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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-targeted VAV2 fusion proteins function independently of Rap1. (A) Schematic representation of HA epitope-tagged DH-PH-CRD VAV2 (VAV2) fused to full-length 63E Rap1a or 17N Rap1a, consisting of the GTPase domain (GTPase), hypervariable domain (HV), and CAAX box. Also shown are VAV2 fused to the COOH terminus of Rap1a encoding the HV and the prenylated (jagged line) CAAX box. The SAAX mutation abolished membrane targeting by preventing prenylation. HeLa cells were transfected with expression vectors for GFP or GFP-Rap1GAP alone (B) or GFP-Rap1GAP together with HA-VAV2 (a), HA-VAV2-63E Rapla (b), HA-VAV2-17N Rapla (c), HA-VAV2-HV-CAAX (d), or HA-VAV2-HV-SAAX (e) vectors (C). Transfected cells were suspended, plated on fibronectin for 1 h, fixed, and labeled with HA epitope tag antibodies. The histogram shows the percentage (average plus the SD) of refractile, poorly spread cells for each condition.
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fig8: Rap1-targeted VAV2 fusion proteins function independently of Rap1. (A) Schematic representation of HA epitope-tagged DH-PH-CRD VAV2 (VAV2) fused to full-length 63E Rap1a or 17N Rap1a, consisting of the GTPase domain (GTPase), hypervariable domain (HV), and CAAX box. Also shown are VAV2 fused to the COOH terminus of Rap1a encoding the HV and the prenylated (jagged line) CAAX box. The SAAX mutation abolished membrane targeting by preventing prenylation. HeLa cells were transfected with expression vectors for GFP or GFP-Rap1GAP alone (B) or GFP-Rap1GAP together with HA-VAV2 (a), HA-VAV2-63E Rapla (b), HA-VAV2-17N Rapla (c), HA-VAV2-HV-CAAX (d), or HA-VAV2-HV-SAAX (e) vectors (C). Transfected cells were suspended, plated on fibronectin for 1 h, fixed, and labeled with HA epitope tag antibodies. The histogram shows the percentage (average plus the SD) of refractile, poorly spread cells for each condition.

Mentions: Collectively, our data suggest that VAV2 requires Rap1.GTP for accumulation at the cell periphery and that this relocalization is required for VAV2 to stimulate cell spreading. A prediction of this model is that Rap1.GTP-dependent Rac GEFs would be able to promote cell spreading in the absence of Rap1 activity if they were targeted to the vicinity of Rap1 by other means. To test this idea, we created fusion proteins of VAV2 and 63E Rap1a, 17N Rap1a, or fragments of Rap1a. The fragments used for these fusions consisted of the 20-residue membrane-targeting domain at the COOH terminus of Rap1a. This domain includes the prenylated (geranylgeranyl modified) COOH-terminal CAAX box that allows for membrane insertion and 16 adjacent residues NH2-terminal to the CAAX box that make up the HV/polybasic domain and dictate what type of membrane is targeted (Hancock, 2003). As a control, a C/S mutation was generated in the CAAX box to prevent prenylation and thus abolish membrane targeting. The ability of the Rap1a-targeted VAV2 fusions to stimulate cell spreading was analyzed in cells in which Rap1 was inhibited with Rap1GAP (Fig. 8). As in Fig. 4, expression of Rap1GAP led to poor spreading, and this phenotype was not reverted by coexpression of the DH-PH-CRD module of VAV2. In contrast, the refractile, poorly spread phenotype was reverted to a highly spread morphology when a fusion of the DH-PH-CRD to 63E Rap1a was coexpressed with Rap1GAP. Because 63E Rap1a is resistant to Rap1GAP, this result could be either because the DH-PH-CRD is correctly localized or because Rap1a is not inactivated. However, a fusion of DH-PH-CRD to inactive 17N Rap1a or to just the HV-CAAX region of Rap1a also reverted cells expressing Rap1GAP from a poorly spread morphology. In contrast, a fusion of the DH-PH-CRD to the nonprenylated HV-SAAX region of Rap1a did not rescue. Thus, fusing the localization signals present in the HV-CAAX region of Rap1a onto VAV2 is sufficient to bypass the requirement for Rap1.GTP for cell spreading. The effect of the Rap1a CAAX region was specific because fusing VAV2 DH-PH-CRD to the K-Ras HV-CAAX region did not induce Rap1-independent spreading (unpublished data). These findings are consistent with a model in which Rap1.GTP relocalizes VAV2 and, likely, other Rac1GEFs including Tiam1 to the cell periphery where it stimulates Rac1-dependent cell spreading.


