Limits...
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.

Show MeSH

Related in: MedlinePlus

Vav2 is not necessary for PDGF-dependent ruffling or EGF-dependent Jnk activation. (A) NIH3T3 cells were stimulated with PDGF or COS7 cells were stimulated with EGF and immunoprecipitations were done with preimmune serum (PI) or Vav2 antibody (Vav2). The proteins were separated by SDS-PAGE and tyrosine phosphorylation of Vav2 was determined by Western blotting. (B) NIH3T3 or COS7 cells were transfected with L342R/L343SVav2, serum-starved, and then NIH3T3 cells were stimulated with PDGF (top) and COS7 cells were stimulated with EGF (bottom). Cells were stained for Vav2 expression with T7 antibody and with rhodamine-phalloidin. The left panels are quiescent cells expressing L342R/L343SVav2, the middle panels are growth factor–stimulated cells expressing L342R/L343SVav2, and the right panels are untransfected cells stimulated with growth factor. (C) COS7 cells were transfected with L342R/L343SVav2 and FLAG-Jnk. The cells were serum starved and stimulated with EGF and Jnk activity ([32P]GST-jun) was determined as described in Material and methods. Cell lysates were also western blotted with FLAG antibody to determined Jnk expression and with T7 antibody to determine Vav2 expression. These results are representative of four experiments. Bar = 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196856&req=5

fig7: Vav2 is not necessary for PDGF-dependent ruffling or EGF-dependent Jnk activation. (A) NIH3T3 cells were stimulated with PDGF or COS7 cells were stimulated with EGF and immunoprecipitations were done with preimmune serum (PI) or Vav2 antibody (Vav2). The proteins were separated by SDS-PAGE and tyrosine phosphorylation of Vav2 was determined by Western blotting. (B) NIH3T3 or COS7 cells were transfected with L342R/L343SVav2, serum-starved, and then NIH3T3 cells were stimulated with PDGF (top) and COS7 cells were stimulated with EGF (bottom). Cells were stained for Vav2 expression with T7 antibody and with rhodamine-phalloidin. The left panels are quiescent cells expressing L342R/L343SVav2, the middle panels are growth factor–stimulated cells expressing L342R/L343SVav2, and the right panels are untransfected cells stimulated with growth factor. (C) COS7 cells were transfected with L342R/L343SVav2 and FLAG-Jnk. The cells were serum starved and stimulated with EGF and Jnk activity ([32P]GST-jun) was determined as described in Material and methods. Cell lysates were also western blotted with FLAG antibody to determined Jnk expression and with T7 antibody to determine Vav2 expression. These results are representative of four experiments. Bar = 10 μm.

Mentions: All exchange factors for Rho family of GTPases contain a DH domain that is required to catalyze nucleotide exchange. We wished to make a Vav2 DH mutant that lacked exchange activity and thus might function as a dominant negative to allow us to identify Vav2-dependent functions. Mutations in the DH domain of several RhoGEFs disrupt catalysis of GDP/GTP exchange (Hart et al., 1994; Liu et al., 1998; Ma et al., 1998). Based on these mutations, we mutated R335 to G and the L342/L343 residues to R342/S343 in the DH domain of Vav2. The R335G mutant functioned very much like wild-type Vav2 in assays of Rac activation, stimulation of Jnk and induction of lamellipodia (data not shown), which is somewhat surprising since a similar mutant in the Trio DH domain markedly inhibited exchange activity in vitro (Liu et al., 1998). The L342R/L343S mutant did not activate Rac (Fig. 5 A), or Rho (data not shown) and did not cause lamellipodia in transfected NIH3T3 cells (Fig. 5 B) or Jnk activation in transfected COS7 cells (data not shown, see Fig. 7 C).


Vav2 is required for cell spreading.

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

Vav2 is not necessary for PDGF-dependent ruffling or EGF-dependent Jnk activation. (A) NIH3T3 cells were stimulated with PDGF or COS7 cells were stimulated with EGF and immunoprecipitations were done with preimmune serum (PI) or Vav2 antibody (Vav2). The proteins were separated by SDS-PAGE and tyrosine phosphorylation of Vav2 was determined by Western blotting. (B) NIH3T3 or COS7 cells were transfected with L342R/L343SVav2, serum-starved, and then NIH3T3 cells were stimulated with PDGF (top) and COS7 cells were stimulated with EGF (bottom). Cells were stained for Vav2 expression with T7 antibody and with rhodamine-phalloidin. The left panels are quiescent cells expressing L342R/L343SVav2, the middle panels are growth factor–stimulated cells expressing L342R/L343SVav2, and the right panels are untransfected cells stimulated with growth factor. (C) COS7 cells were transfected with L342R/L343SVav2 and FLAG-Jnk. The cells were serum starved and stimulated with EGF and Jnk activity ([32P]GST-jun) was determined as described in Material and methods. Cell lysates were also western blotted with FLAG antibody to determined Jnk expression and with T7 antibody to determine Vav2 expression. These results are representative of four experiments. Bar = 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Vav2 is not necessary for PDGF-dependent ruffling or EGF-dependent Jnk activation. (A) NIH3T3 cells were stimulated with PDGF or COS7 cells were stimulated with EGF and immunoprecipitations were done with preimmune serum (PI) or Vav2 antibody (Vav2). The proteins were separated by SDS-PAGE and tyrosine phosphorylation of Vav2 was determined by Western blotting. (B) NIH3T3 or COS7 cells were transfected with L342R/L343SVav2, serum-starved, and then NIH3T3 cells were stimulated with PDGF (top) and COS7 cells were stimulated with EGF (bottom). Cells were stained for Vav2 expression with T7 antibody and with rhodamine-phalloidin. The left panels are quiescent cells expressing L342R/L343SVav2, the middle panels are growth factor–stimulated cells expressing L342R/L343SVav2, and the right panels are untransfected cells stimulated with growth factor. (C) COS7 cells were transfected with L342R/L343SVav2 and FLAG-Jnk. The cells were serum starved and stimulated with EGF and Jnk activity ([32P]GST-jun) was determined as described in Material and methods. Cell lysates were also western blotted with FLAG antibody to determined Jnk expression and with T7 antibody to determine Vav2 expression. These results are representative of four experiments. Bar = 10 μm.
Mentions: All exchange factors for Rho family of GTPases contain a DH domain that is required to catalyze nucleotide exchange. We wished to make a Vav2 DH mutant that lacked exchange activity and thus might function as a dominant negative to allow us to identify Vav2-dependent functions. Mutations in the DH domain of several RhoGEFs disrupt catalysis of GDP/GTP exchange (Hart et al., 1994; Liu et al., 1998; Ma et al., 1998). Based on these mutations, we mutated R335 to G and the L342/L343 residues to R342/S343 in the DH domain of Vav2. The R335G mutant functioned very much like wild-type Vav2 in assays of Rac activation, stimulation of Jnk and induction of lamellipodia (data not shown), which is somewhat surprising since a similar mutant in the Trio DH domain markedly inhibited exchange activity in vitro (Liu et al., 1998). The L342R/L343S mutant did not activate Rac (Fig. 5 A), or Rho (data not shown) and did not cause lamellipodia in transfected NIH3T3 cells (Fig. 5 B) or Jnk activation in transfected COS7 cells (data not shown, see Fig. 7 C).

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