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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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Activation of Vav2 is Src-dependent. (A) HEK293T cells were transfected with Vav2 or Vav2 and KD and harvested after 24 h. Activation of endogenous Rac (PBD) was assayed as described in Materials and methods. Total cells lysates (TCL) were also Western blotted with T7 antibody for Vav2 expression. (B) NIH3T3 cells were transfected with Vav2 and KD Src or Vav2 transfected cells were treated with PP2 (2 μM) for 30 min or left untreated. The cells were fixed and then stained for Vav2 expression with T7 antibody and for actin with rhodamine-phalloidin. The arrow indicates the transfected cell. These results are representative of three experiments.
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fig3: Activation of Vav2 is Src-dependent. (A) HEK293T cells were transfected with Vav2 or Vav2 and KD and harvested after 24 h. Activation of endogenous Rac (PBD) was assayed as described in Materials and methods. Total cells lysates (TCL) were also Western blotted with T7 antibody for Vav2 expression. (B) NIH3T3 cells were transfected with Vav2 and KD Src or Vav2 transfected cells were treated with PP2 (2 μM) for 30 min or left untreated. The cells were fixed and then stained for Vav2 expression with T7 antibody and for actin with rhodamine-phalloidin. The arrow indicates the transfected cell. These results are representative of three experiments.

Mentions: Coexpression of activated Lck with wild-type Vav2 promotes colony formation and induces lamellipodia (Schuebel et al., 1998), and the exchange activity of Vav proteins is activated by Src-like kinases in vitro, but it is not known whether Src kinases are necessary for Vav2 activation in vivo. To determine whether Src family members are necessary for Vav2 activity, HEK293T cells were transfected with Vav2 and kinase-dead (KD) Src, and Rac activation was compared with cells transfected with Vav2 alone. As shown in Fig. 3 A, KD Src blocked Vav2-dependent Rac activation. We did not detect increased Rac activation when Vav2 was cotransfected with activated Src, although Vav2 tyrosine phosphorylation was increased (data not shown), indicating that endogenous Src activity is sufficient to fully activate Vav2. We also determined the effect of KD Src and the Src inhibitor, PP2, on the formation of Vav2-dependent lamellipodia. NIH3T3 were transfected with Vav2 and KD Src or with Vav2 alone and treated with PP2. PP2 treatment or cotransfection of Vav2 and KD Src reduced the number of Vav2 transfected cells with lamellipodia by 80 and 70%, respectively, and in those cells in which lamellipodia were present, they were much less prominent (Fig. 3 B). These data strongly suggest that Src or an Src family member is necessary for Vav2 activation of Rac in vivo.


Vav2 is required for cell spreading.

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

Activation of Vav2 is Src-dependent. (A) HEK293T cells were transfected with Vav2 or Vav2 and KD and harvested after 24 h. Activation of endogenous Rac (PBD) was assayed as described in Materials and methods. Total cells lysates (TCL) were also Western blotted with T7 antibody for Vav2 expression. (B) NIH3T3 cells were transfected with Vav2 and KD Src or Vav2 transfected cells were treated with PP2 (2 μM) for 30 min or left untreated. The cells were fixed and then stained for Vav2 expression with T7 antibody and for actin with rhodamine-phalloidin. The arrow indicates the transfected cell. These results are representative of three experiments.
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Related In: Results  -  Collection

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fig3: Activation of Vav2 is Src-dependent. (A) HEK293T cells were transfected with Vav2 or Vav2 and KD and harvested after 24 h. Activation of endogenous Rac (PBD) was assayed as described in Materials and methods. Total cells lysates (TCL) were also Western blotted with T7 antibody for Vav2 expression. (B) NIH3T3 cells were transfected with Vav2 and KD Src or Vav2 transfected cells were treated with PP2 (2 μM) for 30 min or left untreated. The cells were fixed and then stained for Vav2 expression with T7 antibody and for actin with rhodamine-phalloidin. The arrow indicates the transfected cell. These results are representative of three experiments.
Mentions: Coexpression of activated Lck with wild-type Vav2 promotes colony formation and induces lamellipodia (Schuebel et al., 1998), and the exchange activity of Vav proteins is activated by Src-like kinases in vitro, but it is not known whether Src kinases are necessary for Vav2 activation in vivo. To determine whether Src family members are necessary for Vav2 activity, HEK293T cells were transfected with Vav2 and kinase-dead (KD) Src, and Rac activation was compared with cells transfected with Vav2 alone. As shown in Fig. 3 A, KD Src blocked Vav2-dependent Rac activation. We did not detect increased Rac activation when Vav2 was cotransfected with activated Src, although Vav2 tyrosine phosphorylation was increased (data not shown), indicating that endogenous Src activity is sufficient to fully activate Vav2. We also determined the effect of KD Src and the Src inhibitor, PP2, on the formation of Vav2-dependent lamellipodia. NIH3T3 were transfected with Vav2 and KD Src or with Vav2 alone and treated with PP2. PP2 treatment or cotransfection of Vav2 and KD Src reduced the number of Vav2 transfected cells with lamellipodia by 80 and 70%, respectively, and in those cells in which lamellipodia were present, they were much less prominent (Fig. 3 B). These data strongly suggest that Src or an Src family member is necessary for Vav2 activation of 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