A dominant negative mutation suppresses the function of normal epidermal growth factor receptors by heterodimerization.

Recent studies provide evidence that defective receptors can function as a dominant negative mutation suppressing the action of wild-type receptors. This causes various diminished responses in cell culture and developmental disorders in murine embryogenesis. Here, we describe a model system and a potential mechanism underlying the dominant suppressing response caused by defective epidermal growth factor (EGF) receptors. We used cultured 3T3 cells coexpressing human wild-type receptors and an inactive deletion mutant lacking most of the cytoplasmic domain. When expressed alone, EGF was able to stimulate the dimerization of either wild-type or mutant receptors in living cells as revealed by chemical covalent cross-linking experiments. In response to EGF, heterodimers and homodimers of wild-type and mutant receptors were observed in cells coexpressing both receptor species. However, only homodimers of wild-type EGF receptors underwent EGF-induced tyrosine autophosphorylation in living cells. These results indicate that the integrity of both receptor moieties within receptor dimers is essential for kinase activation and autophosphorylation. Moreover, the presence of mutant receptors in cells expressing wild-type receptors diminished the number of high-affinity binding sites for EGF, reduced the rate of receptor endocytosis and degradation, and diminished biological signalling via EGF receptors. We propose that heterodimerization with defective EGF receptors functions as a dominant negative mutation suppressing the activation and response of normal receptors by formation of unproductive heterodimers.

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A site of tyrosine phosphorylation in the C terminus of the epidermal growth factor receptor is required to activate phospholipase C

Molecular and Cellular Biology ◽

10.1128/mcb.12.1.128-135.1992 ◽

1992 ◽

Vol 12 (1) ◽

pp. 128-135

Author(s):

Q C Vega ◽

C Cochet ◽

O Filhol ◽

C P Chang ◽

S G Rhee ◽

...

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Phospholipase C ◽

Tyrosine Phosphorylation ◽

Inositol Phosphate ◽

Enzyme Activation ◽

Egf Receptors ◽

Wild Type ◽

Epidermal Growth ◽

Mutant Receptors

Cells expressing mutant epidermal growth factor (EGF) receptors have been used to study mechanisms through which EGF increases phospholipase C (PLC) activity. C-terminal truncation mutant EGF receptors are markedly impaired in their ability to increase inositol phosphate formation compared with wild-type EGF receptors. Mutation of the single tyrosine self-phosphorylation site at residue 992 to phenylalanine in an EGF receptor truncated at residue 1000 abolished the ability of EGF to increase inositol phosphate formation. C-terminal deletion mutant receptors that are impaired in their ability to increase inositol phosphate formation effectively phosphorylate PLC-gamma at the same tyrosine residues as do wild-type EGF receptors. EGF enhances PLC-gamma association with wild-type EGF receptors but not with mutant receptors lacking sites of tyrosine phosphorylation. These results indicate that formation of a complex between self-phosphorylated EGF receptors and PLC-gamma is necessary for enzyme activation in vivo. We propose that both binding of PLC-gamma to activated EGF receptors and tyrosine phosphorylation of the enzyme are necessary to elicit biological responses. Kinase-active EGF receptors lacking sites of tyrosine phosphorylation are unable to signal increased inositol phosphate formation and increases in cytosolic Ca2+ concentration.

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A site of tyrosine phosphorylation in the C terminus of the epidermal growth factor receptor is required to activate phospholipase C.

Molecular and Cellular Biology ◽

10.1128/mcb.12.1.128 ◽

1992 ◽

Vol 12 (1) ◽

pp. 128-135 ◽

Cited By ~ 65

Author(s):

Q C Vega ◽

C Cochet ◽

O Filhol ◽

C P Chang ◽

S G Rhee ◽

...

