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Vav2 is required for cell spreading.

Marignani PA, Carpenter CL - J. Cell Biol. (2001)

Bottom Line: Vav2 also catalyzes exchange for RhoA, but does not cause morphologic changes indicative of RhoA activation.A mutation in the pleckstrin homology (PH) domain eliminates exchange activity and this construct does not induce lamellipodia, indicating the PH domain is necessary to catalyze nucleotide exchange.These findings indicate that in fibroblasts Vav2 is necessary for integrin, but not growth factor-dependent activation of Rac leading to lamellipodia.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215, USA.

ABSTRACT
Vav2 is a widely expressed Rho family guanine nucleotide exchange factor highly homologous to Vav1 and Vav3. Activated versions of Vav2 are transforming, but the normal function of Vav2 and how it is regulated are not known. We investigated the pathways that regulate Vav2 exchange activity in vivo and characterized its function. Overexpression of Vav2 activates Rac as assessed by both direct measurement of Rac-GTP and cell morphology. Vav2 also catalyzes exchange for RhoA, but does not cause morphologic changes indicative of RhoA activation. Vav2 nucleotide exchange is Src-dependent in vivo, since the coexpression of Vav2 and dominant negative Src, or treatment with the Src inhibitor PP2, blocks both Vav2-dependent Rac activation and lamellipodia formation. A mutation in the pleckstrin homology (PH) domain eliminates exchange activity and this construct does not induce lamellipodia, indicating the PH domain is necessary to catalyze nucleotide exchange. To further investigate the function of Vav2, we mutated the dbl homology (DH) domain and asked whether this mutant would function as a dominant negative to block Rac-dependent events. Studies using this mutant indicate that Vav2 is not necessary for platelet-derived growth factor- or epidermal growth factor-dependent activation of Rac. The Vav2 DH mutant did act as a dominant negative to inhibit spreading of NIH3T3 cells on fibronectin, specifically by blocking lamellipodia formation. These findings indicate that in fibroblasts Vav2 is necessary for integrin, but not growth factor-dependent activation of Rac leading to lamellipodia.

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L342R/L343SVav2 does not inhibit Vav1. (A) HEK293T cells were transfected with OncoVav1 or OncoVav1 and L342R/L343SVav2 at a DNA ratio of 1:3 (Vav1/Vav2). 24 h later activation of endogenous Rac was determined as described in Materials and methods using the PBD assay. Total cell lysate (TCL) was also Western blotted with Vav2 antibody to determine protein expression. (B) NIH3T3 cells were transfected with OncoVav1 and L342R/L343SVav2 at a DNA ratio of 1:3 (Vav1/Vav2) and fixed 24 h later. The cells were stained for Vav2 expression with T7 antibody and with rhodamine-phalloidin. These results are representative of three experiments.
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fig6: L342R/L343SVav2 does not inhibit Vav1. (A) HEK293T cells were transfected with OncoVav1 or OncoVav1 and L342R/L343SVav2 at a DNA ratio of 1:3 (Vav1/Vav2). 24 h later activation of endogenous Rac was determined as described in Materials and methods using the PBD assay. Total cell lysate (TCL) was also Western blotted with Vav2 antibody to determine protein expression. (B) NIH3T3 cells were transfected with OncoVav1 and L342R/L343SVav2 at a DNA ratio of 1:3 (Vav1/Vav2) and fixed 24 h later. The cells were stained for Vav2 expression with T7 antibody and with rhodamine-phalloidin. These results are representative of three experiments.

Mentions: The purpose of designing a Vav2 DH domain mutant was to use this reagent as a dominant negative in order to identify a function for Vav2. L342R/L343SVav2 should be recruited to sites where Rac would be activated by Vav2 and thereby block recruitment of endogenous Vav2 and thus Rac activation by endogenous Vav2. However, this construct could act nonspecifically, particularly if it sequestered Rac. To be certain that any inhibitory effects of L342R/L343SVav2 were specific for Vav2, we investigated whether it would block Vav1-mediated Rac activation or lamellipodia formation. HEK293T cells were transfected with OncoVav1 or OncoVav1 and L342R/L343SVav2, and activation of endogenous Rac was determined using the PBD assay. L342R/L343SVav2 did not block Vav1-dependent Rac activation (Fig. 6 A). Similarly, L342R/L343SVav2 coexpression did not affect the frequency or extent of lamellipodia or stress fibers in OncoVav1 transfected cells (Fig. 6 B). Thus, the L342R/L343SVav2 mutant could function as a dominant negative specific for Vav2.


Vav2 is required for cell spreading.

