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

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.14.3.1575-1581.1994 ◽

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.14.3.1575 ◽

1994 ◽

Vol 14 (3) ◽

pp. 1575-1581 ◽

Cited By ~ 117

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.

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Biological and biochemical activities of a chimeric epidermal growth factor-Elk receptor tyrosine kinase.

Molecular and Cellular Biology ◽

10.1128/mcb.13.11.7071 ◽

1993 ◽

Vol 13 (11) ◽

pp. 7071-7079 ◽

Cited By ~ 34

Author(s):

V Lhoták ◽

T Pawson

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Dna Synthesis ◽

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Egf Receptor ◽

Transmembrane Protein ◽

Chimeric Receptors ◽

Extracellular Region ◽

Epidermal Growth

Eph, Elk, and Eck are prototypes of a large family of transmembrane protein-tyrosine kinases, which are characterized by a highly conserved cysteine-rich domain and two fibronectin type III repeats in their extracellular regions. Despite the extent of the Eph family, no extracellular ligands for any family member have been identified, and hence, little is known about the biological and biochemical properties of these receptor-like tyrosine kinases. In the absence of a physiological ligand for the Elk receptor, we constructed chimeric receptor molecules, in which the extracellular region of the Elk receptor is replaced by the extracellular, ligand-binding domain of the epidermal growth factor (EGF) receptor. These chimeric receptors were expressed in NIH 3T3 cells that lack endogenous EGF receptors to analyze their signaling properties. The chimeric EGF-Elk receptors became glycosylated, were correctly localized to the plasma membrane, and bound EGF with high affinity. The chimeric receptors underwent autophosphorylation and induced the tyrosine phosphorylation of a specific set of cellular proteins in response to EGF. EGF stimulation also induced DNA synthesis in fibroblasts stably expressing the EGF-Elk receptors. In contrast, EGF stimulation of these cells did not lead to visible changes in cellular morphology, nor did it induce loss of contact inhibition in confluent monolayers or growth in semisolid media. The Elk cytoplasmic domain is therefore able to induce tyrosine phosphorylation and DNA synthesis in response to an extracellular ligand, suggesting that Elk and related polypeptides function as ligand-dependent receptor tyrosine kinases.

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Biological and biochemical activities of a chimeric epidermal growth factor-Elk receptor tyrosine kinase

Molecular and Cellular Biology ◽

10.1128/mcb.13.11.7071-7079.1993 ◽

1993 ◽

Vol 13 (11) ◽

pp. 7071-7079

Author(s):

V Lhoták ◽

T Pawson

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Dna Synthesis ◽

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Egf Receptor ◽

Transmembrane Protein ◽

Chimeric Receptors ◽

Extracellular Region ◽

Epidermal Growth

Eph, Elk, and Eck are prototypes of a large family of transmembrane protein-tyrosine kinases, which are characterized by a highly conserved cysteine-rich domain and two fibronectin type III repeats in their extracellular regions. Despite the extent of the Eph family, no extracellular ligands for any family member have been identified, and hence, little is known about the biological and biochemical properties of these receptor-like tyrosine kinases. In the absence of a physiological ligand for the Elk receptor, we constructed chimeric receptor molecules, in which the extracellular region of the Elk receptor is replaced by the extracellular, ligand-binding domain of the epidermal growth factor (EGF) receptor. These chimeric receptors were expressed in NIH 3T3 cells that lack endogenous EGF receptors to analyze their signaling properties. The chimeric EGF-Elk receptors became glycosylated, were correctly localized to the plasma membrane, and bound EGF with high affinity. The chimeric receptors underwent autophosphorylation and induced the tyrosine phosphorylation of a specific set of cellular proteins in response to EGF. EGF stimulation also induced DNA synthesis in fibroblasts stably expressing the EGF-Elk receptors. In contrast, EGF stimulation of these cells did not lead to visible changes in cellular morphology, nor did it induce loss of contact inhibition in confluent monolayers or growth in semisolid media. The Elk cytoplasmic domain is therefore able to induce tyrosine phosphorylation and DNA synthesis in response to an extracellular ligand, suggesting that Elk and related polypeptides function as ligand-dependent receptor tyrosine kinases.

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

Biochemical Journal ◽

10.1042/bj2930507 ◽

1993 ◽

Vol 293 (2) ◽

pp. 507-511 ◽

Cited By ~ 20

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.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e05-06-0560 ◽

2005 ◽

Vol 16 (11) ◽

pp. 5163-5174 ◽

Cited By ~ 177

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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Interactions of Drosophila Cbl with epidermal growth factor receptors and role of Cbl in R7 photoreceptor cell development.

Molecular and Cellular Biology ◽

10.1128/mcb.17.4.2217 ◽

1997 ◽

Vol 17 (4) ◽

pp. 2217-2225 ◽

Cited By ~ 88

Author(s):

H Meisner ◽

A Daga ◽

J Buxton ◽

B Fernández ◽

A Chawla ◽

...

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Tyrosine Phosphorylation ◽

Egf Receptor ◽

Sh2 Domain ◽

Terminal Region ◽

Receptor Expression ◽

Negative Regulator ◽

Egf Receptors ◽

Epidermal Growth

The human proto-oncogene product c-Cbl and a similar protein in Caenorhabditis elegans (Sli-1) contain a proline-rich COOH-terminal region that binds Src homology 3 (SH3) domains of proteins such as the adapter Grb2. Cb1-Grb2 complexes can be recruited to tyrosine-phosphorylated epidermal growth factor (EGF) receptors through the SH2 domain of Grb2. Here we identify by molecular cloning a Drosophila cDNA encoding a protein (Drosophila Cbl [D-Cbl]) that shows high sequence similarity to the N-terminal region of human c-Cbl but lacks proline-rich sequences and fails to bind Grb2. Nonetheless, in COS-1 cells, expression of hemagglutinin epitope-tagged D-Cbl results in its coimmunoprecipitation with EGF receptors in response to EGF. EGF also caused tyrosine phosphorylation of D-Cbl in such cells, but no association of phosphatidylinositol 3-kinase was detected in assays using anti-p85 antibody. A point mutation in D-Cbl (G305E) that suppresses the negative regulation of LET-23 by the Cbl homolog Sli-1 in C. elegans prevented tyrosine phosphorylation of D-Cbl as well as binding to the liganded EGF receptor in COS-1 cells. Colocalization of EGF receptors with both endogenous c-Cbl or expressed D-Cbl in endosomes of EGF-treated COS-1 cells is also demonstrated by immunofluorescence microscopy. In lysates of adult transgenic Drosophila melanogaster, GST-DCbl binds to the tyrosine-phosphorylated 150-kDa torso-DER chimeric receptor. Expression of D-Cbl directed by the sevenless enhancer in intact Drosophila compromises severely the development of the R7 photoreceptor neuron. These data suggest that despite the lack of Grb2 binding sites, D-Cbl functions as a negative regulator of receptor tyrosine kinase signaling in the Drosophila eye by a mechanism that involves its association with EGF receptors or other tyrosine kinases.

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