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Phospholipase C ◽

Tyrosine Phosphorylation ◽

Inositol Phosphate ◽

Enzyme Activation ◽

Egf Receptors ◽

Wild Type ◽

Epidermal Growth ◽

Mutant Receptors

Cells expressing mutant epidermal growth factor (EGF) receptors have been used to study mechanisms through which EGF increases phospholipase C (PLC) activity. C-terminal truncation mutant EGF receptors are markedly impaired in their ability to increase inositol phosphate formation compared with wild-type EGF receptors. Mutation of the single tyrosine self-phosphorylation site at residue 992 to phenylalanine in an EGF receptor truncated at residue 1000 abolished the ability of EGF to increase inositol phosphate formation. C-terminal deletion mutant receptors that are impaired in their ability to increase inositol phosphate formation effectively phosphorylate PLC-gamma at the same tyrosine residues as do wild-type EGF receptors. EGF enhances PLC-gamma association with wild-type EGF receptors but not with mutant receptors lacking sites of tyrosine phosphorylation. These results indicate that formation of a complex between self-phosphorylated EGF receptors and PLC-gamma is necessary for enzyme activation in vivo. We propose that both binding of PLC-gamma to activated EGF receptors and tyrosine phosphorylation of the enzyme are necessary to elicit biological responses. Kinase-active EGF receptors lacking sites of tyrosine phosphorylation are unable to signal increased inositol phosphate formation and increases in cytosolic Ca2+ concentration.

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Localization of Phospholipase D1 to Caveolin-enriched Membrane via Palmitoylation: Implications for Epidermal Growth Factor Signaling

Molecular Biology of the Cell ◽

10.1091/mbc.e02-02-0100 ◽

2002 ◽

Vol 13 (11) ◽

pp. 3976-3988 ◽

Cited By ~ 40

Author(s):

Jung Min Han ◽

Yong Kim ◽

Jun Sung Lee ◽

Chang Sup Lee ◽

Byoung Dae Lee ◽

...

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Egf Receptor ◽

Point Mutations ◽

Dominant Negative ◽

Growth Factor Signaling ◽

Wild Type ◽

Caveolin 1 ◽

Epidermal Growth ◽

Egf Signaling

Phospholipase D (PLD) has been suggested to mediate epidermal growth factor (EGF) signaling. However, the molecular mechanism of EGF-induced PLD activation has not yet been elucidated. We investigated the importance of the phosphorylation and compartmentalization of PLD1 in EGF signaling. EGF treatment of COS-7 cells transiently expressing PLD1 stimulated PLD1 activity and induced PLD1 phosphorylation. The EGF-induced phosphorylation of threonine147 was completely blocked and the activity of PLD1 attenuated by point mutations (S2A/T147A/S561A) of PLD1 phosphorylation sites. The expression of a dominant negative PKCα mutant by adenovirus-mediated gene transfer greatly inhibited the phosphorylation and activation of PLD1 induced by EGF in PLD1-transfected COS-7 cells. EGF-induced PLD1 phosphorylation occurred primarily in the caveolin-enriched membrane (CEM) fraction, and the kinetics of PLD1 phosphorylation in the CEM were strongly correlated with PLD1 phosphorylation in the total membrane. Interestingly, EGF-induced PLD1 phosphorylation and activation and the coimmunoprecipitation of PLD1 with caveolin-1 and the EGF receptor in the CEM were significantly attenuated in the palmitoylation-deficient C240S/C241S mutant, which did not localize to the CEM. Immunocytochemical analysis revealed that wild-type PLD1 colocalized with caveolin-1 and the EGF receptor and that phosphorylated PLD1 was localized exclusively in the plasma membrane, although some PLD1 was also detected in vesicular structures. Transfection of wild-type PLD1 but not of C240S/C241S mutant increased EGF-induced raf-1 translocation to the CEM and ERK phosphorylation. This study shows, for the first time, that EGF-induced PLD1 phosphorylation and activation occur in the CEM and that the correct localization of PLD1 to the CEM via palmitoylation is critical for EGF signaling.

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

Molecular and Cellular Biology ◽

10.1128/mcb.10.8.4035-4044.1990 ◽

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.

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Vav2 Activates Rac1, Cdc42, and RhoA Downstream from Growth Factor Receptors but Not β1 Integrins

Molecular and Cellular Biology ◽

10.1128/mcb.20.19.7160-7169.2000 ◽

2000 ◽

Vol 20 (19) ◽

pp. 7160-7169 ◽

Cited By ~ 145

Author(s):

