Limits...
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.

Show MeSH

Related in: MedlinePlus

Haptotactic migration toward FN. (A) Wild-type fibroblasts (+/+) and two independently derived lines of PTPα−/− fibroblasts (−/−) were analyzed for migration toward FN as described in Materials and methods. Each bar represents the average of four independent experiments, each experiment with three wells per cell type, ± S.D. The number of migrating wild-type cells was taken as 100%, and other values were calculated relative to this. (B) In other experiments, wild-type fibroblasts (+/+), line 2 PTPα−/− fibroblasts (−/−), and line 2 PTPα−/− fibroblasts infected with adenovirus expressing PTPα (α-wt) or catalytically inactive double mutant PTPα (α-dm) were analyzed for migration toward FN. Each bar represents the average of two independent experiments, each experiment with two wells per cell type, ± S.D.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172736&req=5

fig2: Haptotactic migration toward FN. (A) Wild-type fibroblasts (+/+) and two independently derived lines of PTPα−/− fibroblasts (−/−) were analyzed for migration toward FN as described in Materials and methods. Each bar represents the average of four independent experiments, each experiment with three wells per cell type, ± S.D. The number of migrating wild-type cells was taken as 100%, and other values were calculated relative to this. (B) In other experiments, wild-type fibroblasts (+/+), line 2 PTPα−/− fibroblasts (−/−), and line 2 PTPα−/− fibroblasts infected with adenovirus expressing PTPα (α-wt) or catalytically inactive double mutant PTPα (α-dm) were analyzed for migration toward FN. Each bar represents the average of two independent experiments, each experiment with two wells per cell type, ± S.D.

Mentions: The migration abilities of wild-type and PTPα−/− fibroblasts were examined in a cell culture wound healing assay. Confluent dishes of cells were “wounded” by scraping with a pipette tip, creating a space free of cells. A significant delay in the ability of the PTPα−/− cells to migrate into the empty space was observed (Fig. 1 A), with the wild-type cells closing the gap by 15 h, and the PTPα−/− cells still not able to completely fill the gap even after 24 h. Examination of the leading edge of the migrating cells revealed multiple protrusions and extensions in wild-type cells, but a relatively uniform flat edge on the PTPα−/− cells (Fig. 1, B and C). ECM-integrin signaling events are prominently involved in regulating cell migration, and FAK is a key mediator of this process. As our subsequent investigation of integrin-stimulated FAK phosphorylation revealed defects in FAK Tyr-397 phosphorylation in PTPα-deficient cells (see Results), we examined this in cells at the leading edge of the wound (Fig. 1 C). At a time just before wound closure (about a one-cell wide gap remaining between wild-type cells), the wild-type cells appeared to have more phospho-Tyr397 FAK than PTPα−/− cells. The phospho-Tyr397 FAK was localized within elongated, well-defined structures (likely focal adhesions) in multiple protrusions from the wild-type cells, whereas in PTPα−/− cells, the phospho-Tyr397 FAK appeared to be localized in smaller, round structures. We also analyzed haptotactic migration of the above cells and another independently derived PTPα−/− fibroblast line to the integrin ligand FN in a Transwell chamber assay. Both PTPα−/− lines of cells showed significantly impaired migration (67 and 49%) relative to the PTPα+/+ control cell line (Fig. 2 A), indicating a defect in FN-stimulated responses. To determine whether reintroduction of PTPα would restore migration ability, PTPα or a catalytically defective mutant of PTPα lacking the active site cysteine residues (C414S/C704S) were introduced into PTPα−/− cells by adenoviral infection. Expression of PTPα increased the percentage of migrating cells from 49 to 64% (Fig. 2 B). The extent of restored migration may be low because Western blotting revealed that the introduced PTPα was always less than that present in wild-type cells (unpublished data); nevertheless, this represents a significant (P < 0.005) 1.3-fold increase in haptotaxis. Expression of inactive PTPα had no effect on the number of migrating cells (Fig. 2 B). Together, the above results indicate that efficient cell migration from a wounded cell monolayer and in haptotaxis requires catalytically active PTPα, and that PTPα may function in these processes to promote FAK Tyr-397 phosphorylation and the formation of membrane extensions characteristic of migrating 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)

