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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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Membrane targeting of SHP-2. (A) SHP-2 and GM1 distribution was analyzed by confocal microscopy in nonattached cytSHP-2, SRC-SHP2, or LCK-SHP2 cells by costaining with FITC-CTx (green) and anti–SHP-2 antibody (red); yellow indicates SHP-2 and GM1 colocalization. Single-color images are available online at http://www.jcb.org/cgi/content/full/jcb.200109031/DC1. Bar, 5 μm. (B) cytSHP-2, SRC-SHP2, or LCK-SHP2 cells maintained in suspension or replated on Fn-coated dishes for 5 min were fractionated to analyze DRM partitioning of SHP-2 (DRM [I] and soluble [S] fractions). The lower band corresponds to endogenous SHP-2. Representative data are shown (n = 4). (C) Lysates of mock, cytSHP-2, SRC-SHP2, LCK-SHP2, SH2-truncated ΔSHP-2, or ΔSHP-2C/S cells were immunoprecipitated with anti-6His antibody and PTP activity determined in pellets using pNPP as substrate. Values are expressed as picomoles of phosphate released per minute. Background level is indicated with a dashed line. As a control of protein quantity, part of the immunoprecipitate was analyzed in Western blot using anti–SHP-2 antibody. (D) Lysates from serum-starved 293T cells expressing the catalytic active or inactive SHP-2 chimeras indicated were immunoprecipitated with a polyclonal anti–SHP-2 antibody and the pellets used to analyze PTP activity as in (C). Western blot analysis of pellets with anti–SHP-2 antibody is shown.
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fig4: Membrane targeting of SHP-2. (A) SHP-2 and GM1 distribution was analyzed by confocal microscopy in nonattached cytSHP-2, SRC-SHP2, or LCK-SHP2 cells by costaining with FITC-CTx (green) and anti–SHP-2 antibody (red); yellow indicates SHP-2 and GM1 colocalization. Single-color images are available online at http://www.jcb.org/cgi/content/full/jcb.200109031/DC1. Bar, 5 μm. (B) cytSHP-2, SRC-SHP2, or LCK-SHP2 cells maintained in suspension or replated on Fn-coated dishes for 5 min were fractionated to analyze DRM partitioning of SHP-2 (DRM [I] and soluble [S] fractions). The lower band corresponds to endogenous SHP-2. Representative data are shown (n = 4). (C) Lysates of mock, cytSHP-2, SRC-SHP2, LCK-SHP2, SH2-truncated ΔSHP-2, or ΔSHP-2C/S cells were immunoprecipitated with anti-6His antibody and PTP activity determined in pellets using pNPP as substrate. Values are expressed as picomoles of phosphate released per minute. Background level is indicated with a dashed line. As a control of protein quantity, part of the immunoprecipitate was analyzed in Western blot using anti–SHP-2 antibody. (D) Lysates from serum-starved 293T cells expressing the catalytic active or inactive SHP-2 chimeras indicated were immunoprecipitated with a polyclonal anti–SHP-2 antibody and the pellets used to analyze PTP activity as in (C). Western blot analysis of pellets with anti–SHP-2 antibody is shown.

Mentions: If integrin signaling takes places in rafts, targeting these intermediates to rafts would provide a kinetic advantage to integrin signaling by increasing the interaction efficiency of downstream targets. Double palmitoylation signals of p56LCK are a major determinant in targeting this protein to rafts (Kabouridis et al., 1997). We constructed a SHP-2 chimera by adding the 12 amino acids corresponding to the Lck unique domain, which bears the double S-acylation signal, to the SHP-2 N terminus. A membrane-bound SHP-2 chimera with the human c-SRC myristoylation tag was also used (Zhao and Zhao, 1999). Confocal analysis showed that whereas LCK-SHP2 and SRC-SHP2 are targeted to the plasma membrane, only LCK-SHP2 shows extensive colocalization with the raft marker GM1 (Fig. 4 A). In agreement, flotation gradients showed that only LCK-SHP2 partitions constitutively in DRM, whereas SRC-SHP2 recruitment to DRM requires integrin engagement (Fig. 4 B).


