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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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Reintroduction of PTPα into PTPα−/− cells restores integrin-stimulated FAK Tyr-397 phosphorylation. (A) PTPα−/− cells (line 2) were infected with adenovirus expressing wild-type PTPα (α-wt) or a catalytic mutant of PTPα (α-dm), and were plated on FN-coated dishes for 30 min. FAK immunoprecipitates were prepared from lysates of these cells or of uninfected PTPα+/+ and PTPα−/− cells and probed with anti-phospho-Tyr397–specific antibodies (top panel) or anti-FAK antibodies (middle panel). The expression of endogenous and introduced PTPα was examined in cell lysates (WCL) by probing with anti-PTPα antibodies (bottom panel). (B) FAK Tyr-397 phosphorylation was quantitated from at least three independent experiments such as that presented in A and is shown as the mean ± S.D. FAK Tyr-397 phosphorylation in the PTPα+/+ cells was taken as 100%, and the other data was calculated relative to this.
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fig6: Reintroduction of PTPα into PTPα−/− cells restores integrin-stimulated FAK Tyr-397 phosphorylation. (A) PTPα−/− cells (line 2) were infected with adenovirus expressing wild-type PTPα (α-wt) or a catalytic mutant of PTPα (α-dm), and were plated on FN-coated dishes for 30 min. FAK immunoprecipitates were prepared from lysates of these cells or of uninfected PTPα+/+ and PTPα−/− cells and probed with anti-phospho-Tyr397–specific antibodies (top panel) or anti-FAK antibodies (middle panel). The expression of endogenous and introduced PTPα was examined in cell lysates (WCL) by probing with anti-PTPα antibodies (bottom panel). (B) FAK Tyr-397 phosphorylation was quantitated from at least three independent experiments such as that presented in A and is shown as the mean ± S.D. FAK Tyr-397 phosphorylation in the PTPα+/+ cells was taken as 100%, and the other data was calculated relative to this.

Mentions: To confirm that the defective FAK autophosphorylation in the PTPα−/− cells was a direct result of the absence of PTPα, we examined the consequences of PTPα reexpression. We were unable to generate PTPα-expressing cells through stable transfection and selection, as reported by others and possibly due to toxic effects of high levels of the wild-type phosphatase (Lammers et al., 2000). To obtain high efficiency expression in the PTPα−/− cells, we used adenovirus-mediated expression of wild-type or a catalytically inactive mutant form of PTPα with the active site cysteine residues mutated to serine in both catalytic domains. As shown in Fig. 6 A, PTPα+/+ cells contain three immunodetectable forms of PTPα, all of which are absent in the PTPα−/− cells. The proteins with apparent molecular masses of 130 and 100 kD represent the fully glycosylated and unglycosylated forms of PTPα (Daum et al., 1994), whereas the ∼80-kD form may be a proteolyzed or processed product of 130-kD PTPα (Su et al., 1994, Lammers et al., 2000; Gill-Henn et al., 2001). After adenovirus infection of the PTPα−/− cells, we could detect low levels of the fully glycosylated 130-kD PTPα, trace amounts of the unglycosylated 100-kD PTPα, and amounts of the ∼80-kD PTPα comparable to that in the PTPα+/+ cells (Fig. 6 A). The contribution of the ∼80 kD form to PTPα-dependent integrin signaling events is unknown. In three independent experiments (Fig. 6 B), integrin-stimulation of the PTPα−/− cells induced FAK Tyr-397 phosphorylation to 56% (±2) of that quantitated in PTPα+/+ cells, and reintroduction of wild-type PTPα significantly increased FAK Tyr-397 phosphorylation to 85% (±11). However, expression of catalytically inactive PTPα in the PTPα−/− cells did not significantly affect FAK Tyr-397 phosphorylation (52% ± 17). This confirms that it is the lack of PTPα that causes this defect in integrin signaling, and that the catalytic activity of PTPα is required for FAK Tyr-397 phosphorylation to occur efficiently. The somewhat more efficient restoration of FAK Tyr-397 phosphorylation than of migration affected by reintroduction of PTPα into PTPα-deficient cells (a 1.5-fold vs. a 1.3-fold increase, respectively) may reflect PTPα expression differences, either in total PTPα or in the relative amounts of the three PTPα forms.


