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

2000 ◽  
Vol 20 (19) ◽  
pp. 7160-7169 ◽  
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.

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

2002 ◽  
Vol 13 (11) ◽  
pp. 3976-3988 ◽  
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.

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.

Effect of Dominant-Negative Epidermal Growth Factor Receptors on Cardiomyocyte Hypertrophy

2006 ◽  
Vol 26 (5-6) ◽  
pp. 659-677 ◽  
Author(s):  
HSIU-WEN CHAN ◽  
ANNA JENKINS ◽  
LUISA PIPOLO ◽  
ROSS D. HANNAN ◽  
WALTER G. THOMAS ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Growth Factor Receptors ◽  
Dominant Negative ◽  
Cardiomyocyte Hypertrophy ◽  
Epidermal Growth Factor Receptors ◽  
Epidermal Growth

scholarly journals Epidermal growth factor induces tyrosine phosphorylation of epidermal growth factor receptors not occupied with ligand in intact A431 cells

FEBS Letters ◽  
1990 ◽  
Vol 268 (1) ◽  
pp. 121-124 ◽  
Author(s):  
A.B. Sorokin ◽  
G.F. Reshetnikova ◽  
N.V. Kudryavtseva ◽  
N.N. Nikolsky
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tyrosine Phosphorylation ◽  
Growth Factor Receptors ◽  
Epidermal Growth Factor Receptors ◽  
A431 Cells ◽  
Epidermal Growth

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

1991 ◽  
Vol 11 (3) ◽  
pp. 1454-1463 ◽  
Author(s):  
O Kashles ◽  
Y Yarden ◽  
R Fischer ◽  
A Ullrich ◽  
J Schlessinger
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Living Cells ◽  
Dominant Negative ◽  
Egf Receptors ◽  
Wild Type ◽  
Dominant Negative Mutation ◽  
Epidermal Growth ◽  
Mutant Receptors ◽  
In Cells

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.

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.

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

1991 ◽  
Vol 11 (3) ◽  
pp. 1454-1463
Author(s):  
O Kashles ◽  
Y Yarden ◽  
R Fischer ◽  
A Ullrich ◽  
J Schlessinger
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Living Cells ◽  
Dominant Negative ◽  
Egf Receptors ◽  
Wild Type ◽  
Dominant Negative Mutation ◽  
Epidermal Growth ◽  
Mutant Receptors ◽  
In Cells

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.

scholarly journals Shc Isoform-specific Tyrosine Phosphorylation by the Insulin and Epidermal Growth Factor Receptors

1995 ◽  
Vol 270 (35) ◽  
pp. 20737-20741 ◽  
Author(s):  
Shuichi Okada ◽  
Keishi Yamauchi ◽  
Jeffrey E. Pessin
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tyrosine Phosphorylation ◽  
Growth Factor Receptors ◽  
Epidermal Growth Factor Receptors ◽  
Epidermal Growth
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