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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.

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.

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Interaction between MT1 and FAK is mediated by p130Cas. (a) To test if p130Cas adapts MT1 to FAK, 293T cells were transfected with MT1-GFP alone or together with HA-p130Cas, and the homogenates were incubated with the GST-PRR domain of FAK. Although GST-PRR exhibited modest interaction with MT1, this was markedly increased when p130Cas was coexpressed. (b) To further test p130Cas as an adaptor between MT1 and FAK, HT-1080 cells were treated with control or p130Cas siRNA, then lysed and IP with a FAK antibody. Although both p130Cas and MT1 were coprecipitated in control cells, MT1 coIP was significantly reduced in p130Cas knockdown cells, suggesting p130Cas links MT1 to FAK. (c) Illustration of the domains of full-length Cas and the truncation mutants used to study MT1 interaction. SP, SH3 and proline rich domain; SD, substrate domain; SC, serine-rich region and C terminus. (d and e) MT1-GFP was coexpressed with the indicated SFB-tagged Cas mutants in 293T cells, and IP with S-beads. The substrate domain alone is sufficient to interact with MT1 (d), whereas deletion of this domain abolished Cas–MT1 interaction (e). To test if p130Cas is required for ECM degradation at FAs, HT-1080 cells were treated with p130Cas siRNA (f) and plated on gelatin for 16 h. p130Cas knockdown significantly reduced degradation at FAs (g). Re-expression of Cas WT (h) but not the MT1-binding mutant (i) rescued degradation at FAs. (j) The percentages of cells (g–i) that degraded ECM at FAs were quantified. Values represent the mean of three independent experiments ± SD. Bars, 10 µm.
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fig4: Interaction between MT1 and FAK is mediated by p130Cas. (a) To test if p130Cas adapts MT1 to FAK, 293T cells were transfected with MT1-GFP alone or together with HA-p130Cas, and the homogenates were incubated with the GST-PRR domain of FAK. Although GST-PRR exhibited modest interaction with MT1, this was markedly increased when p130Cas was coexpressed. (b) To further test p130Cas as an adaptor between MT1 and FAK, HT-1080 cells were treated with control or p130Cas siRNA, then lysed and IP with a FAK antibody. Although both p130Cas and MT1 were coprecipitated in control cells, MT1 coIP was significantly reduced in p130Cas knockdown cells, suggesting p130Cas links MT1 to FAK. (c) Illustration of the domains of full-length Cas and the truncation mutants used to study MT1 interaction. SP, SH3 and proline rich domain; SD, substrate domain; SC, serine-rich region and C terminus. (d and e) MT1-GFP was coexpressed with the indicated SFB-tagged Cas mutants in 293T cells, and IP with S-beads. The substrate domain alone is sufficient to interact with MT1 (d), whereas deletion of this domain abolished Cas–MT1 interaction (e). To test if p130Cas is required for ECM degradation at FAs, HT-1080 cells were treated with p130Cas siRNA (f) and plated on gelatin for 16 h. p130Cas knockdown significantly reduced degradation at FAs (g). Re-expression of Cas WT (h) but not the MT1-binding mutant (i) rescued degradation at FAs. (j) The percentages of cells (g–i) that degraded ECM at FAs were quantified. Values represent the mean of three independent experiments ± SD. Bars, 10 µm.

Mentions: Interaction between MT1-MMP and FAK is essential for FA-centric matrix degradation; however, whether this interaction is direct was unclear. To test this, we purified His-MT1 protein from Escherichia coli, and performed direct binding analysis with GST-PRR of FAK. No direct interaction was detected (unpublished data), suggesting the participation of an adaptor. One candidate known to bind directly to both MT1 (Gingras et al., 2008; Gonzalo et al., 2010) and to the second and third PXXP motifs of FAK is p130Cas (Harte et al., 1996; Mitra et al., 2005). However, whether these three proteins form a functional complex is not clear. To test this possibility, we transfected 293T cells with either MT1 alone or together with p130Cas and then performed pull-down assays with the purified GST-PRR of FAK. As shown in Fig. 4 a, p130Cas strongly binds to GST-PRR, and overexpression of p130Cas significantly enhances the amount of MT1-MMP in the pulldown, suggesting that p130Cas facilitates MT1–FAK interaction. To test if these three proteins interact endogenously, FAK was immunoprecipitated from HT-1080 cells, and both p130Cas and MT1 were detected in the Co-IP (Fig. 4 b). Importantly, the FAK–MT1 interaction was attenuated in p130Cas knockdown cells (Fig. 4 b), suggesting that p130Cas mediates the interaction between MT1-MMP and FAK.


