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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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Integrin-stimulated FAK Tyr-397 phosphorylation is impaired in PTPα- cells. (A) Reduced tyrosine phosphorylation of FAK in PTPα−/− cells. FAK immunoprecipitates from PTPα+/+ and PTPα−/− cells adhering to plastic dishes (on dish), retained in suspension (susp), or plated onto FN-coated dishes for 30 min (FN30), were probed with anti-phosphotyrosine antibodies (top panel) or with anti-FAK antibodies (bottom panel). (B) Reduced FAK Tyr-397 phosphorylation in PTPα−/− cells. FAK immunoprecipitates from PTPα+/+ and PTPα−/− cells retained in suspension (susp) or plated onto FN-coated dishes for 30 min (FN30) or 60 min (FN60) were probed with anti-phospho-Tyr397–specific antibodies (top panel), or anti-FAK antibodies (bottom panel). (C) Integrin-stimulated FAK Tyr-397 phosphorylation was quantitated from at least seven independent experiments such as that presented in B and is shown as the mean ± S.D. FAK Tyr-397 phosphorylation in PTPα+/+ cells plated on FN for 30 min was taken as 100%, and the other data was calculated relative to this.
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fig4: Integrin-stimulated FAK Tyr-397 phosphorylation is impaired in PTPα- cells. (A) Reduced tyrosine phosphorylation of FAK in PTPα−/− cells. FAK immunoprecipitates from PTPα+/+ and PTPα−/− cells adhering to plastic dishes (on dish), retained in suspension (susp), or plated onto FN-coated dishes for 30 min (FN30), were probed with anti-phosphotyrosine antibodies (top panel) or with anti-FAK antibodies (bottom panel). (B) Reduced FAK Tyr-397 phosphorylation in PTPα−/− cells. FAK immunoprecipitates from PTPα+/+ and PTPα−/− cells retained in suspension (susp) or plated onto FN-coated dishes for 30 min (FN30) or 60 min (FN60) were probed with anti-phospho-Tyr397–specific antibodies (top panel), or anti-FAK antibodies (bottom panel). (C) Integrin-stimulated FAK Tyr-397 phosphorylation was quantitated from at least seven independent experiments such as that presented in B and is shown as the mean ± S.D. FAK Tyr-397 phosphorylation in PTPα+/+ cells plated on FN for 30 min was taken as 100%, and the other data was calculated relative to this.

Mentions: As src and fyn bind to phospho-Tyr397 of FAK, reduced FAK Tyr-397 autophosphorylation in the PTPα−/− cells could account for less src/fyn binding. The overall phosphotyrosine content of FAK was less in PTPα−/− cells than in PTPα+/+ cells, both under normal culture conditions and after plating on FN (Fig. 4 A). The phosphorylation status of FAK Tyr-397 was examined using an anti-FAK phospho-Tyr397–specific antibody. No phosphorylation of FAK Tyr-397 was detected in any cells in suspension. The phosphorylation of Tyr397 of FAK was consistently observed to be reduced in PTPα−/− cells, compared with wild-type cells, on FN-induced integrin activation (Fig. 4, B and C).


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)

Integrin-stimulated FAK Tyr-397 phosphorylation is impaired in PTPα- cells. (A) Reduced tyrosine phosphorylation of FAK in PTPα−/− cells. FAK immunoprecipitates from PTPα+/+ and PTPα−/− cells adhering to plastic dishes (on dish), retained in suspension (susp), or plated onto FN-coated dishes for 30 min (FN30), were probed with anti-phosphotyrosine antibodies (top panel) or with anti-FAK antibodies (bottom panel). (B) Reduced FAK Tyr-397 phosphorylation in PTPα−/− cells. FAK immunoprecipitates from PTPα+/+ and PTPα−/− cells retained in suspension (susp) or plated onto FN-coated dishes for 30 min (FN30) or 60 min (FN60) were probed with anti-phospho-Tyr397–specific antibodies (top panel), or anti-FAK antibodies (bottom panel). (C) Integrin-stimulated FAK Tyr-397 phosphorylation was quantitated from at least seven independent experiments such as that presented in B and is shown as the mean ± S.D. FAK Tyr-397 phosphorylation in PTPα+/+ cells plated on FN for 30 min was taken as 100%, and the other data was calculated relative to this.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Integrin-stimulated FAK Tyr-397 phosphorylation is impaired in PTPα- cells. (A) Reduced tyrosine phosphorylation of FAK in PTPα−/− cells. FAK immunoprecipitates from PTPα+/+ and PTPα−/− cells adhering to plastic dishes (on dish), retained in suspension (susp), or plated onto FN-coated dishes for 30 min (FN30), were probed with anti-phosphotyrosine antibodies (top panel) or with anti-FAK antibodies (bottom panel). (B) Reduced FAK Tyr-397 phosphorylation in PTPα−/− cells. FAK immunoprecipitates from PTPα+/+ and PTPα−/− cells retained in suspension (susp) or plated onto FN-coated dishes for 30 min (FN30) or 60 min (FN60) were probed with anti-phospho-Tyr397–specific antibodies (top panel), or anti-FAK antibodies (bottom panel). (C) Integrin-stimulated FAK Tyr-397 phosphorylation was quantitated from at least seven independent experiments such as that presented in B and is shown as the mean ± S.D. FAK Tyr-397 phosphorylation in PTPα+/+ cells plated on FN for 30 min was taken as 100%, and the other data was calculated relative to this.
Mentions: As src and fyn bind to phospho-Tyr397 of FAK, reduced FAK Tyr-397 autophosphorylation in the PTPα−/− cells could account for less src/fyn binding. The overall phosphotyrosine content of FAK was less in PTPα−/− cells than in PTPα+/+ cells, both under normal culture conditions and after plating on FN (Fig. 4 A). The phosphorylation status of FAK Tyr-397 was examined using an anti-FAK phospho-Tyr397–specific antibody. No phosphorylation of FAK Tyr-397 was detected in any cells in suspension. The phosphorylation of Tyr397 of FAK was consistently observed to be reduced in PTPα−/− cells, compared with wild-type cells, on FN-induced integrin activation (Fig. 4, B and C).

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