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.

scholarly journals 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.

scholarly journals Epidermal growth factor receptor downregulation in cultured bovine cumulus cells: reconstitution of calcium signaling and stimulated membrane permeabilization

Reproduction ◽  
2005 ◽  
Vol 130 (4) ◽  
pp. 517-528 ◽  
Author(s):  
Zhong Zhao ◽  
Damien Garbett ◽  
Julia L Hill ◽  
David J Gross
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Cumulus Cell ◽  
Growth Factor Receptor ◽  
Cumulus Cells ◽  
Membrane Permeabilization ◽  
Egf Receptors ◽  
Epidermal Growth

Cumulus cell–oocyte complexes (COCs), culturedin vitro, are competent for maturation and fertilization. Inclusion of epidermal growth factor (EGF) in the COC culture medium enhancesin vitromaturation and subsequent embryonic development. It has been shown that isolated COCs exposed to EGF respond with a prolonged and pulsatile release of Ca2+into the extra-cellular medium and that cumulus cells (CCs) of complexes exhibit both a slow rise in intracellular [Ca2+] ([Ca2+]i) and plasma membrane permeabilization in response to EGF. These unusual signaling responses were examined in isolated, cultured bovine CCs. Few individual CCs showed [Ca2+]iincreases; the lack of response was found to be due to decrease of expression of endogenous EGF receptors after dissociation. CCs transfected with a human EGF receptor–GFP fusion protein showed robust, prolonged, EGF-stimulated [Ca2+]ielevations characteristic of CC responses in intact COCs. Many CCs that responded to EGF stimulation with a [Ca2+]irise also released entrapped fura-2 dye at the peak of the [Ca2+]iresponse, suggesting that CC permeabilization and death follows activation of the EGF receptor. The [Ca2+]ielevation due to EGF stimulation and subsequent membrane permeabilization was shown to be mediated by the inositol triphosphate signaling pathway.

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.

scholarly journals The epidermal growth factor receptor phosphorylates GTPase-activating protein (GAP) at Tyr-460, adjacent to the GAP SH2 domains.

1991 ◽  
Vol 11 (5) ◽  
pp. 2511-2516 ◽  
Author(s):  
X Q Liu ◽  
T Pawson
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Egf Receptor ◽  
Sh2 Domain ◽  
Ras Signaling ◽  
Gtpase Activating Protein ◽  
Epidermal Growth

GTPase-activating protein (GAP) stimulates the ability of p21ras to hydrolyze GTP to GDP. Since GAP is phosphorylated by a variety of activated or oncogenic protein-tyrosine kinases, it may couple tyrosine kinases to the Ras signaling pathway. The epidermal growth factor (EGF) receptor cytoplasmic domain phosphorylated human GAP in vitro within a single tryptic phosphopeptide. The same GAP peptide was also apparently phosphorylated on tyrosine in EGF-stimulated rat fibroblasts. Circumstantial evidence suggested that residue 460 might be the site of GAP tyrosine phosphorylation. This possibility was confirmed by phosphorylation of a synthetic peptide corresponding to the predicted tryptic peptide containing Tyr-460. Alteration of Tyr-460 to phenylalanine by site-directed mutagenesis diminished the in vitro phosphorylation of a bacterial GAP polypeptide by the EGF receptor. We conclude that Tyr-460 is a site of GAP tyrosine phosphorylation by the EGF receptor in vitro and likely in vivo. GAP Tyr-460 is located immediately C terminal to the second GAP SH2 domain, suggesting that its phosphorylation might have a role in regulating protein-protein interactions.

The epidermal growth factor receptor network in type 2 pneumocytes exposed to hyperoxia in vitro

1996 ◽  
Vol 270 (2) ◽  
pp. L242-L250 ◽  
Author(s):  
L. Nici ◽  
M. Medina ◽  
A. R. Frackelton
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tyrosine Phosphorylation ◽  
Transforming Growth Factor ◽  
Cell Damage ◽  
Growth Factor Receptor ◽  
Epidermal Growth

Hyperoxia is a well-characterized model of injury and repair of the lung. Type 1 cell damage is followed by type 2 cell proliferation and differentiation which restore normal structure and function. The epidermal growth factor receptor (EGFR) network is known to be a potent modulator of epithelial cell growth. Here we examine the EGFR network on isolated rat type 2 cells and SV40T-T2, a type 2 cell line, under normoxic conditions, after 24 and 48 h of in vitro hyperoxia, and after 24 h of normoxic recovery. EGF induces tyrosine phosphorylation of EGFRs in type 2 cells and SV40T-T2 cells, which decreases with hyperoxia and increases above normoxic levels in recovering cells, suggesting biphasic changes in receptor number or function with injury. The EGFR appears to be stimulated in an autocrine fashion in these cells. There is decreased DNA synthesis and proliferation in SV40T-T2 and isolated type 2 cells treated with tyrphostin B56, a specific EGFR inhibitor. Pretreatment with suramin, which binds to growth factor, results in increased EGFR tyrosine phosphorylation after stimulation, suggesting disruption of normal autocrine receptor downregulation. We have also identified transforming growth factor-alpha (TGF-alpha) in conditioned media (CM) from normoxic and hyperoxic SV40T-T2 and type 2 cells. Finally, we show increased EGF bioactivity in both bronchoalveolar lavage (BAL) from hyperoxic rats and CM from hyperoxic cells compared with normoxic controls. These findings support an integral role for an autocrine EGFR network in the type 2 cell response to injury.

