◽  
Kinase C ◽  
Epidermal Growth ◽  
Nih 3T3

The tumor promoter phorbol ester (TPA) modulates the binding affinity and the mitogenic capacity of the epidermal growth factor (EGF) receptor. Moreover, TPA-induced kinase C phosphorylation occurs mainly on Thr-654 of the EGF receptor, suggesting that the phosphorylation state of this residue regulates ligand-binding affinity and kinase activity of the EGF receptor. To examine the role of this residue, we prepared a Tyr-654 EGF receptor cDNA construct by in vitro site-directed mutagenesis. Like the wild-type receptor, the mutant receptor exhibited typical high- and low-affinity binding sites when expressed on the surface of NIH 3T3 cells. Moreover, TPA regulated the affinity of both wild-type and mutant receptors and stimulated receptor phosphorylation of serine and threonine residues other than Thr-654. The addition of TPA to NIH 3T3 cells expressing a wild-type human EGF receptor blocked the mitogenic capacity of EGF. However, this inhibition did not occur in cells expressing the Tyr-654 EGF receptor mutant. In the latter cells, EGF was able to stimulate DNA synthesis even in the presence of inhibitory concentrations of TPA. While phosphorylation of sites other than Thr-654 may regulate ligand-binding affinity, the phosphorylation of Thr-654 by kinase C appears to provide a negative control mechanism for EGF-induced mitogenesis in mouse NIH 3T3 fibroblasts.

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