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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 necessary for cell spreading promoted by VAV2 and Tiam1, but not SWAP-70 or COOL-1. HeLa cells were transiently transfected with vectors encoding GFP, GFP-Rap1GAP, HA-DH-PH-CRD VAV2, or Myc-C1199 Tiam1, or cotransfected with vectors encoding GFP-Rap1GAP and HA-DH-PH-CRD VAV2, Myc-C1199 Tiam1, HA-SWAP-70, or Myc-COOL-1. Transfected cells were suspended, plated on fibronectin for 1 h, fixed, and labeled with the HA or Myc antibodies. The histogram shows the percentage (average plus the SD) of refractile, poorly spread cells for each condition in the absence (open bars) or presence (closed bars) of GFP-Rap1GAP.
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fig4: Rap1 activity is necessary for cell spreading promoted by VAV2 and Tiam1, but not SWAP-70 or COOL-1. HeLa cells were transiently transfected with vectors encoding GFP, GFP-Rap1GAP, HA-DH-PH-CRD VAV2, or Myc-C1199 Tiam1, or cotransfected with vectors encoding GFP-Rap1GAP and HA-DH-PH-CRD VAV2, Myc-C1199 Tiam1, HA-SWAP-70, or Myc-COOL-1. Transfected cells were suspended, plated on fibronectin for 1 h, fixed, and labeled with the HA or Myc antibodies. The histogram shows the percentage (average plus the SD) of refractile, poorly spread cells for each condition in the absence (open bars) or presence (closed bars) of GFP-Rap1GAP.

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)

Rap1 activity is necessary for cell spreading promoted by VAV2 and Tiam1, but not SWAP-70 or COOL-1. HeLa cells were transiently transfected with vectors encoding GFP, GFP-Rap1GAP, HA-DH-PH-CRD VAV2, or Myc-C1199 Tiam1, or cotransfected with vectors encoding GFP-Rap1GAP and HA-DH-PH-CRD VAV2, Myc-C1199 Tiam1, HA-SWAP-70, or Myc-COOL-1. Transfected cells were suspended, plated on fibronectin for 1 h, fixed, and labeled with the HA or Myc antibodies. The histogram shows the percentage (average plus the SD) of refractile, poorly spread cells for each condition in the absence (open bars) or presence (closed bars) of GFP-Rap1GAP.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Rap1 activity is necessary for cell spreading promoted by VAV2 and Tiam1, but not SWAP-70 or COOL-1. HeLa cells were transiently transfected with vectors encoding GFP, GFP-Rap1GAP, HA-DH-PH-CRD VAV2, or Myc-C1199 Tiam1, or cotransfected with vectors encoding GFP-Rap1GAP and HA-DH-PH-CRD VAV2, Myc-C1199 Tiam1, HA-SWAP-70, or Myc-COOL-1. Transfected cells were suspended, plated on fibronectin for 1 h, fixed, and labeled with the HA or Myc antibodies. The histogram shows the percentage (average plus the SD) of refractile, poorly spread cells for each condition in the absence (open bars) or presence (closed bars) of GFP-Rap1GAP.
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