The epidermal growth factor receptor phosphorylates GTPase-activating protein (GAP) at Tyr-460, adjacent to the GAP SH2 domains

1991 ◽  
Vol 11 (5) ◽  
pp. 2511-2516 ◽  
Author(s):  
X Q Liu ◽  
T Pawson
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Egf Receptor ◽  
Sh2 Domain ◽  
Ras Signaling ◽  
Gtpase Activating Protein ◽  
Epidermal Growth

GTPase-activating protein (GAP) stimulates the ability of p21ras to hydrolyze GTP to GDP. Since GAP is phosphorylated by a variety of activated or oncogenic protein-tyrosine kinases, it may couple tyrosine kinases to the Ras signaling pathway. The epidermal growth factor (EGF) receptor cytoplasmic domain phosphorylated human GAP in vitro within a single tryptic phosphopeptide. The same GAP peptide was also apparently phosphorylated on tyrosine in EGF-stimulated rat fibroblasts. Circumstantial evidence suggested that residue 460 might be the site of GAP tyrosine phosphorylation. This possibility was confirmed by phosphorylation of a synthetic peptide corresponding to the predicted tryptic peptide containing Tyr-460. Alteration of Tyr-460 to phenylalanine by site-directed mutagenesis diminished the in vitro phosphorylation of a bacterial GAP polypeptide by the EGF receptor. We conclude that Tyr-460 is a site of GAP tyrosine phosphorylation by the EGF receptor in vitro and likely in vivo. GAP Tyr-460 is located immediately C terminal to the second GAP SH2 domain, suggesting that its phosphorylation might have a role in regulating protein-protein interactions.

scholarly journals Non-catalytic activation of phospholipase C-γ 1 in vitro by epidermal growth factor receptor

1993 ◽  
Vol 293 (2) ◽  
pp. 507-511 ◽  
Author(s):  
S M T Hernández-Sotomayor ◽  
G Carpenter
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Phospholipase C ◽  
Tyrosine Kinases ◽  
Egf Receptor ◽  
Growth Factor Receptor ◽  
Catalytic Activation ◽  
Epidermal Growth

To investigate the possible functional role of epidermal growth factor (EGF) receptor-phospholipase C-gamma 1 (PLC-gamma 1) complexes, we have measured PLC-gamma 1 activity in vitro in the absence or presence of purified EGF receptor. Immunoprecipitates of PLC-gamma 1 from control A-431 cells were incubated without or with purified EGF receptor in the absence or presence of ATP. Under these conditions the EGF receptor increased non-tyrosine-phosphorylated PLC-gamma 1 activity 3-4-fold in the absence or presence of ATP, but increased tyrosine-phosphorylated and activated PLC-gamma 1 by only 20-50%. Both basal and autophosphorylated forms of the purified EGF receptor increased the activity of the non-tyrosine-phosphorylated PLC-gamma 1, and stoichiometric levels of purified receptor were required to increase PLC activity. Other tyrosine kinases such as the platelet-derived growth factor receptor and erbB-2, but not the insulin receptor, also stimulated PLC-gamma 1 activity. PLC-gamma 1 activity could be activated with the kinase-negative EGF receptor, but a C-terminal truncated receptor was much less effective. Purified EGF receptor could also activate PLC-beta 1, but with a much decreased potency compared with PLC-gamma 1. Our results suggest that in vitro the EGF receptor can increase PLC-gamma 1 activity independently of tyrosine phosphorylation.

scholarly journals Regulation of Epidermal Growth Factor Receptor Down-Regulation by UBPY-mediated Deubiquitination at Endosomes

2005 ◽  
Vol 16 (11) ◽  
pp. 5163-5174 ◽  
Author(s):  
Emi Mizuno ◽  
Takanobu Iura ◽  
Akiko Mukai ◽  
Tamotsu Yoshimori ◽  
Naomi Kitamura ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tyrosine Kinases ◽  
Egf Receptor ◽  
Growth Factor Receptor ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Down Regulation ◽  
Epidermal Growth

Ligand-activated receptor tyrosine kinases undergo endocytosis and are transported via endosomes to lysosomes for degradation. This “receptor down-regulation” process is crucial to terminate the cell proliferation signals produced by activated receptors. During the process, ubiquitination of the receptors serves as a sorting signal for their trafficking from endosomes to lysosomes. Here, we describe the role of a deubiquitinating enzyme UBPY/USP8 in the down-regulation of epidermal growth factor (EGF) receptor (EGFR). Overexpression of UBPY reduced the ubiquitination level of EGFR and delayed its degradation in EGF-stimulated cells. Immunopurified UBPY deubiquitinated EGFR in vitro. In EGF-stimulated cells, UBPY underwent ubiquitination and bound to EGFR. Overexpression of Hrs or a dominant-negative mutant of SKD1, proteins that play roles in the endosomal sorting of ubiquitinated receptors, caused the accumulation of endogenous UBPY on exaggerated endosomes. A catalytically inactive UBPY mutant clearly localized on endosomes, where it overlapped with EGFR when cells were stimulated with EGF. Finally, depletion of endogenous UBPY by RNA interference resulted in elevated ubiquitination and accelerated degradation of EGF-activated EGFR. We conclude that UBPY negatively regulates the rate of EGFR down-regulation by deubiquitinating EGFR on endosomes.

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