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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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Localization of vinculin and appearance of FAK Tyr-397 phosphorylation in integrin-stimulated cells. Cells were allowed to adhere to FN-coated glass coverslips for 30 min (FN30), 60 min (FN60), and 120 min (FN120), and were then fixed and labeled with mouse monoclonal anti–vinculin antibody (red) and rabbit polyclonal antibodies specific to FAK phospho-Tyr397 (green). (A) Wild-type (PTPα+/+) cells. (B) PTPα−/− cells.
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fig5: Localization of vinculin and appearance of FAK Tyr-397 phosphorylation in integrin-stimulated cells. Cells were allowed to adhere to FN-coated glass coverslips for 30 min (FN30), 60 min (FN60), and 120 min (FN120), and were then fixed and labeled with mouse monoclonal anti–vinculin antibody (red) and rabbit polyclonal antibodies specific to FAK phospho-Tyr397 (green). (A) Wild-type (PTPα+/+) cells. (B) PTPα−/− cells.

Mentions: The altered FAK phosphorylation was also confirmed by visualization in cells attached to an FN substratum (Fig. 5). After plating of the cells on FN-coated dishes for 30, 60, and 120 min, the cells were processed for indirect immunofluorescent labeling with anti-vinculin and anti-FAK phosphospecific-Tyr397 antibodies. In wild-type cells, phospho-Tyr397 FAK was localized in focal adhesions present in multiple cell extensions after 30 min on FN. In contrast, PTPα−/− cells possessed membrane ruffles, but virtually no extensions or focal adhesions and mainly nuclear/perinuclear labeling with the anti-FAK Tyr-397 antibody (Fig. 5, A and B; top panels). After 60 min on FN, phospho-Tyr397 FAK was detected in thickened elongated focal adhesions in numerous well-defined cell extensions around the entire periphery of the wild-type cells, and had begun to appear in thin streaky focal adhesions in the outer periphery of PTPα−/− cells, although with less intensity than in the wild-type cells and in the absence of well-defined cell extensions (Fig. 5, A and B; middle panels). After 120 min on FN, the wild-type and PTPα−/− cells appeared similar in terms of FAK Tyr-397 staining and focal adhesion formation (Fig. 5, A and B; bottom panels). These observations suggest that relative to wild-type cells, there is an early delay or impairment of integrin-stimulated FAK Tyr-397 phosphorylation and formation of focal adhesions in the PTPα−/− cells, but that after 120 min on FN, these processes have reached approximately equivalent points in the wild-type and PTPα−/− cells.


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)

Localization of vinculin and appearance of FAK Tyr-397 phosphorylation in integrin-stimulated cells. Cells were allowed to adhere to FN-coated glass coverslips for 30 min (FN30), 60 min (FN60), and 120 min (FN120), and were then fixed and labeled with mouse monoclonal anti–vinculin antibody (red) and rabbit polyclonal antibodies specific to FAK phospho-Tyr397 (green). (A) Wild-type (PTPα+/+) cells. (B) PTPα−/− cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172736&req=5

fig5: Localization of vinculin and appearance of FAK Tyr-397 phosphorylation in integrin-stimulated cells. Cells were allowed to adhere to FN-coated glass coverslips for 30 min (FN30), 60 min (FN60), and 120 min (FN120), and were then fixed and labeled with mouse monoclonal anti–vinculin antibody (red) and rabbit polyclonal antibodies specific to FAK phospho-Tyr397 (green). (A) Wild-type (PTPα+/+) cells. (B) PTPα−/− cells.
Mentions: The altered FAK phosphorylation was also confirmed by visualization in cells attached to an FN substratum (Fig. 5). After plating of the cells on FN-coated dishes for 30, 60, and 120 min, the cells were processed for indirect immunofluorescent labeling with anti-vinculin and anti-FAK phosphospecific-Tyr397 antibodies. In wild-type cells, phospho-Tyr397 FAK was localized in focal adhesions present in multiple cell extensions after 30 min on FN. In contrast, PTPα−/− cells possessed membrane ruffles, but virtually no extensions or focal adhesions and mainly nuclear/perinuclear labeling with the anti-FAK Tyr-397 antibody (Fig. 5, A and B; top panels). After 60 min on FN, phospho-Tyr397 FAK was detected in thickened elongated focal adhesions in numerous well-defined cell extensions around the entire periphery of the wild-type cells, and had begun to appear in thin streaky focal adhesions in the outer periphery of PTPα−/− cells, although with less intensity than in the wild-type cells and in the absence of well-defined cell extensions (Fig. 5, A and B; middle panels). After 120 min on FN, the wild-type and PTPα−/− cells appeared similar in terms of FAK Tyr-397 staining and focal adhesion formation (Fig. 5, A and B; bottom panels). These observations suggest that relative to wild-type cells, there is an early delay or impairment of integrin-stimulated FAK Tyr-397 phosphorylation and formation of focal adhesions in the PTPα−/− cells, but that after 120 min on FN, these processes have reached approximately equivalent points in the wild-type and PTPα−/− cells.

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