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Specific SHP-2 partitioning in raft domains triggers integrin-mediated signaling via Rho activation.

Lacalle RA, Mira E, Gomez-Mouton C, Jimenez-Baranda S, Martinez-A C, Manes S - J. Cell Biol. (2002)

Bottom Line: Cell signaling does not occur randomly over the cell surface, but is integrated within cholesterol-enriched membrane domains, termed rafts.By targeting SHP-2 to raft domains or to a non-raft plasma membrane fraction, we studied the functional role of rafts in signaling.Expression of the dominant negative N19Rho abrogates raft-SHP-2-induced signaling, suggesting that Rho activation is a downstream event in SHP-2 signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, UAM Campus de Cantoblanco, E-28049 Madrid, Spain.

ABSTRACT
Cell signaling does not occur randomly over the cell surface, but is integrated within cholesterol-enriched membrane domains, termed rafts. By targeting SHP-2 to raft domains or to a non-raft plasma membrane fraction, we studied the functional role of rafts in signaling. Serum-depleted, nonattached cells expressing the raft SHP-2 form, but not non-raft SHP-2, display signaling events resembling those observed after fibronectin attachment, such as beta1 integrin clustering, 397Y-FAK phosphorylation, and ERK activation, and also increases Rho-GTP levels. Expression of the dominant negative N19Rho abrogates raft-SHP-2-induced signaling, suggesting that Rho activation is a downstream event in SHP-2 signaling. Expression of a catalytic inactive SHP-2 mutant abrogates the adhesion-induced feedback inhibition of Rho activity, suggesting that SHP-2 contributes to adhesion-induced suppression of Rho activity. Because raft recruitment of SHP-2 occurs physiologically after cell attachment, these results provide a mechanism by which SHP-2 may influence cell adhesion and migration by spatially regulating Rho activity.

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Early integrin signaling is engaged in rafts. (A) Serum-starved 293T cells maintained in suspension or replated on Fn-coated plates were Triton X-100 extracted and fractionated in Optiprep gradients. Fractions were collected from top to bottom of the gradient and analyzed by Western blot with the indicated antibodies. Only the first (I, DRM-enriched) and last fractions (S, Triton X-100–soluble material) are shown. Data are representative of four independent experiments. (B–G) Nonattached (B and E) or Fn-adhered cells for 5 (C and F) or 30 min (D and G) were costained with FITC-CTx (green) and anti–397Y-FAK (B–D) or anti-β1 (E–G), followed by a Cy3-labeled second antibody (red), and analyzed by confocal microscopy. Single-color images are available online at http://www.jcb.org/cgi/content/full/jcb.200109031/DC1. Bar, 5 μm.
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fig2: Early integrin signaling is engaged in rafts. (A) Serum-starved 293T cells maintained in suspension or replated on Fn-coated plates were Triton X-100 extracted and fractionated in Optiprep gradients. Fractions were collected from top to bottom of the gradient and analyzed by Western blot with the indicated antibodies. Only the first (I, DRM-enriched) and last fractions (S, Triton X-100–soluble material) are shown. Data are representative of four independent experiments. (B–G) Nonattached (B and E) or Fn-adhered cells for 5 (C and F) or 30 min (D and G) were costained with FITC-CTx (green) and anti–397Y-FAK (B–D) or anti-β1 (E–G), followed by a Cy3-labeled second antibody (red), and analyzed by confocal microscopy. Single-color images are available online at http://www.jcb.org/cgi/content/full/jcb.200109031/DC1. Bar, 5 μm.

Mentions: Next, we addressed SHP-2 partitioning in rafts after integrin engagement. Serum-starved cells were replated on Fn-coated dishes for different times; after elimination of nonadhered cells, the Triton X-100–insoluble membrane fraction was isolated in flotation gradients. Raft-associated proteins float in density gradients as detergent-resistant membranes (DRMs), whereas the insoluble complexes formed by association with the cytoskeleton, as well as cytosolic and non-raft membrane proteins, copurify at the gradient bottom. Fn attachment triggers early recruitment of SHP-2, as well as of β1 integrin and FAK, to the DRM fraction (Fig. 2 A). SFK proteins, visualized with a pan-SRC antibody, are constitutively associated to DRM. Copurification of caveolin in DRM and the full solubilization of non-raft proteins such as the transferrin receptor (TfR) confirm the quality of the preparation.


