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PTP alpha regulates integrin-stimulated FAK autophosphorylation and cytoskeletal rearrangement in cell spreading and migration.

Zeng L, Si X, Yu WP, Le HT, Ng KP, Teng RM, Ryan K, Wang DZ, Ponniah S, Pallen CJ - J. Cell Biol. (2003)

Bottom Line: Integrin-mediated formation of src-FAK and fyn-FAK complexes was reduced or abolished in PTP alpha-/- cells on FN, concomitant with markedly reduced phosphorylation of FAK at Tyr397.This mimicked the effects of treating wild-type fibroblasts with the src family protein tyrosine kinase (Src-PTK) inhibitor PP2.These results, together with the reduced src/fyn tyrosine kinase activity in PTP alpha-/- fibroblasts (Ponniah et al., 1999; Su et al., 1999), suggest that PTP alpha functions in integrin signaling and cell migration as an Src-PTK activator.

View Article: PubMed Central - PubMed

Affiliation: Cell Regulation Laboratory, Institute of Molecular and Cell Biology, Singapore 117609, Singapore.

ABSTRACT
We investigated the molecular and cellular actions of receptor protein tyrosine phosphatase (PTP) alpha in integrin signaling using immortalized fibroblasts derived from wild-type and PTP alpha-deficient mouse embryos. Defects in PTP alpha-/- migration in a wound healing assay were associated with altered cell shape and focal adhesion kinase (FAK) phosphorylation. The reduced haptotaxis to fibronectin (FN) of PTP alpha-/- cells was increased by expression of active (but not inactive) PTP alpha. Integrin-mediated formation of src-FAK and fyn-FAK complexes was reduced or abolished in PTP alpha-/- cells on FN, concomitant with markedly reduced phosphorylation of FAK at Tyr397. Reintroduction of active (but not inactive) PTP alpha restored FAK Tyr-397 phosphorylation. FN-induced cytoskeletal rearrangement was retarded in PTP alpha-/- cells, with delayed filamentous actin stress fiber assembly and focal adhesion formation. This mimicked the effects of treating wild-type fibroblasts with the src family protein tyrosine kinase (Src-PTK) inhibitor PP2. These results, together with the reduced src/fyn tyrosine kinase activity in PTP alpha-/- fibroblasts (Ponniah et al., 1999; Su et al., 1999), suggest that PTP alpha functions in integrin signaling and cell migration as an Src-PTK activator. Our paper establishes that PTP alpha is required for early integrin-proximal events, acting upstream of FAK to affect the timely and efficient phosphorylation of FAK Tyr-397.

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Similar delays in FN-induced actin stress fiber assembly and focal adhesion formation in PTPα−/− cells and PP2-treated wild-type cells. Wild-type (+/+), PTPα−/− (−/−), PP2-treated (PP2), and PP3-treated (PP3) wild-type cells were plated on FN-coated coverslips for 30 (A; FN30) and 60 (B; FN60) min and stained for F-actin (green) and vinculin (red). The arrows highlight focal adhesions. Bars, 10 μm. (C) PP2- or PP3-treated wild-type fibroblasts were plated on FN-coated coverslips for 30 (FN30) and 60 (FN60) min and stained for F-actin (green) and FAK (red). The arrows highlight some putative focal adhesions visualized by high FAK immunoreactivity. Bar, 10 μm.
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fig7: Similar delays in FN-induced actin stress fiber assembly and focal adhesion formation in PTPα−/− cells and PP2-treated wild-type cells. Wild-type (+/+), PTPα−/− (−/−), PP2-treated (PP2), and PP3-treated (PP3) wild-type cells were plated on FN-coated coverslips for 30 (A; FN30) and 60 (B; FN60) min and stained for F-actin (green) and vinculin (red). The arrows highlight focal adhesions. Bars, 10 μm. (C) PP2- or PP3-treated wild-type fibroblasts were plated on FN-coated coverslips for 30 (FN30) and 60 (FN60) min and stained for F-actin (green) and FAK (red). The arrows highlight some putative focal adhesions visualized by high FAK immunoreactivity. Bar, 10 μm.

