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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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Reduced association of Src-PTKs and FAK in PTPα−/− cells. (A) Reduced src/fyn-FAK interaction in PTPα−/− cells cultured on plastic dishes. Lysates (WCL) of PTPα+/+ and PTPα−/− cells cultured on plastic tissues culture dishes in serum-containing medium were resolved by SDS-PAGE and immunoblotted with anti-src antibodies (top left panel) or anti-fyn antibodies (top right panel). FAK immunoprecipitates were probed with anti-src antibodies (middle left panel), anti-fyn antibodies (middle right panel), or anti-FAK antibodies (bottom panels). HC indicates the antibody heavy chain. (B) Reduced src/fyn-FAK interaction in PTPα−/− cells plated on FN-coated plastic dishes. Lysates (WCL) were prepared from PTPα+/+ and PTPα−/− cells in suspension (susp) or after plating onto FN-coated dishes for 30 min (FN30) and probed with anti-FAK antibodies (top panel). Src (middle and bottom left panels) and fyn (middle and bottom right panels) immunoprecipitates prepared from the cell lysates were probed for the presence of FAK (middle panel), src (bottom left panel), or fyn (bottom right panel). HC indicates the antibody heavy chain.
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fig3: Reduced association of Src-PTKs and FAK in PTPα−/− cells. (A) Reduced src/fyn-FAK interaction in PTPα−/− cells cultured on plastic dishes. Lysates (WCL) of PTPα+/+ and PTPα−/− cells cultured on plastic tissues culture dishes in serum-containing medium were resolved by SDS-PAGE and immunoblotted with anti-src antibodies (top left panel) or anti-fyn antibodies (top right panel). FAK immunoprecipitates were probed with anti-src antibodies (middle left panel), anti-fyn antibodies (middle right panel), or anti-FAK antibodies (bottom panels). HC indicates the antibody heavy chain. (B) Reduced src/fyn-FAK interaction in PTPα−/− cells plated on FN-coated plastic dishes. Lysates (WCL) were prepared from PTPα+/+ and PTPα−/− cells in suspension (susp) or after plating onto FN-coated dishes for 30 min (FN30) and probed with anti-FAK antibodies (top panel). Src (middle and bottom left panels) and fyn (middle and bottom right panels) immunoprecipitates prepared from the cell lysates were probed for the presence of FAK (middle panel), src (bottom left panel), or fyn (bottom right panel). HC indicates the antibody heavy chain.

Mentions: Formation of a FAK–src complex is essential for full phosphorylation and activation of FAK (Owen et al., 1999; Schlaepfer et al., 1999; Ruest et al., 2000) and phosphorylation of FAK-associated substrates such as p130cas, which are known to be required for cell motility (Vuori et al., 1996; Klemke et al., 1998). FAK has also been found to associate with the Src-PTK fyn (Cobb et al., 1994; Cary et al., 1996). Thus, we examined the association of FAK with src and fyn in adherent PTPα−/− cells. Both src and fyn were complexed with FAK in wild-type cells, as evidenced by probing FAK immunoprecipitates with anti-src or -fyn antibodies (Fig. 3 A). However, a greatly decreased amount of src was found in association with FAK in PTPα−/− cells, amounting to 38% ± 8 (n = 3) of the src detected in association with FAK in wild-type cells. Furthermore, the association of fyn with FAK was abolished in PTPα−/− cells (Fig. 3 A). FAK-src and FAK-fyn association was also examined in cells after plating on FN. This time, the src or fyn was immunoprecipitated and immunoblotted to detect associated FAK. FAK was not complexed with src or fyn in either wild-type or PTPα−/− cells held in suspension. Plating of wild-type fibroblasts on the FN-coated dishes induced the association of src and fyn with FAK, but in PTPα−/− cells there was a reduced amount of FAK, or no FAK, detected in association with src or fyn, respectively (Fig. 3 B).