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)

Membrane targeting of SHP-2. (A) SHP-2 and GM1 distribution was analyzed by confocal microscopy in nonattached cytSHP-2, SRC-SHP2, or LCK-SHP2 cells by costaining with FITC-CTx (green) and anti–SHP-2 antibody (red); yellow indicates SHP-2 and GM1 colocalization. Single-color images are available online at http://www.jcb.org/cgi/content/full/jcb.200109031/DC1. Bar, 5 μm. (B) cytSHP-2, SRC-SHP2, or LCK-SHP2 cells maintained in suspension or replated on Fn-coated dishes for 5 min were fractionated to analyze DRM partitioning of SHP-2 (DRM [I] and soluble [S] fractions). The lower band corresponds to endogenous SHP-2. Representative data are shown (n = 4). (C) Lysates of mock, cytSHP-2, SRC-SHP2, LCK-SHP2, SH2-truncated ΔSHP-2, or ΔSHP-2C/S cells were immunoprecipitated with anti-6His antibody and PTP activity determined in pellets using pNPP as substrate. Values are expressed as picomoles of phosphate released per minute. Background level is indicated with a dashed line. As a control of protein quantity, part of the immunoprecipitate was analyzed in Western blot using anti–SHP-2 antibody. (D) Lysates from serum-starved 293T cells expressing the catalytic active or inactive SHP-2 chimeras indicated were immunoprecipitated with a polyclonal anti–SHP-2 antibody and the pellets used to analyze PTP activity as in (C). Western blot analysis of pellets with anti–SHP-2 antibody is shown.
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Related In: Results  -  Collection

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fig4: Membrane targeting of SHP-2. (A) SHP-2 and GM1 distribution was analyzed by confocal microscopy in nonattached cytSHP-2, SRC-SHP2, or LCK-SHP2 cells by costaining with FITC-CTx (green) and anti–SHP-2 antibody (red); yellow indicates SHP-2 and GM1 colocalization. Single-color images are available online at http://www.jcb.org/cgi/content/full/jcb.200109031/DC1. Bar, 5 μm. (B) cytSHP-2, SRC-SHP2, or LCK-SHP2 cells maintained in suspension or replated on Fn-coated dishes for 5 min were fractionated to analyze DRM partitioning of SHP-2 (DRM [I] and soluble [S] fractions). The lower band corresponds to endogenous SHP-2. Representative data are shown (n = 4). (C) Lysates of mock, cytSHP-2, SRC-SHP2, LCK-SHP2, SH2-truncated ΔSHP-2, or ΔSHP-2C/S cells were immunoprecipitated with anti-6His antibody and PTP activity determined in pellets using pNPP as substrate. Values are expressed as picomoles of phosphate released per minute. Background level is indicated with a dashed line. As a control of protein quantity, part of the immunoprecipitate was analyzed in Western blot using anti–SHP-2 antibody. (D) Lysates from serum-starved 293T cells expressing the catalytic active or inactive SHP-2 chimeras indicated were immunoprecipitated with a polyclonal anti–SHP-2 antibody and the pellets used to analyze PTP activity as in (C). Western blot analysis of pellets with anti–SHP-2 antibody is shown.
Mentions: If integrin signaling takes places in rafts, targeting these intermediates to rafts would provide a kinetic advantage to integrin signaling by increasing the interaction efficiency of downstream targets. Double palmitoylation signals of p56LCK are a major determinant in targeting this protein to rafts (Kabouridis et al., 1997). We constructed a SHP-2 chimera by adding the 12 amino acids corresponding to the Lck unique domain, which bears the double S-acylation signal, to the SHP-2 N terminus. A membrane-bound SHP-2 chimera with the human c-SRC myristoylation tag was also used (Zhao and Zhao, 1999). Confocal analysis showed that whereas LCK-SHP2 and SRC-SHP2 are targeted to the plasma membrane, only LCK-SHP2 shows extensive colocalization with the raft marker GM1 (Fig. 4 A). In agreement, flotation gradients showed that only LCK-SHP2 partitions constitutively in DRM, whereas SRC-SHP2 recruitment to DRM requires integrin engagement (Fig. 4 B).

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