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Invasive matrix degradation at focal adhesions occurs via protease recruitment by a FAK-p130Cas complex.

Wang Y, McNiven MA - J. Cell Biol. (2012)

Bottom Line: Importantly, we have found that this MT1-MMP targeting is dependent on an association with a FAK-p130Cas complex situated at FAs and is regulated by Src-mediated phosphorylation of Tyr 573 at the cytoplasmic tail of MT1.Disrupting the FAK-p130Cas-MT1 complex significantly impairs FA-mediated degradation and tumor cell invasion yet does not appear to affect invadopodia formation or function.These findings demonstrate a novel function for FAs and also provide molecular insights into MT1-MMP targeting and function.

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Affiliation: Department of Biochemistry and Molecular Biology, and the Center for Basic Research in Digestive Diseases, Mayo Clinic and Graduate School, Rochester, MN 55905, USA.

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ECM degradation at FAs and invadopodia are differentially regulated. (a–c) To test if MT1-MMP, FAK, and p130Cas are required for invadopodia formation and function, BxPC-3 cells were treated with siRNA against these proteins for 72 h, followed by plating on gelatin for 16 h. Cells were stained for cortactin as an invadopodia marker. (a) MT1-MMP knockdown (dashed line) significantly impaired invadopodia formation and ECM degradation. Importantly, in contrast to FA-based degradation, FAK (b) or p130Cas (c) knockdown had no effect on invadopodia function. (d and e) To confirm that Src kinase regulates invadopodia formation and function, BxPC-3 cells were plated on gelatin in the presence of DMSO or 10 µM PP2 for 16 h. Cells were then stained for actin as an invadopodia marker. PP2 completely abolished invadopodia formation and degradation. (f) The percentages of cells that degrade ECM at invadopodia were quantified. Data represent results from three independent experiments ± SD. Bars, 10 µm.
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fig6: ECM degradation at FAs and invadopodia are differentially regulated. (a–c) To test if MT1-MMP, FAK, and p130Cas are required for invadopodia formation and function, BxPC-3 cells were treated with siRNA against these proteins for 72 h, followed by plating on gelatin for 16 h. Cells were stained for cortactin as an invadopodia marker. (a) MT1-MMP knockdown (dashed line) significantly impaired invadopodia formation and ECM degradation. Importantly, in contrast to FA-based degradation, FAK (b) or p130Cas (c) knockdown had no effect on invadopodia function. (d and e) To confirm that Src kinase regulates invadopodia formation and function, BxPC-3 cells were plated on gelatin in the presence of DMSO or 10 µM PP2 for 16 h. Cells were then stained for actin as an invadopodia marker. PP2 completely abolished invadopodia formation and degradation. (f) The percentages of cells that degrade ECM at invadopodia were quantified. Data represent results from three independent experiments ± SD. Bars, 10 µm.

Mentions: The observations described above suggest that Src-mediated formation of a FAK–p130Cas–MT1 complex regulates ECM degradation at FAs. It is intriguing to test if invadopodia are regulated by the same mechanism. It is well established that Src kinase is a central regulator of invadopodia (Courtneidge et al., 2005). Surprisingly the numerous, centrally located degradation sites generated by the HT-1080 cells were not attenuated by PP2 treatment (Fig. 5 e). Importantly, these central sites did not co-stain for invadopodial markers such as actin and cortactin (Fig. S5, a and c), suggesting that these are distinct structures. In contrast, BxPC-3 cells, which do form numerous classical invadopodia (Fig. S5, b and d), exhibited a marked inhibition in the formation of centrally located ECM degradation sites by PP2 treatment (Fig. 6, d and e). Therefore, we tested if a disruption of the FAK–p130Cas–MT1 complex in BxPC-3 cells affects ECM degradation at invadopodia as it did at FAs (Figs. 2 and 4). BxPC3 cells were treated with siRNAs against MT1, FAK, or p130Cas, then plated on gelatin for 16 h, and stained for the invadopodia marker cortactin (Fig. 6, a–c). As expected, the MT1 knockdown affected invadopodia-type degradation due to an inhibition of invadopodia formation (Fig. 6 a). In contrast, FAK or p130Cas knockdown did not affect invadopodia function (Fig. 6, b and c), suggesting that, unlike that observed at FAs, MT1-MMP’s function at invadopodia is independent of the FAK–p130Cas complex.