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

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

Interaction between MT1 and FAK is mediated by p130Cas. (a) To test if p130Cas adapts MT1 to FAK, 293T cells were transfected with MT1-GFP alone or together with HA-p130Cas, and the homogenates were incubated with the GST-PRR domain of FAK. Although GST-PRR exhibited modest interaction with MT1, this was markedly increased when p130Cas was coexpressed. (b) To further test p130Cas as an adaptor between MT1 and FAK, HT-1080 cells were treated with control or p130Cas siRNA, then lysed and IP with a FAK antibody. Although both p130Cas and MT1 were coprecipitated in control cells, MT1 coIP was significantly reduced in p130Cas knockdown cells, suggesting p130Cas links MT1 to FAK. (c) Illustration of the domains of full-length Cas and the truncation mutants used to study MT1 interaction. SP, SH3 and proline rich domain; SD, substrate domain; SC, serine-rich region and C terminus. (d and e) MT1-GFP was coexpressed with the indicated SFB-tagged Cas mutants in 293T cells, and IP with S-beads. The substrate domain alone is sufficient to interact with MT1 (d), whereas deletion of this domain abolished Cas–MT1 interaction (e). To test if p130Cas is required for ECM degradation at FAs, HT-1080 cells were treated with p130Cas siRNA (f) and plated on gelatin for 16 h. p130Cas knockdown significantly reduced degradation at FAs (g). Re-expression of Cas WT (h) but not the MT1-binding mutant (i) rescued degradation at FAs. (j) The percentages of cells (g–i) that degraded ECM at FAs were quantified. Values represent the mean of three independent experiments ± SD. Bars, 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig4: Interaction between MT1 and FAK is mediated by p130Cas. (a) To test if p130Cas adapts MT1 to FAK, 293T cells were transfected with MT1-GFP alone or together with HA-p130Cas, and the homogenates were incubated with the GST-PRR domain of FAK. Although GST-PRR exhibited modest interaction with MT1, this was markedly increased when p130Cas was coexpressed. (b) To further test p130Cas as an adaptor between MT1 and FAK, HT-1080 cells were treated with control or p130Cas siRNA, then lysed and IP with a FAK antibody. Although both p130Cas and MT1 were coprecipitated in control cells, MT1 coIP was significantly reduced in p130Cas knockdown cells, suggesting p130Cas links MT1 to FAK. (c) Illustration of the domains of full-length Cas and the truncation mutants used to study MT1 interaction. SP, SH3 and proline rich domain; SD, substrate domain; SC, serine-rich region and C terminus. (d and e) MT1-GFP was coexpressed with the indicated SFB-tagged Cas mutants in 293T cells, and IP with S-beads. The substrate domain alone is sufficient to interact with MT1 (d), whereas deletion of this domain abolished Cas–MT1 interaction (e). To test if p130Cas is required for ECM degradation at FAs, HT-1080 cells were treated with p130Cas siRNA (f) and plated on gelatin for 16 h. p130Cas knockdown significantly reduced degradation at FAs (g). Re-expression of Cas WT (h) but not the MT1-binding mutant (i) rescued degradation at FAs. (j) The percentages of cells (g–i) that degraded ECM at FAs were quantified. Values represent the mean of three independent experiments ± SD. Bars, 10 µm.
Mentions: Interaction between MT1-MMP and FAK is essential for FA-centric matrix degradation; however, whether this interaction is direct was unclear. To test this, we purified His-MT1 protein from Escherichia coli, and performed direct binding analysis with GST-PRR of FAK. No direct interaction was detected (unpublished data), suggesting the participation of an adaptor. One candidate known to bind directly to both MT1 (Gingras et al., 2008; Gonzalo et al., 2010) and to the second and third PXXP motifs of FAK is p130Cas (Harte et al., 1996; Mitra et al., 2005). However, whether these three proteins form a functional complex is not clear. To test this possibility, we transfected 293T cells with either MT1 alone or together with p130Cas and then performed pull-down assays with the purified GST-PRR of FAK. As shown in Fig. 4 a, p130Cas strongly binds to GST-PRR, and overexpression of p130Cas significantly enhances the amount of MT1-MMP in the pulldown, suggesting that p130Cas facilitates MT1–FAK interaction. To test if these three proteins interact endogenously, FAK was immunoprecipitated from HT-1080 cells, and both p130Cas and MT1 were detected in the Co-IP (Fig. 4 b). Importantly, the FAK–MT1 interaction was attenuated in p130Cas knockdown cells (Fig. 4 b), suggesting that p130Cas mediates the interaction between MT1-MMP and FAK.

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