Betty P. Liu ◽

Keith Burridge

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Tyrosine Phosphorylation ◽

G Proteins ◽

Growth Factor Receptors ◽

Dominant Negative ◽

Wild Type ◽

Epidermal Growth ◽

Rho Family ◽

Constitutively Active

ABSTRACT The Rho family of GTPases plays a major role in the organization of the actin cytoskeleton. These G proteins are activated by guanine nucleotide exchange factors that stimulate the exchange of bound GDP for GTP. In their GTP-bound state, these G proteins interact with downstream effectors. Vav2 is an exchange factor for Rho family GTPases. It is a ubiquitously expressed homologue of Vav1, and like Vav1, it has previously been shown to be activated by tyrosine phosphorylation. Because Vav1 becomes tyrosine phosphorylated and activated following integrin engagement in hematopoietic cells, we investigated the tyrosine phosphorylation of Vav2 in response to integrin-mediated adhesion in fibroblasts and epithelial cells. However, no tyrosine phosphorylation of Vav2 was detected in response to integrin engagement. In contrast, treating cells with either epidermal growth factor or platelet-derived growth factor stimulated tyrosine phosphorylation of Vav2. We have examined the effects of overexpressing either wild-type or amino-terminally truncated (constitutively active) forms of Vav2 as fusion proteins with green fluorescent protein. Overexpression of either wild-type or constitutively active Vav2 resulted in prominent membrane ruffles and enhanced stress fibers. These cells revealed elevated rates of cell migration that were inhibited by expression of dominant negative forms of Rac1 and Cdc42. Using a binding assay to measure the activity of Rac1, Cdc42, and RhoA, we found that overexpression of Vav2 resulted in increased activity of each of these G proteins. Expression of a carboxy-terminal fragment of Vav2 decreased the elevation of Rac1 activity induced by epidermal growth factor, consistent with Vav2 mediating activation of Rac1 downstream from growth factor receptors.

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Shc Phosphotyrosine-Binding Domain Dominantly Interacts with Epidermal Growth Factor Receptors and Mediates Ras Activation in Intact Cells

Molecular Endocrinology ◽

10.1210/mend.12.4.0094 ◽

1998 ◽

Vol 12 (4) ◽

pp. 536-543 ◽

Cited By ~ 35

Author(s):

Kazuhiko Sakaguchi ◽

Yoshinori Okabayashi ◽

Yoshiaki Kido ◽

Sachiko Kimura ◽

Yoko Matsumura ◽

...

Keyword(s):

Growth Factor ◽

Egf Receptor ◽

Sh2 Domain ◽

Wild Type ◽

Intact Cells ◽

Sh2 Domains ◽

Epidermal Growth ◽

Mutant Receptors ◽

Ptb Domain ◽

In Cells

Abstract The adaptor protein Shc contains a phosphotyrosine binding (PTB) domain and a Src homology 2 (SH2) domain, both of which are known to interact with phosphorylated tyrosines. We have shown previously that tyrosine 1148 of the activated epidermal growth factor (EGF) receptor is a major binding site for Shc while tyrosine 1173 is a secondary binding site in intact cells. In the present study, we investigated the interaction between the PTB and SH2 domains of Shc and the activated human EGF receptor. Mutant 52-kDa Shc with an arginine-to-lysine substitution at residue 175 in the PTB domain (Shc R175K) or 397 in the SH2 domain (Shc R397K) was coexpressed in Chinese hamster ovary cells overexpressing the wild-type or mutant EGF receptors that retained only one of the autophosphorylation sites at tyrosine 1148 (QM1148) or 1173 (QM1173). Shc R397K was coprecipitated with the QM1148 and QM1173 receptors, was tyrosine-phosphorylated, and associated with Grb2 and Sos. In contrast, coprecipitation of Shc R175K with the mutant receptors was barely detectable. In cells expressing the QM1173 receptor, Shc R175K was tyrosine-phosphorylated and associated with Grb2, while association of Sos was barely detectable. In cells expressing the QM1148 receptor, tyrosine phosphorylation of Shc R175K was markedly reduced. When both Shc R175K and 46-kDa Shc R397K were coexpressed with the mutant receptors, p46 Shc R397K was dominantly tyrosine-phosphorylated. In cells expressing the wild-type receptor, Shc R397K, but not Shc R175K, translocated to the membrane in an EGF-dependent manner. In addition, Ras activity stimulated by the immunoprecipitates of Shc R397K was significantly higher than that by the immunoprecipitates of Shc R175K. The present results indicate that tyrosine 1148 of the activated EGF receptor mainly interacts with the Shc PTB domain in intact cells. Tyrosine 1173 interacts with both the PTB and SH2 domains, although the interaction with the PTB domain is dominant. In addition, Shc bound to the activated EGF receptor via the PTB domain dominantly interacts with Grb2-Sos complex and plays a major role in the Ras-signaling pathway.

