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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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Rac1 acts downstream of Rap1 in spreading cells. (A) Rac1 is necessary for cell spreading by active Rap1. HeLa cells were transfected with vectors encoding GFP or constitutively active GFP-63E Rap1a alone or together with inhibitory effector fragments that block signaling downstream from Rho family proteins. Myc-RBD POSH, Myc-GBD N-WASP, and Myc-RBD Rhotekin specifically inhibit signaling from Rac1, Cdc42, and RhoA, respectively. Rac1, Cdc42, and likely other Rho proteins are inhibited by Myc-PBD PAK1. Transfected cells were suspended, plated on fibronectin for 30 min, fixed, and labeled with Myc antibodies. (B) Decreased spreading by Rap1 inactivation is rescued by active Rac1. HeLa cells were transfected with vectors encoding GFP or Flag-Rap1GAP alone or together with constitutively active GFP-61L Rac1, and then allowed to spread on fibronectin for 1 h. (C) Histogram showing the percentage (average plus the SD) of flat, well-spread cells in the experiment described in A. 1, GFP (white bar); 2, GFP-63E Rap1a; 3, GFP-63E Rap1a + Myc-PBD PAK1; 4, GFP-63E Rap1a + Myc-RBD POSH; 5, GFP-63E Rap1a + Myc-GBD N-WASP; 6, GFP-63E Rap1a + Myc-RBD Rhotekin. (D) Histogram showing the percentage (average plus the SD) of refractile, poorly spread cells in the experiment described in B. a, GFP (white bar); b, Flag-Rap1GAP; c, Flag-Rap1GAP + GFP-61L Rac1; d, GFP-61L Rac1.
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fig1: Rac1 acts downstream of Rap1 in spreading cells. (A) Rac1 is necessary for cell spreading by active Rap1. HeLa cells were transfected with vectors encoding GFP or constitutively active GFP-63E Rap1a alone or together with inhibitory effector fragments that block signaling downstream from Rho family proteins. Myc-RBD POSH, Myc-GBD N-WASP, and Myc-RBD Rhotekin specifically inhibit signaling from Rac1, Cdc42, and RhoA, respectively. Rac1, Cdc42, and likely other Rho proteins are inhibited by Myc-PBD PAK1. Transfected cells were suspended, plated on fibronectin for 30 min, fixed, and labeled with Myc antibodies. (B) Decreased spreading by Rap1 inactivation is rescued by active Rac1. HeLa cells were transfected with vectors encoding GFP or Flag-Rap1GAP alone or together with constitutively active GFP-61L Rac1, and then allowed to spread on fibronectin for 1 h. (C) Histogram showing the percentage (average plus the SD) of flat, well-spread cells in the experiment described in A. 1, GFP (white bar); 2, GFP-63E Rap1a; 3, GFP-63E Rap1a + Myc-PBD PAK1; 4, GFP-63E Rap1a + Myc-RBD POSH; 5, GFP-63E Rap1a + Myc-GBD N-WASP; 6, GFP-63E Rap1a + Myc-RBD Rhotekin. (D) Histogram showing the percentage (average plus the SD) of refractile, poorly spread cells in the experiment described in B. a, GFP (white bar); b, Flag-Rap1GAP; c, Flag-Rap1GAP + GFP-61L Rac1; d, GFP-61L Rac1.

Mentions: Rap1 regulates cell spreading and adhesion through an unknown mechanism. Given that Rap1 and Rho family GTPases lead to similar alterations in cell morphology, Rho proteins may function downstream of Rap1. To investigate this possibility, we made use of adherent HeLa cells transiently transfected with various constructs. Cells were suspended with EDTA, held in suspension in serum-free medium for 1 h, plated on fibronectin-coated surfaces, and examined 30 to 60 min later. Under these conditions, Rac1a and Rap1 are both activated, which is consistent with a possible functional relationship (unpublished data). Moreover, HeLa cells expressing activated (63E mutant) Rap1a adopted a flat, isotropic morphology (Fig. 1 A, 2) and resembled cells overexpressing activated Rac1 (Fig. 1 B, d) or Rac GEFs (see Fig. 4).