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

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

Rap1-targeted VAV2 fusion proteins function independently of Rap1. (A) Schematic representation of HA epitope-tagged DH-PH-CRD VAV2 (VAV2) fused to full-length 63E Rap1a or 17N Rap1a, consisting of the GTPase domain (GTPase), hypervariable domain (HV), and CAAX box. Also shown are VAV2 fused to the COOH terminus of Rap1a encoding the HV and the prenylated (jagged line) CAAX box. The SAAX mutation abolished membrane targeting by preventing prenylation. HeLa cells were transfected with expression vectors for GFP or GFP-Rap1GAP alone (B) or GFP-Rap1GAP together with HA-VAV2 (a), HA-VAV2-63E Rapla (b), HA-VAV2-17N Rapla (c), HA-VAV2-HV-CAAX (d), or HA-VAV2-HV-SAAX (e) vectors (C). Transfected cells were suspended, plated on fibronectin for 1 h, fixed, and labeled with HA epitope tag antibodies. The histogram shows the percentage (average plus the SD) of refractile, poorly spread cells for each condition.
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Related In: Results  -  Collection

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fig8: Rap1-targeted VAV2 fusion proteins function independently of Rap1. (A) Schematic representation of HA epitope-tagged DH-PH-CRD VAV2 (VAV2) fused to full-length 63E Rap1a or 17N Rap1a, consisting of the GTPase domain (GTPase), hypervariable domain (HV), and CAAX box. Also shown are VAV2 fused to the COOH terminus of Rap1a encoding the HV and the prenylated (jagged line) CAAX box. The SAAX mutation abolished membrane targeting by preventing prenylation. HeLa cells were transfected with expression vectors for GFP or GFP-Rap1GAP alone (B) or GFP-Rap1GAP together with HA-VAV2 (a), HA-VAV2-63E Rapla (b), HA-VAV2-17N Rapla (c), HA-VAV2-HV-CAAX (d), or HA-VAV2-HV-SAAX (e) vectors (C). Transfected cells were suspended, plated on fibronectin for 1 h, fixed, and labeled with HA epitope tag antibodies. The histogram shows the percentage (average plus the SD) of refractile, poorly spread cells for each condition.
Mentions: Collectively, our data suggest that VAV2 requires Rap1.GTP for accumulation at the cell periphery and that this relocalization is required for VAV2 to stimulate cell spreading. A prediction of this model is that Rap1.GTP-dependent Rac GEFs would be able to promote cell spreading in the absence of Rap1 activity if they were targeted to the vicinity of Rap1 by other means. To test this idea, we created fusion proteins of VAV2 and 63E Rap1a, 17N Rap1a, or fragments of Rap1a. The fragments used for these fusions consisted of the 20-residue membrane-targeting domain at the COOH terminus of Rap1a. This domain includes the prenylated (geranylgeranyl modified) COOH-terminal CAAX box that allows for membrane insertion and 16 adjacent residues NH2-terminal to the CAAX box that make up the HV/polybasic domain and dictate what type of membrane is targeted (Hancock, 2003). As a control, a C/S mutation was generated in the CAAX box to prevent prenylation and thus abolish membrane targeting. The ability of the Rap1a-targeted VAV2 fusions to stimulate cell spreading was analyzed in cells in which Rap1 was inhibited with Rap1GAP (Fig. 8). As in Fig. 4, expression of Rap1GAP led to poor spreading, and this phenotype was not reverted by coexpression of the DH-PH-CRD module of VAV2. In contrast, the refractile, poorly spread phenotype was reverted to a highly spread morphology when a fusion of the DH-PH-CRD to 63E Rap1a was coexpressed with Rap1GAP. Because 63E Rap1a is resistant to Rap1GAP, this result could be either because the DH-PH-CRD is correctly localized or because Rap1a is not inactivated. However, a fusion of DH-PH-CRD to inactive 17N Rap1a or to just the HV-CAAX region of Rap1a also reverted cells expressing Rap1GAP from a poorly spread morphology. In contrast, a fusion of the DH-PH-CRD to the nonprenylated HV-SAAX region of Rap1a did not rescue. Thus, fusing the localization signals present in the HV-CAAX region of Rap1a onto VAV2 is sufficient to bypass the requirement for Rap1.GTP for cell spreading. The effect of the Rap1a CAAX region was specific because fusing VAV2 DH-PH-CRD to the K-Ras HV-CAAX region did not induce Rap1-independent spreading (unpublished data). These findings are consistent with a model in which Rap1.GTP relocalizes VAV2 and, likely, other Rac1GEFs including Tiam1 to the cell periphery where it stimulates Rac1-dependent cell spreading.

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