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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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Raft targeting of LCK-SHP2 regulates actin cytoskeleton in nonattached cells. (A) Indirect immunofluorescence with anti-β1 antibody of nonattached or Fn-attached SRC-SHP2 or LCK-SHP2 cells, as indicated. Bar, 5 μm. (B) Serum-depleted SRC-SHP2 or LCK-SHP2 cells were latrunculin-B-treated, then left in suspension (sus) or Fn-replated for 5 (att) or 30 min (spr). FAK was precipitated and probed with anti–397Y-FAK and -FAK antibodies (n = 3).
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fig8: Raft targeting of LCK-SHP2 regulates actin cytoskeleton in nonattached cells. (A) Indirect immunofluorescence with anti-β1 antibody of nonattached or Fn-attached SRC-SHP2 or LCK-SHP2 cells, as indicated. Bar, 5 μm. (B) Serum-depleted SRC-SHP2 or LCK-SHP2 cells were latrunculin-B-treated, then left in suspension (sus) or Fn-replated for 5 (att) or 30 min (spr). FAK was precipitated and probed with anti–397Y-FAK and -FAK antibodies (n = 3).

Mentions: Next, we studied the mechanism by which LCK-SHP2 regulates FAK phosphorylation. LCK-SHP2 does not affect β1 affinity, as analyzed with the anti-β1 antibody HUTS-21, which binds specifically to activated β1 integrins (unpublished data). Confocal analysis nonetheless indicates large β1 integrin patches on the surface of suspension LCK-SHP2 cells, compared with diffuse β1 staining in cytSHP2- (unpublished data) or SRC-SHP2 cells (Fig. 8 A). Latrunculin-B disruption of actin cytoskeleton inhibits 397Y-FAK phosphorylation of nonattached LCK-SHP2 cells (Fig. 8 B), suggesting that basal LCK-SHP2–induced signaling is achieved by regulating the actin cytoskeleton. Cytoskeleton rearrangements are controlled by Rho GTPase family members (Kjoller and Hall, 1999), of which Rho is particularly prominent, as it influences integrin-induced ERK activation and FAK phosphorylation (Clark et al., 1998). To analyze whether Rho function is needed for LCK-SHP2 signaling in nonattached cells, we coexpressed a green fluorescent protein (GFP)-tagged N19Rho dominant negative mutant with LCK-SHP2. We found that N19Rho, but not GFP-N17Rac, inhibits 397Y-FAK phosphorylation in serum-depleted, nonattached LCK-SHP2 cells (Fig. 9 A).


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)

Raft targeting of LCK-SHP2 regulates actin cytoskeleton in nonattached cells. (A) Indirect immunofluorescence with anti-β1 antibody of nonattached or Fn-attached SRC-SHP2 or LCK-SHP2 cells, as indicated. Bar, 5 μm. (B) Serum-depleted SRC-SHP2 or LCK-SHP2 cells were latrunculin-B-treated, then left in suspension (sus) or Fn-replated for 5 (att) or 30 min (spr). FAK was precipitated and probed with anti–397Y-FAK and -FAK antibodies (n = 3).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199243&req=5

fig8: Raft targeting of LCK-SHP2 regulates actin cytoskeleton in nonattached cells. (A) Indirect immunofluorescence with anti-β1 antibody of nonattached or Fn-attached SRC-SHP2 or LCK-SHP2 cells, as indicated. Bar, 5 μm. (B) Serum-depleted SRC-SHP2 or LCK-SHP2 cells were latrunculin-B-treated, then left in suspension (sus) or Fn-replated for 5 (att) or 30 min (spr). FAK was precipitated and probed with anti–397Y-FAK and -FAK antibodies (n = 3).
Mentions: Next, we studied the mechanism by which LCK-SHP2 regulates FAK phosphorylation. LCK-SHP2 does not affect β1 affinity, as analyzed with the anti-β1 antibody HUTS-21, which binds specifically to activated β1 integrins (unpublished data). Confocal analysis nonetheless indicates large β1 integrin patches on the surface of suspension LCK-SHP2 cells, compared with diffuse β1 staining in cytSHP2- (unpublished data) or SRC-SHP2 cells (Fig. 8 A). Latrunculin-B disruption of actin cytoskeleton inhibits 397Y-FAK phosphorylation of nonattached LCK-SHP2 cells (Fig. 8 B), suggesting that basal LCK-SHP2–induced signaling is achieved by regulating the actin cytoskeleton. Cytoskeleton rearrangements are controlled by Rho GTPase family members (Kjoller and Hall, 1999), of which Rho is particularly prominent, as it influences integrin-induced ERK activation and FAK phosphorylation (Clark et al., 1998). To analyze whether Rho function is needed for LCK-SHP2 signaling in nonattached cells, we coexpressed a green fluorescent protein (GFP)-tagged N19Rho dominant negative mutant with LCK-SHP2. We found that N19Rho, but not GFP-N17Rac, inhibits 397Y-FAK phosphorylation in serum-depleted, nonattached LCK-SHP2 cells (Fig. 9 A).

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