Limits...
Rap1 promotes cell spreading by localizing Rac guanine nucleotide exchange factors.

Arthur WT, Quilliam LA, Cooper JA - J. Cell Biol. (2004)

Bottom Line: Rap1 is necessary for the accumulation of VAV2 in membrane protrusions at the cell periphery.In addition, if VAV2 is artificially localized to the cell edge with the subcellular targeting domain of Rap1a, it increases cell spreading independently of Rap1.These results lead us to propose that Rap1 promotes cell spreading by localizing a subset of Rac GEFs to sites of active lamellipodia extension.

View Article: PubMed Central - PubMed

Affiliation: Fred Hutchison Cancer Research Center, Seattle, WA 98109, USA. barthur@fhcrc.org

ABSTRACT
The Ras-related GTPase Rap1 stimulates integrin-mediated adhesion and spreading in various mammalian cell types. Here, we demonstrate that Rap1 regulates cell spreading by localizing guanine nucleotide exchange factors (GEFs) that act via the Rho family GTPase Rac1. Rap1a activates Rac1 and requires Rac1 to enhance spreading, whereas Rac1 induces spreading independently of Rap1. Active Rap1a binds to a subset of Rac GEFs, including VAV2 and Tiam1 but not others such as SWAP-70 or COOL-1. Overexpressed VAV2 and Tiam1 specifically require Rap1 to promote spreading, even though Rac1 is activated independently of Rap1. Rap1 is necessary for the accumulation of VAV2 in membrane protrusions at the cell periphery. In addition, if VAV2 is artificially localized to the cell edge with the subcellular targeting domain of Rap1a, it increases cell spreading independently of Rap1. These results lead us to propose that Rap1 promotes cell spreading by localizing a subset of Rac GEFs to sites of active lamellipodia extension.

Show MeSH

Related in: MedlinePlus

Rap1 binds to a subset of Rac GEFs. (A) Rap1 and VAV2 coimmunoprecipitate. HA (left) and VAV2 (right) antibodies were used for immunoprecipitation from serum-starved HeLa cells electroporated with vectors encoding HA-CRD VAV2 (negative control), HA-wild-type Rap1a (mostly GDP bound), or HA-63E Rapla (GTP bound). Immunoprecipitates (IP) and total cell lysates (TCL) were then immunoblotted with HA or VAV2 antibodies. (B) Rap1 binds to VAV2 directly. GTP-loaded GST-Rab5, GST-H-Ras, and GST-Rap1a on glutathione beads were incubated in the absence (−) or presence (+) of bacterially expressed His-DH-PH-CRD VAV2 (input). Beads were washed, and the precipitates were immunoblotted with an anti-His antibody (His-VAV2) or stained with Coomassie blue (GST-GTPases). (C) Rap1 interacts with the DH-PH module of VAV2. GST-63E Rapla was used to pulldown HA-DH-PH-CRD VAV2 (truncation a), HA-DH-PH-CRD W503L VAV2 (b), HA-DH-PH VAV2 (c), HA-DH VAV2 (d), and HA-CRD VAV2 (e) from transiently transfected HeLa cells. Pulldowns (GST-63E Rapla) and total cell lysates were immunoblotted with HA antibodies. (D) Rap1 interacts with a subset of Rac GEFs. GST-63E Rapla was used to pulldown Myc-COOL-1, Myc-C1199 Tiam1, HA-DH-PH-CRD VAV2, HA-SWAP-70, and HA-DH-PH Tiam1 from transiently transfected HeLa cells. Pulldowns and total cell lysates were immunoblotted with Myc (left) or HA (right) antibodies.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172522&req=5

fig3: Rap1 binds to a subset of Rac GEFs. (A) Rap1 and VAV2 coimmunoprecipitate. HA (left) and VAV2 (right) antibodies were used for immunoprecipitation from serum-starved HeLa cells electroporated with vectors encoding HA-CRD VAV2 (negative control), HA-wild-type Rap1a (mostly GDP bound), or HA-63E Rapla (GTP bound). Immunoprecipitates (IP) and total cell lysates (TCL) were then immunoblotted with HA or VAV2 antibodies. (B) Rap1 binds to VAV2 directly. GTP-loaded GST-Rab5, GST-H-Ras, and GST-Rap1a on glutathione beads were incubated in the absence (−) or presence (+) of bacterially expressed His-DH-PH-CRD VAV2 (input). Beads were washed, and the precipitates were immunoblotted with an anti-His antibody (His-VAV2) or stained with Coomassie blue (GST-GTPases). (C) Rap1 interacts with the DH-PH module of VAV2. GST-63E Rapla was used to pulldown HA-DH-PH-CRD VAV2 (truncation a), HA-DH-PH-CRD W503L VAV2 (b), HA-DH-PH VAV2 (c), HA-DH VAV2 (d), and HA-CRD VAV2 (e) from transiently transfected HeLa cells. Pulldowns (GST-63E Rapla) and total cell lysates were immunoblotted with HA antibodies. (D) Rap1 interacts with a subset of Rac GEFs. GST-63E Rapla was used to pulldown Myc-COOL-1, Myc-C1199 Tiam1, HA-DH-PH-CRD VAV2, HA-SWAP-70, and HA-DH-PH Tiam1 from transiently transfected HeLa cells. Pulldowns and total cell lysates were immunoblotted with Myc (left) or HA (right) antibodies.

