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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 activates Rac1. HeLa cells were transfected with an empty vector or a vector encoding constitutively active HA-63E Rap1a. Cells were suspended, plated on fibronectin (FN) or poly-l-lysine (PLL) for 3 h, and lysed, and then active GTP-bound Rac1 was precipitated with GST-PBD PAK1. Precipitates (active) and total cell lysates (total) were then immunoblotted with Rac1 or HA antibodies.
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fig2: Rap1 activates Rac1. HeLa cells were transfected with an empty vector or a vector encoding constitutively active HA-63E Rap1a. Cells were suspended, plated on fibronectin (FN) or poly-l-lysine (PLL) for 3 h, and lysed, and then active GTP-bound Rac1 was precipitated with GST-PBD PAK1. Precipitates (active) and total cell lysates (total) were then immunoblotted with Rac1 or HA antibodies.

Mentions: To test whether Rap1 regulates Rac1 GTP levels, we measured the effect of activated Rap1a on Rac1.GTP levels in cells that had been plated on fibronectin or poly-l-lysine for 3 h (Fig. 2). Rac1 activity was measured by precipitating active GTP-bound Rac1 with a GST fusion protein containing the PBD of PAK1 (del Pozo et al., 2000). Rac1 was activated by 63E Rap1a, on both fibronectin and poly-l-lysine surfaces. This finding suggests that Rap1 activates Rac1, and such activation is not secondary to Rap1-induced increases in outside-in signaling through integrins.


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

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

Rap1 activates Rac1. HeLa cells were transfected with an empty vector or a vector encoding constitutively active HA-63E Rap1a. Cells were suspended, plated on fibronectin (FN) or poly-l-lysine (PLL) for 3 h, and lysed, and then active GTP-bound Rac1 was precipitated with GST-PBD PAK1. Precipitates (active) and total cell lysates (total) were then immunoblotted with Rac1 or HA antibodies.
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Related In: Results  -  Collection

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

fig2: Rap1 activates Rac1. HeLa cells were transfected with an empty vector or a vector encoding constitutively active HA-63E Rap1a. Cells were suspended, plated on fibronectin (FN) or poly-l-lysine (PLL) for 3 h, and lysed, and then active GTP-bound Rac1 was precipitated with GST-PBD PAK1. Precipitates (active) and total cell lysates (total) were then immunoblotted with Rac1 or HA antibodies.
Mentions: To test whether Rap1 regulates Rac1 GTP levels, we measured the effect of activated Rap1a on Rac1.GTP levels in cells that had been plated on fibronectin or poly-l-lysine for 3 h (Fig. 2). Rac1 activity was measured by precipitating active GTP-bound Rac1 with a GST fusion protein containing the PBD of PAK1 (del Pozo et al., 2000). Rac1 was activated by 63E Rap1a, on both fibronectin and poly-l-lysine surfaces. This finding suggests that Rap1 activates Rac1, and such activation is not secondary to Rap1-induced increases in outside-in signaling through integrins.

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