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Fast receptor-induced formation of glycerophosphoinositol-4-phosphate, a putative novel intracellular messenger in the Ras pathway.

Molecular Biology of the Cell ◽

10.1091/mbc.8.3.443 ◽

1997 ◽

Vol 8 (3) ◽

pp. 443-453 ◽

Cited By ~ 21

Author(s):

M Falasca ◽

A Carvelli ◽

C Iurisci ◽

R G Qiu ◽

M H Symons ◽

...

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Cyclic Amp ◽

Kinase Inhibitor ◽

Dominant Negative ◽

Ras Pathway ◽

Epidermal Growth ◽

Phosphoinositide 3 Kinase ◽

Intracellular Messenger ◽

In Cells

Glycerophosphoinositols are phosphoinositide metabolites whose levels are constitutively elevated in Ras-transformed cells. Here, we show that one of these compounds, glycerophosphoinositol-4-phosphate (GroPIns-4-P) responds acutely to the stimulation of the epidermal growth factor receptor, with a fast, massive and transient increase. The mechanism leading to GroPIns-4-P formation involves the activation of phosphoinositide-3 kinase and the small GTP-binding protein Rac, since GroPIns-4-P was neither formed in cells expressing the dominant negative form of Rac nor in cells treated with the phosphoinositide-3 kinase inhibitor wortmannin. GroPIns-4-P has been previously shown to inhibit adenylyl cyclase. Accordingly, epidermal growth factor also decreased the basal, cholera toxin-stimulated, and forskolin-stimulated cyclic AMP levels with kinetics similar to those of GroPIns-4-P formation, suggesting that GroPIns-4-P mediates this inhibitory effect. The hormone-induced formation of GroPIns-4-P was detected in several cell lines of various origin, suggesting that GroPIns-4-P is a novel intracellular messenger of the Ras pathway, possibly able to convey information from tyrosine kinase receptors to the cyclic AMP cascade.

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The Mouse SKD1, a Homologue of Yeast Vps4p, Is Required for Normal Endosomal Trafficking and Morphology in Mammalian Cells

Molecular Biology of the Cell ◽

10.1091/mbc.11.2.747 ◽

2000 ◽

Vol 11 (2) ◽

pp. 747-763 ◽

Cited By ~ 144

Author(s):

Tamotsu Yoshimori ◽

Fumi Yamagata ◽

Akitsugu Yamamoto ◽

Noboru Mizushima ◽

Yukiko Kabeya ◽

...

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Mammalian Cells ◽

Secretory Pathway ◽

Density Lipoprotein ◽

Dominant Negative ◽

Mutant Protein ◽

Endosomal Trafficking ◽

Dominant Negative Mutation ◽

Epidermal Growth

The mouse SKD1 is an AAA-type ATPase homologous to the yeast Vps4p implicated in transport from endosomes to the vacuole. To elucidate a possible role of SKD1 in mammalian endocytosis, we generated a mutant SKD1, harboring a mutation (E235Q) that is equivalent to the dominant negative mutation (E233Q) in Vps4p. Overexpression of the mutant SKD1 in cultured mammalian cells caused defect in uptake of transferrin and low-density lipoprotein. This was due to loss of their receptors from the cell surface. The decrease of the surface transferrin receptor (TfR) was correlated with expression levels of the mutant protein. The mutant protein displayed a perinuclear punctate distribution in contrast to a diffuse pattern of the wild-type SKD1. TfR, the lysosomal protein lamp-1, endocytosed dextran, and epidermal growth factor but not markers for the secretory pathway were accumulated in the mutant SKD1–localized compartments. Degradation of epidermal growth factor was inhibited. Electron microscopy revealed that the compartments were exaggerated multivesicular vacuoles with numerous tubulo-vesicular extensions containing TfR and endocytosed horseradish peroxidase. The early endosome antigen EEA1 was also redistributed to these aberrant membranes. Taken together, our findings suggest that SKD1 regulates morphology of endosomes and membrane traffic through them.

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

Molecular and Cellular Biology ◽

10.1128/mcb.24.11.4664-4676.2004 ◽

2004 ◽

Vol 24 (11) ◽

pp. 4664-4676 ◽

Cited By ~ 31

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.

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