Mentions: We first tested whether or not Rap1a associates with VAV2. VAV2 is a widely expressed GEF that reportedly acts on Rac1, RhoA, and Cdc42 in vitro, although overexpression of VAV2 leads to a predominantly Rac1-like morphology (Schuebel et al., 1998; Liu and Burridge, 2000; Marignani and Carpenter, 2001). VAV2 contains NH2-terminal Calponin homology and acidic domains and central Dbl homology (DH) and Pleckstrin homology (PH) domains (Fig. 3 B). The DH domain mediates catalysis of GDP-GTP exchange on Rho proteins, whereas the PH domain binds phospholipids and participates in DH domain regulation (Han et al., 1998; Ma et al., 1998). The VAV2 COOH-terminal region is made up of a cysteine-rich domain (CRD) followed by a Src homology 2 domain flanked by two Src homology 3 domains. The CRD was of particular interest to this work because it is a motif that is also found in Raf and facilitates Ras-Raf binding (Hu et al., 1995). Additionally, the Calponin homology and DH-PH module have been separately implicated in Bud1 binding to Cdc24 (Park et al., 1997; Gulli and Peter, 2001). We conducted coimmunoprecipitation experiments using serum-starved cells expressing HA-tagged wild-type Rap1a (mostly GDP-bound) or constitutively active 63E Rap1a (GTP-bound) to determine if Rap1 binds to VAV2 (Fig. 3 A). We found that endogenous VAV2 coimmunoprecipitated with 63E Rap1a, and to a lesser extent with wild-type Rap1a. Another HA-tagged protein, HA-CRD VAV2, did not coimmunoprecipitate endogenous VAV2. Similar results were obtained in reciprocal immunoprecipitations using VAV2 antibodies. We also found that bacterial GST-Rap1a, but not Rab5 or H-Ras, precipitated bacterially expressed and purified His-DH-PH-CRD VAV2, suggesting that these proteins interact directly (Fig. 3 B).


Rap1 promotes cell spreading by localizing Rac guanine nucleotide exchange factors.

Arthur WT, Quilliam LA, Cooper JA - J. Cell Biol. (2004)

Rap1 binds to a subset of Rac GEFs. (A) Rap1 and VAV2 coimmunoprecipitate. HA (left) and VAV2 (right) antibodies were used for immunoprecipitation from serum-starved HeLa cells electroporated with vectors encoding HA-CRD VAV2 (negative control), HA-wild-type Rap1a (mostly GDP bound), or HA-63E Rapla (GTP bound). Immunoprecipitates (IP) and total cell lysates (TCL) were then immunoblotted with HA or VAV2 antibodies. (B) Rap1 binds to VAV2 directly. GTP-loaded GST-Rab5, GST-H-Ras, and GST-Rap1a on glutathione beads were incubated in the absence (−) or presence (+) of bacterially expressed His-DH-PH-CRD VAV2 (input). Beads were washed, and the precipitates were immunoblotted with an anti-His antibody (His-VAV2) or stained with Coomassie blue (GST-GTPases). (C) Rap1 interacts with the DH-PH module of VAV2. GST-63E Rapla was used to pulldown HA-DH-PH-CRD VAV2 (truncation a), HA-DH-PH-CRD W503L VAV2 (b), HA-DH-PH VAV2 (c), HA-DH VAV2 (d), and HA-CRD VAV2 (e) from transiently transfected HeLa cells. Pulldowns (GST-63E Rapla) and total cell lysates were immunoblotted with HA antibodies. (D) Rap1 interacts with a subset of Rac GEFs. GST-63E Rapla was used to pulldown Myc-COOL-1, Myc-C1199 Tiam1, HA-DH-PH-CRD VAV2, HA-SWAP-70, and HA-DH-PH Tiam1 from transiently transfected HeLa cells. Pulldowns and total cell lysates were immunoblotted with Myc (left) or HA (right) antibodies.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172522&req=5