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)

Reintroduction of PTPα into PTPα−/− cells restores integrin-stimulated FAK Tyr-397 phosphorylation. (A) PTPα−/− cells (line 2) were infected with adenovirus expressing wild-type PTPα (α-wt) or a catalytic mutant of PTPα (α-dm), and were plated on FN-coated dishes for 30 min. FAK immunoprecipitates were prepared from lysates of these cells or of uninfected PTPα+/+ and PTPα−/− cells and probed with anti-phospho-Tyr397–specific antibodies (top panel) or anti-FAK antibodies (middle panel). The expression of endogenous and introduced PTPα was examined in cell lysates (WCL) by probing with anti-PTPα antibodies (bottom panel). (B) FAK Tyr-397 phosphorylation was quantitated from at least three independent experiments such as that presented in A and is shown as the mean ± S.D. FAK Tyr-397 phosphorylation in the PTPα+/+ cells was taken as 100%, and the other data was calculated relative to this.
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Related In: Results  -  Collection

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fig6: Reintroduction of PTPα into PTPα−/− cells restores integrin-stimulated FAK Tyr-397 phosphorylation. (A) PTPα−/− cells (line 2) were infected with adenovirus expressing wild-type PTPα (α-wt) or a catalytic mutant of PTPα (α-dm), and were plated on FN-coated dishes for 30 min. FAK immunoprecipitates were prepared from lysates of these cells or of uninfected PTPα+/+ and PTPα−/− cells and probed with anti-phospho-Tyr397–specific antibodies (top panel) or anti-FAK antibodies (middle panel). The expression of endogenous and introduced PTPα was examined in cell lysates (WCL) by probing with anti-PTPα antibodies (bottom panel). (B) FAK Tyr-397 phosphorylation was quantitated from at least three independent experiments such as that presented in A and is shown as the mean ± S.D. FAK Tyr-397 phosphorylation in the PTPα+/+ cells was taken as 100%, and the other data was calculated relative to this.
Mentions: To confirm that the defective FAK autophosphorylation in the PTPα−/− cells was a direct result of the absence of PTPα, we examined the consequences of PTPα reexpression. We were unable to generate PTPα-expressing cells through stable transfection and selection, as reported by others and possibly due to toxic effects of high levels of the wild-type phosphatase (Lammers et al., 2000). To obtain high efficiency expression in the PTPα−/− cells, we used adenovirus-mediated expression of wild-type or a catalytically inactive mutant form of PTPα with the active site cysteine residues mutated to serine in both catalytic domains. As shown in Fig. 6 A, PTPα+/+ cells contain three immunodetectable forms of PTPα, all of which are absent in the PTPα−/− cells. The proteins with apparent molecular masses of 130 and 100 kD represent the fully glycosylated and unglycosylated forms of PTPα (Daum et al., 1994), whereas the ∼80-kD form may be a proteolyzed or processed product of 130-kD PTPα (Su et al., 1994, Lammers et al., 2000; Gill-Henn et al., 2001). After adenovirus infection of the PTPα−/− cells, we could detect low levels of the fully glycosylated 130-kD PTPα, trace amounts of the unglycosylated 100-kD PTPα, and amounts of the ∼80-kD PTPα comparable to that in the PTPα+/+ cells (Fig. 6 A). The contribution of the ∼80 kD form to PTPα-dependent integrin signaling events is unknown. In three independent experiments (Fig. 6 B), integrin-stimulation of the PTPα−/− cells induced FAK Tyr-397 phosphorylation to 56% (±2) of that quantitated in PTPα+/+ cells, and reintroduction of wild-type PTPα significantly increased FAK Tyr-397 phosphorylation to 85% (±11). However, expression of catalytically inactive PTPα in the PTPα−/− cells did not significantly affect FAK Tyr-397 phosphorylation (52% ± 17). This confirms that it is the lack of PTPα that causes this defect in integrin signaling, and that the catalytic activity of PTPα is required for FAK Tyr-397 phosphorylation to occur efficiently. The somewhat more efficient restoration of FAK Tyr-397 phosphorylation than of migration affected by reintroduction of PTPα into PTPα-deficient cells (a 1.5-fold vs. a 1.3-fold increase, respectively) may reflect PTPα expression differences, either in total PTPα or in the relative amounts of the three PTPα forms.

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