Mentions: The FN-induced phosphorylation of Tyr397 of FAK is reduced on treatment of fibroblasts with the selective Src-PTK inhibitor PP2 (Salazar and Rozengurt, 2001). This suggested that defective Src-PTK function underlies the impaired FAK Tyr-397 phosphorylation in PTPα−/− cells. Integrin-stimulation is reported to activate src and promote its translocation to focal adhesions (Kaplan et al., 1995; Clark et al., 1998; Sieg et al., 1998; Oh et al., 1999), but we were unable to detect either of these events in wild-type fibroblasts and so could not determine whether they were affected by the lack of PTPα. In agreement with Su et al. (1999), we observed that FN-induced cell spreading was delayed in PTPα−/− cells. Spreading involves cytoskeletal rearrangement leading to stress fiber assembly and focal adhesion formation, and is impaired in FAK−/− cells expressing a FAK autophosphorylation site mutant (Owen et al., 1999). To determine if the parallel FAK autophosphorylation defect resulting from the absence of PTPα or the chemical inhibition of Src-PTKs extended to a similar impairment of cell spreading events, the distribution of actin, vinculin, and FAK were visualized and compared in these two situations. Plating of wild-type cells on FN for 30 min led to cell spreading and the formation of long actin stress fibers oriented in bundles at the leading edge of the polarized cells (Fig. 7 A). Putative focal adhesion sites at the end of these stress fibers were visualized by vinculin staining. In contrast, PTPα−/− cells were much less well spread and unpolarized. This was coincident with a virtual lack of actin stress fibers, and the appearance of F-actin–rich membrane ruffles and lamellipodia around the cell periphery. Furthermore, these cells had compacted cytoplasmic vinculin staining consistent with a lack of focal adhesion formation (Fig. 7 A). The PP2-treated wild-type cells appeared very similar, with reduced spreading and F-actin in membrane ruffles, no actin stress fibers, and compacted cytoplasmic vinculin staining that colocalized with what appeared to be perinuclear actin staining (Fig. 7 A). This latter colocalization was also observed in several of the PTPα−/− cells. Treatment of wild-type cells with PP3, an inactive analogue of PP2, did not alter the actin and vinculin localizations (Fig. 7 A). At 60 min on FN, actin staining defined thick longitudinally oriented stress fibers in the untreated and PP3-treated wild-type cells with vinculin present at the ends of the stress fibers in focal adhesion-like structures (Fig. 7 B). At 60 min, both the PTPα−/− cells and the PP2-treated wild-type cells had spread more and developed actin stress fibers with some vinculin colocalizing at the ends of these fibers, appearing similar to wild-type cells plated on FN for 30 min. Retarded FN-induced focal adhesion formation was also evident in PP2-treated wild-type cells stained for FAK (Fig. 7 C), as observed previously with the PTPα−/− cells (Fig. 4). Thus, the absence of PTPα leads to a temporally indistinguishable cell spreading delay and physically similar altered cell morphology as observed with the inhibition of Src-PTK activity. Delayed stress fiber assembly and focal adhesion formation were characteristic of both situations.


PTP alpha regulates integrin-stimulated FAK autophosphorylation and cytoskeletal rearrangement in cell spreading and migration.

Zeng L, Si X, Yu WP, Le HT, Ng KP, Teng RM, Ryan K, Wang DZ, Ponniah S, Pallen CJ - J. Cell Biol. (2003)

Similar delays in FN-induced actin stress fiber assembly and focal adhesion formation in PTPα−/− cells and PP2-treated wild-type cells. Wild-type (+/+), PTPα−/− (−/−), PP2-treated (PP2), and PP3-treated (PP3) wild-type cells were plated on FN-coated coverslips for 30 (A; FN30) and 60 (B; FN60) min and stained for F-actin (green) and vinculin (red). The arrows highlight focal adhesions. Bars, 10 μm. (C) PP2- or PP3-treated wild-type fibroblasts were plated on FN-coated coverslips for 30 (FN30) and 60 (FN60) min and stained for F-actin (green) and FAK (red). The arrows highlight some putative focal adhesions visualized by high FAK immunoreactivity. Bar, 10 μm.
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Related In: Results  -  Collection

