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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 activity is dispensable for Rac1 activation by VAV2 and Tiam1. (left) HeLa cells were transfected with an empty vector (vector) or a vector encoding constitutively active HA-DH-PH-CRD VAV2 (VAV2), FLAG-Rap1GAP (Rap1GAP), or both. (right) In separate experiments, cells were transfected with an empty vector or a vector encoding constitutively active Myc-C1199 Tiam1 (Tiam1), FLAG-Rap1GAP, or both. The cells were lysed, and active GTP-bound Rac was precipitated with GST-PBD PAK1. Precipitates (active) and total cell lysates (total Rac1, HA/Myc, FLAG) were immunoblotted with Rac1, HA, Myc, or FLAG antibodies.
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fig5: Rap1 activity is dispensable for Rac1 activation by VAV2 and Tiam1. (left) HeLa cells were transfected with an empty vector (vector) or a vector encoding constitutively active HA-DH-PH-CRD VAV2 (VAV2), FLAG-Rap1GAP (Rap1GAP), or both. (right) In separate experiments, cells were transfected with an empty vector or a vector encoding constitutively active Myc-C1199 Tiam1 (Tiam1), FLAG-Rap1GAP, or both. The cells were lysed, and active GTP-bound Rac was precipitated with GST-PBD PAK1. Precipitates (active) and total cell lysates (total Rac1, HA/Myc, FLAG) were immunoblotted with Rac1, HA, Myc, or FLAG antibodies.

Mentions: Several potential mechanisms may explain why VAV2 and Tiam1 require Rap1 activity to regulate cell morphology. First, Rap1 could stimulate the GEF activities of VAV2 and Tiam1. Second, Rap1 could localize VAV2 and Tiam1 to specific sites where Rac1 activation is required for cell spreading. We tested whether or not Rap1 regulates Rac1 activation by exogenous VAV2 and Tiam1 (Fig. 5). Expression of truncated, constitutively active variants of DH-PH-CRD VAV2 or C1199 Tiam1 increased Rac1 GTP content even when endogenous Rap1 was inactivated by Rap1GAP. Furthermore, the addition of Rap1.GTP to in vitro exchange reactions did not augment GTP loading of Rac by VAV2 (unpublished data). These data show that Rap1.GTP is dispensable for Rac1 activation by constitutively active VAV2 and Tiam1, but do not exclude the possibility that Rap1 regulates to the catalytic activity of full-length GEFs in physiological conditions. These findings (Figs. 4 and 5) further suggest that increased total cellular levels of Rac1.GTP are not sufficient for cell spreading in the absence of Rap1 activity. Activation of a specific subpopulation of Rac1 may be required for spreading.


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

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

Rap1 activity is dispensable for Rac1 activation by VAV2 and Tiam1. (left) HeLa cells were transfected with an empty vector (vector) or a vector encoding constitutively active HA-DH-PH-CRD VAV2 (VAV2), FLAG-Rap1GAP (Rap1GAP), or both. (right) In separate experiments, cells were transfected with an empty vector or a vector encoding constitutively active Myc-C1199 Tiam1 (Tiam1), FLAG-Rap1GAP, or both. The cells were lysed, and active GTP-bound Rac was precipitated with GST-PBD PAK1. Precipitates (active) and total cell lysates (total Rac1, HA/Myc, FLAG) were immunoblotted with Rac1, HA, Myc, or FLAG antibodies.
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Related In: Results  -  Collection

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fig5: Rap1 activity is dispensable for Rac1 activation by VAV2 and Tiam1. (left) HeLa cells were transfected with an empty vector (vector) or a vector encoding constitutively active HA-DH-PH-CRD VAV2 (VAV2), FLAG-Rap1GAP (Rap1GAP), or both. (right) In separate experiments, cells were transfected with an empty vector or a vector encoding constitutively active Myc-C1199 Tiam1 (Tiam1), FLAG-Rap1GAP, or both. The cells were lysed, and active GTP-bound Rac was precipitated with GST-PBD PAK1. Precipitates (active) and total cell lysates (total Rac1, HA/Myc, FLAG) were immunoblotted with Rac1, HA, Myc, or FLAG antibodies.
Mentions: Several potential mechanisms may explain why VAV2 and Tiam1 require Rap1 activity to regulate cell morphology. First, Rap1 could stimulate the GEF activities of VAV2 and Tiam1. Second, Rap1 could localize VAV2 and Tiam1 to specific sites where Rac1 activation is required for cell spreading. We tested whether or not Rap1 regulates Rac1 activation by exogenous VAV2 and Tiam1 (Fig. 5). Expression of truncated, constitutively active variants of DH-PH-CRD VAV2 or C1199 Tiam1 increased Rac1 GTP content even when endogenous Rap1 was inactivated by Rap1GAP. Furthermore, the addition of Rap1.GTP to in vitro exchange reactions did not augment GTP loading of Rac by VAV2 (unpublished data). These data show that Rap1.GTP is dispensable for Rac1 activation by constitutively active VAV2 and Tiam1, but do not exclude the possibility that Rap1 regulates to the catalytic activity of full-length GEFs in physiological conditions. These findings (Figs. 4 and 5) further suggest that increased total cellular levels of Rac1.GTP are not sufficient for cell spreading in the absence of Rap1 activity. Activation of a specific subpopulation of Rac1 may be required for 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