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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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SHP-2 function is needed for cell adhesion and spreading on Fn. (A) Serum-starved mock- (▵), cytSHP-2– (▪), or cytSHP-2C/S–transfected (▴) 293T cells were added to Fn-coated plates. Images were recorded by phase-contrast microscopy (200×) at distinct times after replating. Representative fields are shown (n = 5). (B) Quantification of random fields in five experiments in (A). (C) Mock- (▵), cytSHP-2– (▪), cytSHP-2C/S– (▴), FRNK- (•), or cytSHP-2C/S+FRNK–transfected (□) cells were replated on dishes coated with several Fn concentrations; adhered cells were estimated after 15 min incubation (n = 4; **, P < 0.01, two-tailed t test).
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fig1: SHP-2 function is needed for cell adhesion and spreading on Fn. (A) Serum-starved mock- (▵), cytSHP-2– (▪), or cytSHP-2C/S–transfected (▴) 293T cells were added to Fn-coated plates. Images were recorded by phase-contrast microscopy (200×) at distinct times after replating. Representative fields are shown (n = 5). (B) Quantification of random fields in five experiments in (A). (C) Mock- (▵), cytSHP-2– (▪), cytSHP-2C/S– (▴), FRNK- (•), or cytSHP-2C/S+FRNK–transfected (□) cells were replated on dishes coated with several Fn concentrations; adhered cells were estimated after 15 min incubation (n = 4; **, P < 0.01, two-tailed t test).

Mentions: We analyzed the role of SHP-2 downstream of β1 integrins in human embryonic kidney 293T cells by expressing a catalytic inactive dominant mutant of this enzyme (cytSHP-2C/S). SHP-2C/S overexpression drastically delays 293T cell spreading on Fn compared to mock-transfected cells or cells expressing the wild-type SHP-2 (cytSHP-2) form (Fig. 1, A and B). Even at long adhesion times (60 min), SHP-2C/S cells show defects in polarized extension, with a rounded morphology (Fig. 1 A). Cells expressing the mutant also showed decreased adhesion to this ECM substrate (Fig. 1 C). To study the role of SHP-2 in integrin function, we coexpressed SHP-2C/S with the FAK C-terminal domain (FAK-related nonkinase [FRNK]). FRNK overexpression interferes with FAK function in a dominant inhibitory manner, reducing FAK tyrosine phosphorylation and focal adhesion assembly (Schlaepfer et al., 1999). FRNK expression reduces cell adhesion to Fn; FRNK and SHP-2C/S coexpression further decreases cell adhesion to this substrate (Fig. 1 C). These results show that SHP-2 activity is required for integrin function.


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)

SHP-2 function is needed for cell adhesion and spreading on Fn. (A) Serum-starved mock- (▵), cytSHP-2– (▪), or cytSHP-2C/S–transfected (▴) 293T cells were added to Fn-coated plates. Images were recorded by phase-contrast microscopy (200×) at distinct times after replating. Representative fields are shown (n = 5). (B) Quantification of random fields in five experiments in (A). (C) Mock- (▵), cytSHP-2– (▪), cytSHP-2C/S– (▴), FRNK- (•), or cytSHP-2C/S+FRNK–transfected (□) cells were replated on dishes coated with several Fn concentrations; adhered cells were estimated after 15 min incubation (n = 4; **, P < 0.01, two-tailed t test).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199243&req=5

fig1: SHP-2 function is needed for cell adhesion and spreading on Fn. (A) Serum-starved mock- (▵), cytSHP-2– (▪), or cytSHP-2C/S–transfected (▴) 293T cells were added to Fn-coated plates. Images were recorded by phase-contrast microscopy (200×) at distinct times after replating. Representative fields are shown (n = 5). (B) Quantification of random fields in five experiments in (A). (C) Mock- (▵), cytSHP-2– (▪), cytSHP-2C/S– (▴), FRNK- (•), or cytSHP-2C/S+FRNK–transfected (□) cells were replated on dishes coated with several Fn concentrations; adhered cells were estimated after 15 min incubation (n = 4; **, P < 0.01, two-tailed t test).
Mentions: We analyzed the role of SHP-2 downstream of β1 integrins in human embryonic kidney 293T cells by expressing a catalytic inactive dominant mutant of this enzyme (cytSHP-2C/S). SHP-2C/S overexpression drastically delays 293T cell spreading on Fn compared to mock-transfected cells or cells expressing the wild-type SHP-2 (cytSHP-2) form (Fig. 1, A and B). Even at long adhesion times (60 min), SHP-2C/S cells show defects in polarized extension, with a rounded morphology (Fig. 1 A). Cells expressing the mutant also showed decreased adhesion to this ECM substrate (Fig. 1 C). To study the role of SHP-2 in integrin function, we coexpressed SHP-2C/S with the FAK C-terminal domain (FAK-related nonkinase [FRNK]). FRNK overexpression interferes with FAK function in a dominant inhibitory manner, reducing FAK tyrosine phosphorylation and focal adhesion assembly (Schlaepfer et al., 1999). FRNK expression reduces cell adhesion to Fn; FRNK and SHP-2C/S coexpression further decreases cell adhesion to this substrate (Fig. 1 C). These results show that SHP-2 activity is required for integrin function.

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