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fig7: Similar delays in FN-induced actin stress fiber assembly and focal adhesion formation in PTPα−/− cells and PP2-treated wild-type cells. Wild-type (+/+), PTPα−/− (−/−), PP2-treated (PP2), and PP3-treated (PP3) wild-type cells were plated on FN-coated coverslips for 30 (A; FN30) and 60 (B; FN60) min and stained for F-actin (green) and vinculin (red). The arrows highlight focal adhesions. Bars, 10 μm. (C) PP2- or PP3-treated wild-type fibroblasts were plated on FN-coated coverslips for 30 (FN30) and 60 (FN60) min and stained for F-actin (green) and FAK (red). The arrows highlight some putative focal adhesions visualized by high FAK immunoreactivity. Bar, 10 μm.
Mentions: The FN-induced phosphorylation of Tyr397 of FAK is reduced on treatment of fibroblasts with the selective Src-PTK inhibitor PP2 (Salazar and Rozengurt, 2001). This suggested that defective Src-PTK function underlies the impaired FAK Tyr-397 phosphorylation in PTPα−/− cells. Integrin-stimulation is reported to activate src and promote its translocation to focal adhesions (Kaplan et al., 1995; Clark et al., 1998; Sieg et al., 1998; Oh et al., 1999), but we were unable to detect either of these events in wild-type fibroblasts and so could not determine whether they were affected by the lack of PTPα. In agreement with Su et al. (1999), we observed that FN-induced cell spreading was delayed in PTPα−/− cells. Spreading involves cytoskeletal rearrangement leading to stress fiber assembly and focal adhesion formation, and is impaired in FAK−/− cells expressing a FAK autophosphorylation site mutant (Owen et al., 1999). To determine if the parallel FAK autophosphorylation defect resulting from the absence of PTPα or the chemical inhibition of Src-PTKs extended to a similar impairment of cell spreading events, the distribution of actin, vinculin, and FAK were visualized and compared in these two situations. Plating of wild-type cells on FN for 30 min led to cell spreading and the formation of long actin stress fibers oriented in bundles at the leading edge of the polarized cells (Fig. 7 A). Putative focal adhesion sites at the end of these stress fibers were visualized by vinculin staining. In contrast, PTPα−/− cells were much less well spread and unpolarized. This was coincident with a virtual lack of actin stress fibers, and the appearance of F-actin–rich membrane ruffles and lamellipodia around the cell periphery. Furthermore, these cells had compacted cytoplasmic vinculin staining consistent with a lack of focal adhesion formation (Fig. 7 A). The PP2-treated wild-type cells appeared very similar, with reduced spreading and F-actin in membrane ruffles, no actin stress fibers, and compacted cytoplasmic vinculin staining that colocalized with what appeared to be perinuclear actin staining (Fig. 7 A). This latter colocalization was also observed in several of the PTPα−/− cells. Treatment of wild-type cells with PP3, an inactive analogue of PP2, did not alter the actin and vinculin localizations (Fig. 7 A). At 60 min on FN, actin staining defined thick longitudinally oriented stress fibers in the untreated and PP3-treated wild-type cells with vinculin present at the ends of the stress fibers in focal adhesion-like structures (Fig. 7 B). At 60 min, both the PTPα−/− cells and the PP2-treated wild-type cells had spread more and developed actin stress fibers with some vinculin colocalizing at the ends of these fibers, appearing similar to wild-type cells plated on FN for 30 min. Retarded FN-induced focal adhesion formation was also evident in PP2-treated wild-type cells stained for FAK (Fig. 7 C), as observed previously with the PTPα−/− cells (Fig. 4). Thus, the absence of PTPα leads to a temporally indistinguishable cell spreading delay and physically similar altered cell morphology as observed with the inhibition of Src-PTK activity. Delayed stress fiber assembly and focal adhesion formation were characteristic of both situations.

Bottom Line: Integrin-mediated formation of src-FAK and fyn-FAK complexes was reduced or abolished in PTP alpha-/- cells on FN, concomitant with markedly reduced phosphorylation of FAK at Tyr397.This mimicked the effects of treating wild-type fibroblasts with the src family protein tyrosine kinase (Src-PTK) inhibitor PP2.These results, together with the reduced src/fyn tyrosine kinase activity in PTP alpha-/- fibroblasts (Ponniah et al., 1999; Su et al., 1999), suggest that PTP alpha functions in integrin signaling and cell migration as an Src-PTK activator.

View Article: PubMed Central - PubMed

Affiliation: Cell Regulation Laboratory, Institute of Molecular and Cell Biology, Singapore 117609, Singapore.

ABSTRACT
We investigated the molecular and cellular actions of receptor protein tyrosine phosphatase (PTP) alpha in integrin signaling using immortalized fibroblasts derived from wild-type and PTP alpha-deficient mouse embryos. Defects in PTP alpha-/- migration in a wound healing assay were associated with altered cell shape and focal adhesion kinase (FAK) phosphorylation. The reduced haptotaxis to fibronectin (FN) of PTP alpha-/- cells was increased by expression of active (but not inactive) PTP alpha. Integrin-mediated formation of src-FAK and fyn-FAK complexes was reduced or abolished in PTP alpha-/- cells on FN, concomitant with markedly reduced phosphorylation of FAK at Tyr397. Reintroduction of active (but not inactive) PTP alpha restored FAK Tyr-397 phosphorylation. FN-induced cytoskeletal rearrangement was retarded in PTP alpha-/- cells, with delayed filamentous actin stress fiber assembly and focal adhesion formation. This mimicked the effects of treating wild-type fibroblasts with the src family protein tyrosine kinase (Src-PTK) inhibitor PP2. These results, together with the reduced src/fyn tyrosine kinase activity in PTP alpha-/- fibroblasts (Ponniah et al., 1999; Su et al., 1999), suggest that PTP alpha functions in integrin signaling and cell migration as an Src-PTK activator. Our paper establishes that PTP alpha is required for early integrin-proximal events, acting upstream of FAK to affect the timely and efficient phosphorylation of FAK Tyr-397.

Show MeSH
Related in: MedlinePlus