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)

Reduced association of Src-PTKs and FAK in PTPα−/− cells. (A) Reduced src/fyn-FAK interaction in PTPα−/− cells cultured on plastic dishes. Lysates (WCL) of PTPα+/+ and PTPα−/− cells cultured on plastic tissues culture dishes in serum-containing medium were resolved by SDS-PAGE and immunoblotted with anti-src antibodies (top left panel) or anti-fyn antibodies (top right panel). FAK immunoprecipitates were probed with anti-src antibodies (middle left panel), anti-fyn antibodies (middle right panel), or anti-FAK antibodies (bottom panels). HC indicates the antibody heavy chain. (B) Reduced src/fyn-FAK interaction in PTPα−/− cells plated on FN-coated plastic dishes. Lysates (WCL) were prepared from PTPα+/+ and PTPα−/− cells in suspension (susp) or after plating onto FN-coated dishes for 30 min (FN30) and probed with anti-FAK antibodies (top panel). Src (middle and bottom left panels) and fyn (middle and bottom right panels) immunoprecipitates prepared from the cell lysates were probed for the presence of FAK (middle panel), src (bottom left panel), or fyn (bottom right panel). HC indicates the antibody heavy chain.
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fig3: Reduced association of Src-PTKs and FAK in PTPα−/− cells. (A) Reduced src/fyn-FAK interaction in PTPα−/− cells cultured on plastic dishes. Lysates (WCL) of PTPα+/+ and PTPα−/− cells cultured on plastic tissues culture dishes in serum-containing medium were resolved by SDS-PAGE and immunoblotted with anti-src antibodies (top left panel) or anti-fyn antibodies (top right panel). FAK immunoprecipitates were probed with anti-src antibodies (middle left panel), anti-fyn antibodies (middle right panel), or anti-FAK antibodies (bottom panels). HC indicates the antibody heavy chain. (B) Reduced src/fyn-FAK interaction in PTPα−/− cells plated on FN-coated plastic dishes. Lysates (WCL) were prepared from PTPα+/+ and PTPα−/− cells in suspension (susp) or after plating onto FN-coated dishes for 30 min (FN30) and probed with anti-FAK antibodies (top panel). Src (middle and bottom left panels) and fyn (middle and bottom right panels) immunoprecipitates prepared from the cell lysates were probed for the presence of FAK (middle panel), src (bottom left panel), or fyn (bottom right panel). HC indicates the antibody heavy chain.
Mentions: Formation of a FAK–src complex is essential for full phosphorylation and activation of FAK (Owen et al., 1999; Schlaepfer et al., 1999; Ruest et al., 2000) and phosphorylation of FAK-associated substrates such as p130cas, which are known to be required for cell motility (Vuori et al., 1996; Klemke et al., 1998). FAK has also been found to associate with the Src-PTK fyn (Cobb et al., 1994; Cary et al., 1996). Thus, we examined the association of FAK with src and fyn in adherent PTPα−/− cells. Both src and fyn were complexed with FAK in wild-type cells, as evidenced by probing FAK immunoprecipitates with anti-src or -fyn antibodies (Fig. 3 A). However, a greatly decreased amount of src was found in association with FAK in PTPα−/− cells, amounting to 38% ± 8 (n = 3) of the src detected in association with FAK in wild-type cells. Furthermore, the association of fyn with FAK was abolished in PTPα−/− cells (Fig. 3 A). FAK-src and FAK-fyn association was also examined in cells after plating on FN. This time, the src or fyn was immunoprecipitated and immunoblotted to detect associated FAK. FAK was not complexed with src or fyn in either wild-type or PTPα−/− cells held in suspension. Plating of wild-type fibroblasts on the FN-coated dishes induced the association of src and fyn with FAK, but in PTPα−/− cells there was a reduced amount of FAK, or no FAK, detected in association with src or fyn, respectively (Fig. 3 B).

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