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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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The PH domain is necessary for Vav2 exchange activity. (A) HEK293T cells were transfected with R425GVav2 and exchange activity for endogenous Rac (PBD) was determined as described in Materials and methods. Total cell lysates (TCL) were also Western blotted for Rac and Vav2 expression with Rac and T7 antibodies, respectively. (B) NIH3T3 cells were transfected with R425GVav2, fixed 24 h later, and stained for R425GVav2 expression with T7 antibody and for actin with rho-damine-phalloidin. The arrow indicates the transfected cell. (C) HEK293T cells were transfected with Vav2 and treated after 24 h with wortmannin (200 nM) for 1 h. Activation of endogenous Rac (PBD) was determined as described in Materials and methods. Total cell lysates were Western blotted for Rac and Vav2 expression using Rac and Vav2 antibodies, respectively. (D) NIH3T3 cells were transfected with Vav2, and after 24 h the cells were treated with wortmannin (200 nM) for 1 h and then fixed and stained for Vav2 expression with T7 antibody and for actin with rhodamine-phalloidin. These results are representative of three experiments.
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fig4: The PH domain is necessary for Vav2 exchange activity. (A) HEK293T cells were transfected with R425GVav2 and exchange activity for endogenous Rac (PBD) was determined as described in Materials and methods. Total cell lysates (TCL) were also Western blotted for Rac and Vav2 expression with Rac and T7 antibodies, respectively. (B) NIH3T3 cells were transfected with R425GVav2, fixed 24 h later, and stained for R425GVav2 expression with T7 antibody and for actin with rho-damine-phalloidin. The arrow indicates the transfected cell. (C) HEK293T cells were transfected with Vav2 and treated after 24 h with wortmannin (200 nM) for 1 h. Activation of endogenous Rac (PBD) was determined as described in Materials and methods. Total cell lysates were Western blotted for Rac and Vav2 expression using Rac and Vav2 antibodies, respectively. (D) NIH3T3 cells were transfected with Vav2, and after 24 h the cells were treated with wortmannin (200 nM) for 1 h and then fixed and stained for Vav2 expression with T7 antibody and for actin with rhodamine-phalloidin. These results are representative of three experiments.

Mentions: The activity of Vav1 is stimulated by phosphoinositide products of phosphoinositide 3 (PI-3) kinase binding to the PH domain, however, mutations in the PH domain of Vav1 do not completely inhibit exchange activity and a mutation in the PH domain of Vav3 does not impair its function (Han et al., 1998; Movilla and Bustelo, 1999). The role of the PH domain in Vav2 function is not known. To determine whether the PH domain is necessary for Vav2 activity we mutated R425 to G, based on mutations in Vav1 that disrupt the binding of phosphoinositides (Han et al., 1998). HEK293T cells were transfected with R425G Vav2 and Rac1 activation was measured. This construct did not activate Rac (Fig. 4 A) and or cause lamellipodia (compare Fig. 4 B with 2 A), indicating that unlike Vav1 and Vav3, the PH domain is required for Vav2 function.


Vav2 is required for cell spreading.

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

The PH domain is necessary for Vav2 exchange activity. (A) HEK293T cells were transfected with R425GVav2 and exchange activity for endogenous Rac (PBD) was determined as described in Materials and methods. Total cell lysates (TCL) were also Western blotted for Rac and Vav2 expression with Rac and T7 antibodies, respectively. (B) NIH3T3 cells were transfected with R425GVav2, fixed 24 h later, and stained for R425GVav2 expression with T7 antibody and for actin with rho-damine-phalloidin. The arrow indicates the transfected cell. (C) HEK293T cells were transfected with Vav2 and treated after 24 h with wortmannin (200 nM) for 1 h. Activation of endogenous Rac (PBD) was determined as described in Materials and methods. Total cell lysates were Western blotted for Rac and Vav2 expression using Rac and Vav2 antibodies, respectively. (D) NIH3T3 cells were transfected with Vav2, and after 24 h the cells were treated with wortmannin (200 nM) for 1 h and then fixed and stained for Vav2 expression with T7 antibody and for actin with rhodamine-phalloidin. These results are representative of three experiments.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: The PH domain is necessary for Vav2 exchange activity. (A) HEK293T cells were transfected with R425GVav2 and exchange activity for endogenous Rac (PBD) was determined as described in Materials and methods. Total cell lysates (TCL) were also Western blotted for Rac and Vav2 expression with Rac and T7 antibodies, respectively. (B) NIH3T3 cells were transfected with R425GVav2, fixed 24 h later, and stained for R425GVav2 expression with T7 antibody and for actin with rho-damine-phalloidin. The arrow indicates the transfected cell. (C) HEK293T cells were transfected with Vav2 and treated after 24 h with wortmannin (200 nM) for 1 h. Activation of endogenous Rac (PBD) was determined as described in Materials and methods. Total cell lysates were Western blotted for Rac and Vav2 expression using Rac and Vav2 antibodies, respectively. (D) NIH3T3 cells were transfected with Vav2, and after 24 h the cells were treated with wortmannin (200 nM) for 1 h and then fixed and stained for Vav2 expression with T7 antibody and for actin with rhodamine-phalloidin. These results are representative of three experiments.
Mentions: The activity of Vav1 is stimulated by phosphoinositide products of phosphoinositide 3 (PI-3) kinase binding to the PH domain, however, mutations in the PH domain of Vav1 do not completely inhibit exchange activity and a mutation in the PH domain of Vav3 does not impair its function (Han et al., 1998; Movilla and Bustelo, 1999). The role of the PH domain in Vav2 function is not known. To determine whether the PH domain is necessary for Vav2 activity we mutated R425 to G, based on mutations in Vav1 that disrupt the binding of phosphoinositides (Han et al., 1998). HEK293T cells were transfected with R425G Vav2 and Rac1 activation was measured. This construct did not activate Rac (Fig. 4 A) and or cause lamellipodia (compare Fig. 4 B with 2 A), indicating that unlike Vav1 and Vav3, the PH domain is required for Vav2 function.

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