fig3: Rap1 binds to a subset of Rac GEFs. (A) Rap1 and VAV2 coimmunoprecipitate. HA (left) and VAV2 (right) antibodies were used for immunoprecipitation from serum-starved HeLa cells electroporated with vectors encoding HA-CRD VAV2 (negative control), HA-wild-type Rap1a (mostly GDP bound), or HA-63E Rapla (GTP bound). Immunoprecipitates (IP) and total cell lysates (TCL) were then immunoblotted with HA or VAV2 antibodies. (B) Rap1 binds to VAV2 directly. GTP-loaded GST-Rab5, GST-H-Ras, and GST-Rap1a on glutathione beads were incubated in the absence (−) or presence (+) of bacterially expressed His-DH-PH-CRD VAV2 (input). Beads were washed, and the precipitates were immunoblotted with an anti-His antibody (His-VAV2) or stained with Coomassie blue (GST-GTPases). (C) Rap1 interacts with the DH-PH module of VAV2. GST-63E Rapla was used to pulldown HA-DH-PH-CRD VAV2 (truncation a), HA-DH-PH-CRD W503L VAV2 (b), HA-DH-PH VAV2 (c), HA-DH VAV2 (d), and HA-CRD VAV2 (e) from transiently transfected HeLa cells. Pulldowns (GST-63E Rapla) and total cell lysates were immunoblotted with HA antibodies. (D) Rap1 interacts with a subset of Rac GEFs. GST-63E Rapla was used to pulldown Myc-COOL-1, Myc-C1199 Tiam1, HA-DH-PH-CRD VAV2, HA-SWAP-70, and HA-DH-PH Tiam1 from transiently transfected HeLa cells. Pulldowns and total cell lysates were immunoblotted with Myc (left) or HA (right) antibodies.
Mentions: We first tested whether or not Rap1a associates with VAV2. VAV2 is a widely expressed GEF that reportedly acts on Rac1, RhoA, and Cdc42 in vitro, although overexpression of VAV2 leads to a predominantly Rac1-like morphology (Schuebel et al., 1998; Liu and Burridge, 2000; Marignani and Carpenter, 2001). VAV2 contains NH2-terminal Calponin homology and acidic domains and central Dbl homology (DH) and Pleckstrin homology (PH) domains (Fig. 3 B). The DH domain mediates catalysis of GDP-GTP exchange on Rho proteins, whereas the PH domain binds phospholipids and participates in DH domain regulation (Han et al., 1998; Ma et al., 1998). The VAV2 COOH-terminal region is made up of a cysteine-rich domain (CRD) followed by a Src homology 2 domain flanked by two Src homology 3 domains. The CRD was of particular interest to this work because it is a motif that is also found in Raf and facilitates Ras-Raf binding (Hu et al., 1995). Additionally, the Calponin homology and DH-PH module have been separately implicated in Bud1 binding to Cdc24 (Park et al., 1997; Gulli and Peter, 2001). We conducted coimmunoprecipitation experiments using serum-starved cells expressing HA-tagged wild-type Rap1a (mostly GDP-bound) or constitutively active 63E Rap1a (GTP-bound) to determine if Rap1 binds to VAV2 (Fig. 3 A). We found that endogenous VAV2 coimmunoprecipitated with 63E Rap1a, and to a lesser extent with wild-type Rap1a. Another HA-tagged protein, HA-CRD VAV2, did not coimmunoprecipitate endogenous VAV2. Similar results were obtained in reciprocal immunoprecipitations using VAV2 antibodies. We also found that bacterial GST-Rap1a, but not Rab5 or H-Ras, precipitated bacterially expressed and purified His-DH-PH-CRD VAV2, suggesting that these proteins interact directly (Fig. 3 B).

Bottom Line: Rap1 is necessary for the accumulation of VAV2 in membrane protrusions at the cell periphery.In addition, if VAV2 is artificially localized to the cell edge with the subcellular targeting domain of Rap1a, it increases cell spreading independently of Rap1.These results lead us to propose that Rap1 promotes cell spreading by localizing a subset of Rac GEFs to sites of active lamellipodia extension.

View Article: PubMed Central - PubMed

Affiliation: Fred Hutchison Cancer Research Center, Seattle, WA 98109, USA. barthur@fhcrc.org

ABSTRACT
The Ras-related GTPase Rap1 stimulates integrin-mediated adhesion and spreading in various mammalian cell types. Here, we demonstrate that Rap1 regulates cell spreading by localizing guanine nucleotide exchange factors (GEFs) that act via the Rho family GTPase Rac1. Rap1a activates Rac1 and requires Rac1 to enhance spreading, whereas Rac1 induces spreading independently of Rap1. Active Rap1a binds to a subset of Rac GEFs, including VAV2 and Tiam1 but not others such as SWAP-70 or COOL-1. Overexpressed VAV2 and Tiam1 specifically require Rap1 to promote spreading, even though Rac1 is activated independently of Rap1. Rap1 is necessary for the accumulation of VAV2 in membrane protrusions at the cell periphery. In addition, if VAV2 is artificially localized to the cell edge with the subcellular targeting domain of Rap1a, it increases cell spreading independently of Rap1. These results lead us to propose that Rap1 promotes cell spreading by localizing a subset of Rac GEFs to sites of active lamellipodia extension.

Show MeSH
Related in: MedlinePlus