Invasive matrix degradation at focal adhesions occurs via protease recruitment by a FAK-p130Cas complex.

Wang Y, McNiven MA - J. Cell Biol. (2012)

ECM degradation at FAs and invadopodia are differentially regulated. (a–c) To test if MT1-MMP, FAK, and p130Cas are required for invadopodia formation and function, BxPC-3 cells were treated with siRNA against these proteins for 72 h, followed by plating on gelatin for 16 h. Cells were stained for cortactin as an invadopodia marker. (a) MT1-MMP knockdown (dashed line) significantly impaired invadopodia formation and ECM degradation. Importantly, in contrast to FA-based degradation, FAK (b) or p130Cas (c) knockdown had no effect on invadopodia function. (d and e) To confirm that Src kinase regulates invadopodia formation and function, BxPC-3 cells were plated on gelatin in the presence of DMSO or 10 µM PP2 for 16 h. Cells were then stained for actin as an invadopodia marker. PP2 completely abolished invadopodia formation and degradation. (f) The percentages of cells that degrade ECM at invadopodia were quantified. Data represent results from three independent experiments ± SD. Bars, 10 µm.
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fig6: ECM degradation at FAs and invadopodia are differentially regulated. (a–c) To test if MT1-MMP, FAK, and p130Cas are required for invadopodia formation and function, BxPC-3 cells were treated with siRNA against these proteins for 72 h, followed by plating on gelatin for 16 h. Cells were stained for cortactin as an invadopodia marker. (a) MT1-MMP knockdown (dashed line) significantly impaired invadopodia formation and ECM degradation. Importantly, in contrast to FA-based degradation, FAK (b) or p130Cas (c) knockdown had no effect on invadopodia function. (d and e) To confirm that Src kinase regulates invadopodia formation and function, BxPC-3 cells were plated on gelatin in the presence of DMSO or 10 µM PP2 for 16 h. Cells were then stained for actin as an invadopodia marker. PP2 completely abolished invadopodia formation and degradation. (f) The percentages of cells that degrade ECM at invadopodia were quantified. Data represent results from three independent experiments ± SD. Bars, 10 µm.
Mentions: The observations described above suggest that Src-mediated formation of a FAK–p130Cas–MT1 complex regulates ECM degradation at FAs. It is intriguing to test if invadopodia are regulated by the same mechanism. It is well established that Src kinase is a central regulator of invadopodia (Courtneidge et al., 2005). Surprisingly the numerous, centrally located degradation sites generated by the HT-1080 cells were not attenuated by PP2 treatment (Fig. 5 e). Importantly, these central sites did not co-stain for invadopodial markers such as actin and cortactin (Fig. S5, a and c), suggesting that these are distinct structures. In contrast, BxPC-3 cells, which do form numerous classical invadopodia (Fig. S5, b and d), exhibited a marked inhibition in the formation of centrally located ECM degradation sites by PP2 treatment (Fig. 6, d and e). Therefore, we tested if a disruption of the FAK–p130Cas–MT1 complex in BxPC-3 cells affects ECM degradation at invadopodia as it did at FAs (Figs. 2 and 4). BxPC3 cells were treated with siRNAs against MT1, FAK, or p130Cas, then plated on gelatin for 16 h, and stained for the invadopodia marker cortactin (Fig. 6, a–c). As expected, the MT1 knockdown affected invadopodia-type degradation due to an inhibition of invadopodia formation (Fig. 6 a). In contrast, FAK or p130Cas knockdown did not affect invadopodia function (Fig. 6, b and c), suggesting that, unlike that observed at FAs, MT1-MMP’s function at invadopodia is independent of the FAK–p130Cas complex.

Bottom Line: Importantly, we have found that this MT1-MMP targeting is dependent on an association with a FAK-p130Cas complex situated at FAs and is regulated by Src-mediated phosphorylation of Tyr 573 at the cytoplasmic tail of MT1.Disrupting the FAK-p130Cas-MT1 complex significantly impairs FA-mediated degradation and tumor cell invasion yet does not appear to affect invadopodia formation or function.These findings demonstrate a novel function for FAs and also provide molecular insights into MT1-MMP targeting and function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, and the Center for Basic Research in Digestive Diseases, Mayo Clinic and Graduate School, Rochester, MN 55905, USA.

Show MeSH
Related in: MedlinePlus