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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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Vav2 stimulates lamellipodia and Jnk activity. (A) NIH3T3 cells were transfected with Vav2, fixed after 24 h, and stained for Vav2 expression with the T7 antibody and for actin with rhodamine-phalloidin. (B) COS7 cells were transfected with Vav2 and FLAG epitope–tagged Jnk followed by immunoprecipitation of Jnk with FLAG antibody. Jnk activity was determined as described in Materials and methods by phosphorylation of GST-jun (top). Cell lysates were Western blotted to determine Jnk (middle) and Vav2 (bottom) expression. These results are representative of four experiments.
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fig2: Vav2 stimulates lamellipodia and Jnk activity. (A) NIH3T3 cells were transfected with Vav2, fixed after 24 h, and stained for Vav2 expression with the T7 antibody and for actin with rhodamine-phalloidin. (B) COS7 cells were transfected with Vav2 and FLAG epitope–tagged Jnk followed by immunoprecipitation of Jnk with FLAG antibody. Jnk activity was determined as described in Materials and methods by phosphorylation of GST-jun (top). Cell lysates were Western blotted to determine Jnk (middle) and Vav2 (bottom) expression. These results are representative of four experiments.

Mentions: Since we detected activation of Rac and Rho by Vav2 we investigated the effects of overexpressing Vav2 on actin structures in NIH3T3 cells to determine whether the phenotype suggested a predominance of either Rac or Rho activation. Overexpression of Vav2 resulted in extensive lamellipodia in 80% of transfected cells (Fig. 2 A). No untransfected cells had the extensive lamellipodia seen in the Vav2 transfected cells and less than 20% of untransfected cells had any lamellipodia. These results are similar to Moores et al. (2000), but in contrast to Schuebel et al. (1998), who reported that overexpression of wild-type Vav2 had no effect on actin. We detected few cells that had prominent stress fibers or were contracted, suggesting that under these conditions there was little activation of RhoA relative to Rac. We also assayed Jnk activity in COS7 cells transfected with Vav2 and found that overexpression of Vav2-activated Jnk (Fig. 2 B). Since activation of Jnk in COS7 cells is dependent on Rac or Cdc42 but not Rho (Coso et al., 1995), and we detect minimal activation of Cdc42 by Vav2, stimulation of Jnk by Vav2 in COS7 cells indicates that Vav2 activates Rac in vivo.


Vav2 is required for cell spreading.

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

Vav2 stimulates lamellipodia and Jnk activity. (A) NIH3T3 cells were transfected with Vav2, fixed after 24 h, and stained for Vav2 expression with the T7 antibody and for actin with rhodamine-phalloidin. (B) COS7 cells were transfected with Vav2 and FLAG epitope–tagged Jnk followed by immunoprecipitation of Jnk with FLAG antibody. Jnk activity was determined as described in Materials and methods by phosphorylation of GST-jun (top). Cell lysates were Western blotted to determine Jnk (middle) and Vav2 (bottom) expression. These results are representative of four experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196856&req=5

fig2: Vav2 stimulates lamellipodia and Jnk activity. (A) NIH3T3 cells were transfected with Vav2, fixed after 24 h, and stained for Vav2 expression with the T7 antibody and for actin with rhodamine-phalloidin. (B) COS7 cells were transfected with Vav2 and FLAG epitope–tagged Jnk followed by immunoprecipitation of Jnk with FLAG antibody. Jnk activity was determined as described in Materials and methods by phosphorylation of GST-jun (top). Cell lysates were Western blotted to determine Jnk (middle) and Vav2 (bottom) expression. These results are representative of four experiments.
Mentions: Since we detected activation of Rac and Rho by Vav2 we investigated the effects of overexpressing Vav2 on actin structures in NIH3T3 cells to determine whether the phenotype suggested a predominance of either Rac or Rho activation. Overexpression of Vav2 resulted in extensive lamellipodia in 80% of transfected cells (Fig. 2 A). No untransfected cells had the extensive lamellipodia seen in the Vav2 transfected cells and less than 20% of untransfected cells had any lamellipodia. These results are similar to Moores et al. (2000), but in contrast to Schuebel et al. (1998), who reported that overexpression of wild-type Vav2 had no effect on actin. We detected few cells that had prominent stress fibers or were contracted, suggesting that under these conditions there was little activation of RhoA relative to Rac. We also assayed Jnk activity in COS7 cells transfected with Vav2 and found that overexpression of Vav2-activated Jnk (Fig. 2 B). Since activation of Jnk in COS7 cells is dependent on Rac or Cdc42 but not Rho (Coso et al., 1995), and we detect minimal activation of Cdc42 by Vav2, stimulation of Jnk by Vav2 in COS7 cells indicates that Vav2 activates Rac in vivo.

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