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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 GTPases by Vav2 expression in vivo. NIH3T3 fibroblasts (A, C, and D) or HEK293T cells (B) were transfected with Vav2, Vav1, or Dbl plus myc-Rac (A), HA-Cdc42 (C), or HA-RhoA (D) at a ratio of 3:1 (RhoGEF/GTPase). The effect of Vav1 and Vav2 on endogenous Rac was determined in HEK293T cells (B). Cells were harvested after 24 h and lysates incubated with GST-PBD (A–C) or GST-TRBD (D) bound to glutathione beads for 40 min at 4°C and then washed and the proteins separated by SDS-PAGE. Activation of Rac, RhoA, or Cdc42 was determined by Western blotting with Rac, myc, or HA antibodies. Total cell lysates (TCL) were also Western blotted for expression of Vav2 with Vav2 antibody and GTPases as described in Material and methods. These results are representative of three experiments.
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fig1: Activation of GTPases by Vav2 expression in vivo. NIH3T3 fibroblasts (A, C, and D) or HEK293T cells (B) were transfected with Vav2, Vav1, or Dbl plus myc-Rac (A), HA-Cdc42 (C), or HA-RhoA (D) at a ratio of 3:1 (RhoGEF/GTPase). The effect of Vav1 and Vav2 on endogenous Rac was determined in HEK293T cells (B). Cells were harvested after 24 h and lysates incubated with GST-PBD (A–C) or GST-TRBD (D) bound to glutathione beads for 40 min at 4°C and then washed and the proteins separated by SDS-PAGE. Activation of Rac, RhoA, or Cdc42 was determined by Western blotting with Rac, myc, or HA antibodies. Total cell lysates (TCL) were also Western blotted for expression of Vav2 with Vav2 antibody and GTPases as described in Material and methods. These results are representative of three experiments.

Mentions: An important step in understanding the function of Vav2 is to determine the GTPases it activates. Studies of the exchange activity of Vav2 in vitro have yielded conflicting results, as discussed above. Recently, assays have been described that allow measurement of Rac, Cdc42, and RhoA activation in vivo (Sander et al., 1998; Ren et al., 1999). We used these assays to determine the GTP binding proteins activated by Vav2 expression in vivo. NIH3T3 fibroblasts were transfected with Vav2 and myc-Rac, hemagglutinin (HA)-Cdc42, or HA-RhoA. Cells were harvested after 24 h, lysed, and incubated with glutathione S-transferase (GST)-PBD or GST-RBD bound to glutathione agarose beads. The amount of activated Rac, Cdc42, or RhoA was determined by probing the Western blots for the GTPases associated with the GST-PBD or GST-RBD constructs. Vav2 activated Rac1 (Fig. 1 A) and to a lesser extent RhoA (Fig. 1 D), but we detected minimal activation of Cdc42, although Dbl did activate Cdc42 as expected (Fig. 1 C). Similar results were obtained when the experiments were done using HEK293T or Cos7 cells. We also compared activation of endogenous Rac by Vav2 and Vav1 and detected a similar extent of activation (Fig. 1 B).


Vav2 is required for cell spreading.

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

Activation of GTPases by Vav2 expression in vivo. NIH3T3 fibroblasts (A, C, and D) or HEK293T cells (B) were transfected with Vav2, Vav1, or Dbl plus myc-Rac (A), HA-Cdc42 (C), or HA-RhoA (D) at a ratio of 3:1 (RhoGEF/GTPase). The effect of Vav1 and Vav2 on endogenous Rac was determined in HEK293T cells (B). Cells were harvested after 24 h and lysates incubated with GST-PBD (A–C) or GST-TRBD (D) bound to glutathione beads for 40 min at 4°C and then washed and the proteins separated by SDS-PAGE. Activation of Rac, RhoA, or Cdc42 was determined by Western blotting with Rac, myc, or HA antibodies. Total cell lysates (TCL) were also Western blotted for expression of Vav2 with Vav2 antibody and GTPases as described in Material and methods. These results are representative of three experiments.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Activation of GTPases by Vav2 expression in vivo. NIH3T3 fibroblasts (A, C, and D) or HEK293T cells (B) were transfected with Vav2, Vav1, or Dbl plus myc-Rac (A), HA-Cdc42 (C), or HA-RhoA (D) at a ratio of 3:1 (RhoGEF/GTPase). The effect of Vav1 and Vav2 on endogenous Rac was determined in HEK293T cells (B). Cells were harvested after 24 h and lysates incubated with GST-PBD (A–C) or GST-TRBD (D) bound to glutathione beads for 40 min at 4°C and then washed and the proteins separated by SDS-PAGE. Activation of Rac, RhoA, or Cdc42 was determined by Western blotting with Rac, myc, or HA antibodies. Total cell lysates (TCL) were also Western blotted for expression of Vav2 with Vav2 antibody and GTPases as described in Material and methods. These results are representative of three experiments.
Mentions: An important step in understanding the function of Vav2 is to determine the GTPases it activates. Studies of the exchange activity of Vav2 in vitro have yielded conflicting results, as discussed above. Recently, assays have been described that allow measurement of Rac, Cdc42, and RhoA activation in vivo (Sander et al., 1998; Ren et al., 1999). We used these assays to determine the GTP binding proteins activated by Vav2 expression in vivo. NIH3T3 fibroblasts were transfected with Vav2 and myc-Rac, hemagglutinin (HA)-Cdc42, or HA-RhoA. Cells were harvested after 24 h, lysed, and incubated with glutathione S-transferase (GST)-PBD or GST-RBD bound to glutathione agarose beads. The amount of activated Rac, Cdc42, or RhoA was determined by probing the Western blots for the GTPases associated with the GST-PBD or GST-RBD constructs. Vav2 activated Rac1 (Fig. 1 A) and to a lesser extent RhoA (Fig. 1 D), but we detected minimal activation of Cdc42, although Dbl did activate Cdc42 as expected (Fig. 1 C). Similar results were obtained when the experiments were done using HEK293T or Cos7 cells. We also compared activation of endogenous Rac by Vav2 and Vav1 and detected a similar extent of activation (Fig. 1 B).

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