Limits...
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.

Show MeSH

Related in: MedlinePlus

Model: Rap1 regulates morphology by targeting a subset of Rac GEFs to the edge of spreading cells. (A) Only when Rac GEFs are properly targeted do they locally activate Rac, resulting in cell spreading by the formation of productive membrane protrusions adjacent to the ECM. COOL-1 and SWAP-70, two Rac GEFs that do not interact with Rap, are targeted to the protrusive structures by Rap1-independent mechanisms. In contrast, the Rac GEFs VAV2 and Tiam1 are targeted to ECM-associated plasma membranes by binding to active Rap1 (Rap1.GTP) but not inactive Rap1 (Rap1.GDP), following Rap1 activation by adhesion signals. (B) Our proposed model of Rap1 regulation of spreading through Rac GEFs is comparable to the mechanism by which the yeast protein Bud1/Rsr1 controls budding via Cdc24, a Cdc42 GEF.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172522&req=5

fig9: Model: Rap1 regulates morphology by targeting a subset of Rac GEFs to the edge of spreading cells. (A) Only when Rac GEFs are properly targeted do they locally activate Rac, resulting in cell spreading by the formation of productive membrane protrusions adjacent to the ECM. COOL-1 and SWAP-70, two Rac GEFs that do not interact with Rap, are targeted to the protrusive structures by Rap1-independent mechanisms. In contrast, the Rac GEFs VAV2 and Tiam1 are targeted to ECM-associated plasma membranes by binding to active Rap1 (Rap1.GTP) but not inactive Rap1 (Rap1.GDP), following Rap1 activation by adhesion signals. (B) Our proposed model of Rap1 regulation of spreading through Rac GEFs is comparable to the mechanism by which the yeast protein Bud1/Rsr1 controls budding via Cdc24, a Cdc42 GEF.

Mentions: Rap1 regulates the interaction of a variety of cell types with the extracellular matrix or adhesion molecules on adjacent cells (Bos et al., 2001; Caron, 2003). Loss of Rap1 activity in D. melanogaster disrupts adherens junctions and cell–cell adhesion in the embryo, eye disc, and ovary (Hariharan et al., 1991; Asha et al., 1999; Knox and Brown, 2002). Rap1 is activated in animal cells in response to numerous stimuli and is important for cell adhesion, spreading, and migration (Bos et al., 2001). In this work, we examined the mechanism by which Rap1 enhances spreading of HeLa cells on the extracellular matrix protein fibronectin in the absence of serum. We found that Rap1 acts through the Rho family GTPase Rac1 (Fig. 1). Both active and inactive Rap1 are present in pseudopods and at the periphery of spreading cells, where plasma membrane is actively engaging the extracellular matrix (Figs. 6 and 7). When associated with GTP, Rap1 binds to specific Rac GEFs, including VAV2 and Tiam1, via their DH-PH regions (Fig. 3). Rap1 is required to recruit VAV2, and likely other GEFs, to sites of membrane protrusion (Fig. 7). Localization of VAV2 by Rap1 is required to induce Rac1-dependent protrusive activity (Figs. 4 and 8). In addition to localizing VAV2, Rap1 has the potential to also activate VAV2 locally at the cell periphery, but our experiments did not address this possibility. After Rac-dependent protrusive activity, new sites for Rap1 localization are created (Fig. 7 A), continuing the cycle. Rac activation has recently been reported to regulate the localization of a Rap1 GEF, GRP2, to sites of actin polymerization (Caloca et al., 2004). Therefore, Rap1 and Rac may promote membrane protrusion in a highly cooperative manner (Fig. 9 A).


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

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

Model: Rap1 regulates morphology by targeting a subset of Rac GEFs to the edge of spreading cells. (A) Only when Rac GEFs are properly targeted do they locally activate Rac, resulting in cell spreading by the formation of productive membrane protrusions adjacent to the ECM. COOL-1 and SWAP-70, two Rac GEFs that do not interact with Rap, are targeted to the protrusive structures by Rap1-independent mechanisms. In contrast, the Rac GEFs VAV2 and Tiam1 are targeted to ECM-associated plasma membranes by binding to active Rap1 (Rap1.GTP) but not inactive Rap1 (Rap1.GDP), following Rap1 activation by adhesion signals. (B) Our proposed model of Rap1 regulation of spreading through Rac GEFs is comparable to the mechanism by which the yeast protein Bud1/Rsr1 controls budding via Cdc24, a Cdc42 GEF.
© Copyright Policy
Related In: Results  -  Collection

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

fig9: Model: Rap1 regulates morphology by targeting a subset of Rac GEFs to the edge of spreading cells. (A) Only when Rac GEFs are properly targeted do they locally activate Rac, resulting in cell spreading by the formation of productive membrane protrusions adjacent to the ECM. COOL-1 and SWAP-70, two Rac GEFs that do not interact with Rap, are targeted to the protrusive structures by Rap1-independent mechanisms. In contrast, the Rac GEFs VAV2 and Tiam1 are targeted to ECM-associated plasma membranes by binding to active Rap1 (Rap1.GTP) but not inactive Rap1 (Rap1.GDP), following Rap1 activation by adhesion signals. (B) Our proposed model of Rap1 regulation of spreading through Rac GEFs is comparable to the mechanism by which the yeast protein Bud1/Rsr1 controls budding via Cdc24, a Cdc42 GEF.
Mentions: Rap1 regulates the interaction of a variety of cell types with the extracellular matrix or adhesion molecules on adjacent cells (Bos et al., 2001; Caron, 2003). Loss of Rap1 activity in D. melanogaster disrupts adherens junctions and cell–cell adhesion in the embryo, eye disc, and ovary (Hariharan et al., 1991; Asha et al., 1999; Knox and Brown, 2002). Rap1 is activated in animal cells in response to numerous stimuli and is important for cell adhesion, spreading, and migration (Bos et al., 2001). In this work, we examined the mechanism by which Rap1 enhances spreading of HeLa cells on the extracellular matrix protein fibronectin in the absence of serum. We found that Rap1 acts through the Rho family GTPase Rac1 (Fig. 1). Both active and inactive Rap1 are present in pseudopods and at the periphery of spreading cells, where plasma membrane is actively engaging the extracellular matrix (Figs. 6 and 7). When associated with GTP, Rap1 binds to specific Rac GEFs, including VAV2 and Tiam1, via their DH-PH regions (Fig. 3). Rap1 is required to recruit VAV2, and likely other GEFs, to sites of membrane protrusion (Fig. 7). Localization of VAV2 by Rap1 is required to induce Rac1-dependent protrusive activity (Figs. 4 and 8). In addition to localizing VAV2, Rap1 has the potential to also activate VAV2 locally at the cell periphery, but our experiments did not address this possibility. After Rac-dependent protrusive activity, new sites for Rap1 localization are created (Fig. 7 A), continuing the cycle. Rac activation has recently been reported to regulate the localization of a Rap1 GEF, GRP2, to sites of actin polymerization (Caloca et al., 2004). Therefore, Rap1 and Rac may promote membrane protrusion in a highly cooperative manner (Fig. 9 A).

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