Marignani PA, Carpenter CL - J. Cell Biol. (2001)

L342R/L343SVav2 does not inhibit Vav1. (A) HEK293T cells were transfected with OncoVav1 or OncoVav1 and L342R/L343SVav2 at a DNA ratio of 1:3 (Vav1/Vav2). 24 h later activation of endogenous Rac was determined as described in Materials and methods using the PBD assay. Total cell lysate (TCL) was also Western blotted with Vav2 antibody to determine protein expression. (B) NIH3T3 cells were transfected with OncoVav1 and L342R/L343SVav2 at a DNA ratio of 1:3 (Vav1/Vav2) and fixed 24 h later. The cells were stained for Vav2 expression with T7 antibody and with rhodamine-phalloidin. These results are representative of three experiments.
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Related In: Results  -  Collection

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fig6: L342R/L343SVav2 does not inhibit Vav1. (A) HEK293T cells were transfected with OncoVav1 or OncoVav1 and L342R/L343SVav2 at a DNA ratio of 1:3 (Vav1/Vav2). 24 h later activation of endogenous Rac was determined as described in Materials and methods using the PBD assay. Total cell lysate (TCL) was also Western blotted with Vav2 antibody to determine protein expression. (B) NIH3T3 cells were transfected with OncoVav1 and L342R/L343SVav2 at a DNA ratio of 1:3 (Vav1/Vav2) and fixed 24 h later. The cells were stained for Vav2 expression with T7 antibody and with rhodamine-phalloidin. These results are representative of three experiments.
Mentions: The purpose of designing a Vav2 DH domain mutant was to use this reagent as a dominant negative in order to identify a function for Vav2. L342R/L343SVav2 should be recruited to sites where Rac would be activated by Vav2 and thereby block recruitment of endogenous Vav2 and thus Rac activation by endogenous Vav2. However, this construct could act nonspecifically, particularly if it sequestered Rac. To be certain that any inhibitory effects of L342R/L343SVav2 were specific for Vav2, we investigated whether it would block Vav1-mediated Rac activation or lamellipodia formation. HEK293T cells were transfected with OncoVav1 or OncoVav1 and L342R/L343SVav2, and activation of endogenous Rac was determined using the PBD assay. L342R/L343SVav2 did not block Vav1-dependent Rac activation (Fig. 6 A). Similarly, L342R/L343SVav2 coexpression did not affect the frequency or extent of lamellipodia or stress fibers in OncoVav1 transfected cells (Fig. 6 B). Thus, the L342R/L343SVav2 mutant could function as a dominant negative specific for Vav2.

Bottom Line: Vav2 also catalyzes exchange for RhoA, but does not cause morphologic changes indicative of RhoA activation.A mutation in the pleckstrin homology (PH) domain eliminates exchange activity and this construct does not induce lamellipodia, indicating the PH domain is necessary to catalyze nucleotide exchange.These findings indicate that in fibroblasts Vav2 is necessary for integrin, but not growth factor-dependent activation of Rac leading to lamellipodia.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215, USA.

ABSTRACT
Vav2 is a widely expressed Rho family guanine nucleotide exchange factor highly homologous to Vav1 and Vav3. Activated versions of Vav2 are transforming, but the normal function of Vav2 and how it is regulated are not known. We investigated the pathways that regulate Vav2 exchange activity in vivo and characterized its function. Overexpression of Vav2 activates Rac as assessed by both direct measurement of Rac-GTP and cell morphology. Vav2 also catalyzes exchange for RhoA, but does not cause morphologic changes indicative of RhoA activation. Vav2 nucleotide exchange is Src-dependent in vivo, since the coexpression of Vav2 and dominant negative Src, or treatment with the Src inhibitor PP2, blocks both Vav2-dependent Rac activation and lamellipodia formation. A mutation in the pleckstrin homology (PH) domain eliminates exchange activity and this construct does not induce lamellipodia, indicating the PH domain is necessary to catalyze nucleotide exchange. To further investigate the function of Vav2, we mutated the dbl homology (DH) domain and asked whether this mutant would function as a dominant negative to block Rac-dependent events. Studies using this mutant indicate that Vav2 is not necessary for platelet-derived growth factor- or epidermal growth factor-dependent activation of Rac. The Vav2 DH mutant did act as a dominant negative to inhibit spreading of NIH3T3 cells on fibronectin, specifically by blocking lamellipodia formation. These findings indicate that in fibroblasts Vav2 is necessary for integrin, but not growth factor-dependent activation of Rac leading to lamellipodia.

Show MeSH
Related in: MedlinePlus