Haptotactic migration toward FN. (A) Wild-type fibroblasts (+/+) and two independently derived lines of PTPα−/− fibroblasts (−/−) were analyzed for migration toward FN as described in Materials and methods. Each bar represents the average of four independent experiments, each experiment with three wells per cell type, ± S.D. The number of migrating wild-type cells was taken as 100%, and other values were calculated relative to this. (B) In other experiments, wild-type fibroblasts (+/+), line 2 PTPα−/− fibroblasts (−/−), and line 2 PTPα−/− fibroblasts infected with adenovirus expressing PTPα (α-wt) or catalytically inactive double mutant PTPα (α-dm) were analyzed for migration toward FN. Each bar represents the average of two independent experiments, each experiment with two wells per cell type, ± S.D.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172736&req=5

fig2: Haptotactic migration toward FN. (A) Wild-type fibroblasts (+/+) and two independently derived lines of PTPα−/− fibroblasts (−/−) were analyzed for migration toward FN as described in Materials and methods. Each bar represents the average of four independent experiments, each experiment with three wells per cell type, ± S.D. The number of migrating wild-type cells was taken as 100%, and other values were calculated relative to this. (B) In other experiments, wild-type fibroblasts (+/+), line 2 PTPα−/− fibroblasts (−/−), and line 2 PTPα−/− fibroblasts infected with adenovirus expressing PTPα (α-wt) or catalytically inactive double mutant PTPα (α-dm) were analyzed for migration toward FN. Each bar represents the average of two independent experiments, each experiment with two wells per cell type, ± S.D.
Mentions: The migration abilities of wild-type and PTPα−/− fibroblasts were examined in a cell culture wound healing assay. Confluent dishes of cells were “wounded” by scraping with a pipette tip, creating a space free of cells. A significant delay in the ability of the PTPα−/− cells to migrate into the empty space was observed (Fig. 1 A), with the wild-type cells closing the gap by 15 h, and the PTPα−/− cells still not able to completely fill the gap even after 24 h. Examination of the leading edge of the migrating cells revealed multiple protrusions and extensions in wild-type cells, but a relatively uniform flat edge on the PTPα−/− cells (Fig. 1, B and C). ECM-integrin signaling events are prominently involved in regulating cell migration, and FAK is a key mediator of this process. As our subsequent investigation of integrin-stimulated FAK phosphorylation revealed defects in FAK Tyr-397 phosphorylation in PTPα-deficient cells (see Results), we examined this in cells at the leading edge of the wound (Fig. 1 C). At a time just before wound closure (about a one-cell wide gap remaining between wild-type cells), the wild-type cells appeared to have more phospho-Tyr397 FAK than PTPα−/− cells. The phospho-Tyr397 FAK was localized within elongated, well-defined structures (likely focal adhesions) in multiple protrusions from the wild-type cells, whereas in PTPα−/− cells, the phospho-Tyr397 FAK appeared to be localized in smaller, round structures. We also analyzed haptotactic migration of the above cells and another independently derived PTPα−/− fibroblast line to the integrin ligand FN in a Transwell chamber assay. Both PTPα−/− lines of cells showed significantly impaired migration (67 and 49%) relative to the PTPα+/+ control cell line (Fig. 2 A), indicating a defect in FN-stimulated responses. To determine whether reintroduction of PTPα would restore migration ability, PTPα or a catalytically defective mutant of PTPα lacking the active site cysteine residues (C414S/C704S) were introduced into PTPα−/− cells by adenoviral infection. Expression of PTPα increased the percentage of migrating cells from 49 to 64% (Fig. 2 B). The extent of restored migration may be low because Western blotting revealed that the introduced PTPα was always less than that present in wild-type cells (unpublished data); nevertheless, this represents a significant (P < 0.005) 1.3-fold increase in haptotaxis. Expression of inactive PTPα had no effect on the number of migrating cells (Fig. 2 B). Together, the above results indicate that efficient cell migration from a wounded cell monolayer and in haptotaxis requires catalytically active PTPα, and that PTPα may function in these processes to promote FAK Tyr-397 phosphorylation and the formation of membrane extensions characteristic of migrating 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