Specific SHP-2 partitioning in raft domains triggers integrin-mediated signaling via Rho activation.

Lacalle RA, Mira E, Gomez-Mouton C, Jimenez-Baranda S, Martinez-A C, Manes S - J. Cell Biol. (2002)

Early integrin signaling is engaged in rafts. (A) Serum-starved 293T cells maintained in suspension or replated on Fn-coated plates were Triton X-100 extracted and fractionated in Optiprep gradients. Fractions were collected from top to bottom of the gradient and analyzed by Western blot with the indicated antibodies. Only the first (I, DRM-enriched) and last fractions (S, Triton X-100–soluble material) are shown. Data are representative of four independent experiments. (B–G) Nonattached (B and E) or Fn-adhered cells for 5 (C and F) or 30 min (D and G) were costained with FITC-CTx (green) and anti–397Y-FAK (B–D) or anti-β1 (E–G), followed by a Cy3-labeled second antibody (red), and analyzed by confocal microscopy. Single-color images are available online at http://www.jcb.org/cgi/content/full/jcb.200109031/DC1. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199243&req=5

fig2: Early integrin signaling is engaged in rafts. (A) Serum-starved 293T cells maintained in suspension or replated on Fn-coated plates were Triton X-100 extracted and fractionated in Optiprep gradients. Fractions were collected from top to bottom of the gradient and analyzed by Western blot with the indicated antibodies. Only the first (I, DRM-enriched) and last fractions (S, Triton X-100–soluble material) are shown. Data are representative of four independent experiments. (B–G) Nonattached (B and E) or Fn-adhered cells for 5 (C and F) or 30 min (D and G) were costained with FITC-CTx (green) and anti–397Y-FAK (B–D) or anti-β1 (E–G), followed by a Cy3-labeled second antibody (red), and analyzed by confocal microscopy. Single-color images are available online at http://www.jcb.org/cgi/content/full/jcb.200109031/DC1. Bar, 5 μm.
Mentions: Next, we addressed SHP-2 partitioning in rafts after integrin engagement. Serum-starved cells were replated on Fn-coated dishes for different times; after elimination of nonadhered cells, the Triton X-100–insoluble membrane fraction was isolated in flotation gradients. Raft-associated proteins float in density gradients as detergent-resistant membranes (DRMs), whereas the insoluble complexes formed by association with the cytoskeleton, as well as cytosolic and non-raft membrane proteins, copurify at the gradient bottom. Fn attachment triggers early recruitment of SHP-2, as well as of β1 integrin and FAK, to the DRM fraction (Fig. 2 A). SFK proteins, visualized with a pan-SRC antibody, are constitutively associated to DRM. Copurification of caveolin in DRM and the full solubilization of non-raft proteins such as the transferrin receptor (TfR) confirm the quality of the preparation.

Bottom Line: Cell signaling does not occur randomly over the cell surface, but is integrated within cholesterol-enriched membrane domains, termed rafts.By targeting SHP-2 to raft domains or to a non-raft plasma membrane fraction, we studied the functional role of rafts in signaling.Expression of the dominant negative N19Rho abrogates raft-SHP-2-induced signaling, suggesting that Rho activation is a downstream event in SHP-2 signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, UAM Campus de Cantoblanco, E-28049 Madrid, Spain.

ABSTRACT
Cell signaling does not occur randomly over the cell surface, but is integrated within cholesterol-enriched membrane domains, termed rafts. By targeting SHP-2 to raft domains or to a non-raft plasma membrane fraction, we studied the functional role of rafts in signaling. Serum-depleted, nonattached cells expressing the raft SHP-2 form, but not non-raft SHP-2, display signaling events resembling those observed after fibronectin attachment, such as beta1 integrin clustering, 397Y-FAK phosphorylation, and ERK activation, and also increases Rho-GTP levels. Expression of the dominant negative N19Rho abrogates raft-SHP-2-induced signaling, suggesting that Rho activation is a downstream event in SHP-2 signaling. Expression of a catalytic inactive SHP-2 mutant abrogates the adhesion-induced feedback inhibition of Rho activity, suggesting that SHP-2 contributes to adhesion-induced suppression of Rho activity. Because raft recruitment of SHP-2 occurs physiologically after cell attachment, these results provide a mechanism by which SHP-2 may influence cell adhesion and migration by spatially regulating Rho activity.

Show MeSH
Related in: MedlinePlus