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

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

Rac1 acts downstream of Rap1 in spreading cells. (A) Rac1 is necessary for cell spreading by active Rap1. HeLa cells were transfected with vectors encoding GFP or constitutively active GFP-63E Rap1a alone or together with inhibitory effector fragments that block signaling downstream from Rho family proteins. Myc-RBD POSH, Myc-GBD N-WASP, and Myc-RBD Rhotekin specifically inhibit signaling from Rac1, Cdc42, and RhoA, respectively. Rac1, Cdc42, and likely other Rho proteins are inhibited by Myc-PBD PAK1. Transfected cells were suspended, plated on fibronectin for 30 min, fixed, and labeled with Myc antibodies. (B) Decreased spreading by Rap1 inactivation is rescued by active Rac1. HeLa cells were transfected with vectors encoding GFP or Flag-Rap1GAP alone or together with constitutively active GFP-61L Rac1, and then allowed to spread on fibronectin for 1 h. (C) Histogram showing the percentage (average plus the SD) of flat, well-spread cells in the experiment described in A. 1, GFP (white bar); 2, GFP-63E Rap1a; 3, GFP-63E Rap1a + Myc-PBD PAK1; 4, GFP-63E Rap1a + Myc-RBD POSH; 5, GFP-63E Rap1a + Myc-GBD N-WASP; 6, GFP-63E Rap1a + Myc-RBD Rhotekin. (D) Histogram showing the percentage (average plus the SD) of refractile, poorly spread cells in the experiment described in B. a, GFP (white bar); b, Flag-Rap1GAP; c, Flag-Rap1GAP + GFP-61L Rac1; d, GFP-61L Rac1.
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Related In: Results  -  Collection

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fig1: Rac1 acts downstream of Rap1 in spreading cells. (A) Rac1 is necessary for cell spreading by active Rap1. HeLa cells were transfected with vectors encoding GFP or constitutively active GFP-63E Rap1a alone or together with inhibitory effector fragments that block signaling downstream from Rho family proteins. Myc-RBD POSH, Myc-GBD N-WASP, and Myc-RBD Rhotekin specifically inhibit signaling from Rac1, Cdc42, and RhoA, respectively. Rac1, Cdc42, and likely other Rho proteins are inhibited by Myc-PBD PAK1. Transfected cells were suspended, plated on fibronectin for 30 min, fixed, and labeled with Myc antibodies. (B) Decreased spreading by Rap1 inactivation is rescued by active Rac1. HeLa cells were transfected with vectors encoding GFP or Flag-Rap1GAP alone or together with constitutively active GFP-61L Rac1, and then allowed to spread on fibronectin for 1 h. (C) Histogram showing the percentage (average plus the SD) of flat, well-spread cells in the experiment described in A. 1, GFP (white bar); 2, GFP-63E Rap1a; 3, GFP-63E Rap1a + Myc-PBD PAK1; 4, GFP-63E Rap1a + Myc-RBD POSH; 5, GFP-63E Rap1a + Myc-GBD N-WASP; 6, GFP-63E Rap1a + Myc-RBD Rhotekin. (D) Histogram showing the percentage (average plus the SD) of refractile, poorly spread cells in the experiment described in B. a, GFP (white bar); b, Flag-Rap1GAP; c, Flag-Rap1GAP + GFP-61L Rac1; d, GFP-61L Rac1.
Mentions: Rap1 regulates cell spreading and adhesion through an unknown mechanism. Given that Rap1 and Rho family GTPases lead to similar alterations in cell morphology, Rho proteins may function downstream of Rap1. To investigate this possibility, we made use of adherent HeLa cells transiently transfected with various constructs. Cells were suspended with EDTA, held in suspension in serum-free medium for 1 h, plated on fibronectin-coated surfaces, and examined 30 to 60 min later. Under these conditions, Rac1a and Rap1 are both activated, which is consistent with a possible functional relationship (unpublished data). Moreover, HeLa cells expressing activated (63E mutant) Rap1a adopted a flat, isotropic morphology (Fig. 1 A, 2) and resembled cells overexpressing activated Rac1 (Fig. 1 B, d) or Rac GEFs (see Fig. 4).

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