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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.

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Rap1 targets VAV2 to matrix-associated membrane protrusions. Pseudopodia and total cell extracts were prepared with Transwell filters from HeLa cells electrotransfected with vectors encoding GFP or GFP-Rap1GAP. Extracts were adjusted for equal content of ERK, a protein found at equal levels in pseudopodia and total cell extracts (Cho and Klemke, 2002). Pseudopodial (pseud.) and total cell (total) extracts were immunoblotted with antibodies for Rap1, VAV2, Paxillin, ERK, or GFP.
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fig6: Rap1 targets VAV2 to matrix-associated membrane protrusions. Pseudopodia and total cell extracts were prepared with Transwell filters from HeLa cells electrotransfected with vectors encoding GFP or GFP-Rap1GAP. Extracts were adjusted for equal content of ERK, a protein found at equal levels in pseudopodia and total cell extracts (Cho and Klemke, 2002). Pseudopodial (pseud.) and total cell (total) extracts were immunoblotted with antibodies for Rap1, VAV2, Paxillin, ERK, or GFP.

Mentions: We used two approaches to test whether or not Rap1 can regulate the subcellular localization of VAV2. First, Cho and Klemke (2002) have developed a technique for identifying proteins that are enriched in pseudopodia engaging a fibronectin-coated substrate. We prepared pseudopodial extracts and total extracts from cells with normal or attenuated Rap1 activity and used Western blotting to examine the distribution of endogenous proteins (Fig. 6). As reported previously (Cho and Klemke, 2002), Paxillin was concentrated in pseudopodia and ERK was distributed equally in the pseudopodia and total extracts. Relative to ERK, Rap1 was also concentrated in pseudopodia. However, Rap1 localization was not affected by Rap1GAP overexpression. Like Rap1, VAV2 accumulated in pseudopodia under control conditions. However, the targeting of VAV2 to pseudopodia was blocked by expression of Rap1GAP. These data demonstrate that both GDP- and GTP-bound forms of Rap1 are concentrated in matrix-associated membrane protrusions and suggest that the active form of Rap1 mediates the localization of VAV2 to these structures.


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

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

Rap1 targets VAV2 to matrix-associated membrane protrusions. Pseudopodia and total cell extracts were prepared with Transwell filters from HeLa cells electrotransfected with vectors encoding GFP or GFP-Rap1GAP. Extracts were adjusted for equal content of ERK, a protein found at equal levels in pseudopodia and total cell extracts (Cho and Klemke, 2002). Pseudopodial (pseud.) and total cell (total) extracts were immunoblotted with antibodies for Rap1, VAV2, Paxillin, ERK, or GFP.
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Related In: Results  -  Collection

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

fig6: Rap1 targets VAV2 to matrix-associated membrane protrusions. Pseudopodia and total cell extracts were prepared with Transwell filters from HeLa cells electrotransfected with vectors encoding GFP or GFP-Rap1GAP. Extracts were adjusted for equal content of ERK, a protein found at equal levels in pseudopodia and total cell extracts (Cho and Klemke, 2002). Pseudopodial (pseud.) and total cell (total) extracts were immunoblotted with antibodies for Rap1, VAV2, Paxillin, ERK, or GFP.
Mentions: We used two approaches to test whether or not Rap1 can regulate the subcellular localization of VAV2. First, Cho and Klemke (2002) have developed a technique for identifying proteins that are enriched in pseudopodia engaging a fibronectin-coated substrate. We prepared pseudopodial extracts and total extracts from cells with normal or attenuated Rap1 activity and used Western blotting to examine the distribution of endogenous proteins (Fig. 6). As reported previously (Cho and Klemke, 2002), Paxillin was concentrated in pseudopodia and ERK was distributed equally in the pseudopodia and total extracts. Relative to ERK, Rap1 was also concentrated in pseudopodia. However, Rap1 localization was not affected by Rap1GAP overexpression. Like Rap1, VAV2 accumulated in pseudopodia under control conditions. However, the targeting of VAV2 to pseudopodia was blocked by expression of Rap1GAP. These data demonstrate that both GDP- and GTP-bound forms of Rap1 are concentrated in matrix-associated membrane protrusions and suggest that the active form of Rap1 mediates the localization of VAV2 to these structures.

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