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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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FA-mediated matrix degradation promotes tumor cell invasion. PANC-1 cells were transfected with MT1-MMP WT or mutants (a), and invasive potential was analyzed by the Boyden chamber assay in which filters were coated with 1 mg/ml Matrigel. Constructs that facilitate FA-type degradation (MT1-WT, ΔCP-FAT) significantly promoted invasion compared with the GFP vector or the MT1-Y573F mutant that cannot bind p130Cas (d). To test if a reduction of FAK and p130Cas exerts an inhibitory effect, HT-1080 cells were transfected with siRNA twice, and then rescued with either WT or mutant proteins (b and c). Although knockdown of either FAK or p130Cas significantly impaired invasion (e), this could be reversed by expression of WT proteins. Mutant proteins that cannot reconstitute the FAK–p130Cas–MT1 complex did not provide a functional rescue (e). The graphed data represent results from three independent experiments ± SD, and were normalized to the average of control cells (as 1). (f) Illustration depicting the central theme of this study: a FAK–p130Cas complex mediates the docking of MT1-MMP at FAs. MT1-MMP is trafficked to the surface via exocytotic vesicles for fusion with the membrane to release MT1 that can be recruited by the FAK–p130Cas complex at FAs. In addition, MT1 is recruited to lamellipodia by CD44 that may diffuse along the membrane for recruitment to FAs. Src kinase activity at FAs mediates MT1 phosphorylation at Y573 that facilitates an interaction with the FAK–p130Cas complex at FAs.
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fig7: FA-mediated matrix degradation promotes tumor cell invasion. PANC-1 cells were transfected with MT1-MMP WT or mutants (a), and invasive potential was analyzed by the Boyden chamber assay in which filters were coated with 1 mg/ml Matrigel. Constructs that facilitate FA-type degradation (MT1-WT, ΔCP-FAT) significantly promoted invasion compared with the GFP vector or the MT1-Y573F mutant that cannot bind p130Cas (d). To test if a reduction of FAK and p130Cas exerts an inhibitory effect, HT-1080 cells were transfected with siRNA twice, and then rescued with either WT or mutant proteins (b and c). Although knockdown of either FAK or p130Cas significantly impaired invasion (e), this could be reversed by expression of WT proteins. Mutant proteins that cannot reconstitute the FAK–p130Cas–MT1 complex did not provide a functional rescue (e). The graphed data represent results from three independent experiments ± SD, and were normalized to the average of control cells (as 1). (f) Illustration depicting the central theme of this study: a FAK–p130Cas complex mediates the docking of MT1-MMP at FAs. MT1-MMP is trafficked to the surface via exocytotic vesicles for fusion with the membrane to release MT1 that can be recruited by the FAK–p130Cas complex at FAs. In addition, MT1 is recruited to lamellipodia by CD44 that may diffuse along the membrane for recruitment to FAs. Src kinase activity at FAs mediates MT1 phosphorylation at Y573 that facilitates an interaction with the FAK–p130Cas complex at FAs.

Mentions: The observations described above have implicated a novel role for a FAK–p130Cas–MT1 complex in the degradation of ECM at FAs. To test if this FA-centric protein complex contributes to tumor cell invasion, an in vitro Boyden chamber invasion assay was implemented. As PANC-1 cells do not express endogenous MT1-MMP, cells were transfected to express MT1 WT, Y573F, or MT1ΔCP-FAT (Fig. 7 a). Cells were suspended in serum-free medium and plated in the upper chamber 24 h after transfection, then challenged to invade through a Matrigel-coated filter into the lower chamber containing full serum (10% FBS) medium. After a 36-h period, cells were fixed and the number of invaded cells per field was quantified. Importantly, the protein components that facilitate ECM degradation at FAs (MT1 WT and MT1ΔCP-FAT, Fig. 3 e) also significantly enhanced invasion through the filter compared with MT1 Y573F or GFP vector (Fig. 7 d). As a reciprocal approach, HT-1080 cells were transfected twice with siRNA against FAK or p130Cas, and subsequently rescued with either WT or mutant constructs (Fig. 7, b and c). Interestingly, cells with reduced levels of FAK or p130Cas exhibited significantly reduced invasion compared with siRNA control cells, which is consistent with previous reports (Ueda et al., 2003; Chan et al., 2009; Cunningham-Edmondson and Hanks, 2009). Moreover, reexpression of the respective WT proteins rescued invasion whereas expression of the mutants FAK P2/3A and CasΔSD that perturb formation of the trimeric complex did not (Fig. 7 e). Taken together, these data suggest that the targeting of MT1-MMP to FAs by the FAK–p130Cas complex is important for cancer cell invasion.


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

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

FA-mediated matrix degradation promotes tumor cell invasion. PANC-1 cells were transfected with MT1-MMP WT or mutants (a), and invasive potential was analyzed by the Boyden chamber assay in which filters were coated with 1 mg/ml Matrigel. Constructs that facilitate FA-type degradation (MT1-WT, ΔCP-FAT) significantly promoted invasion compared with the GFP vector or the MT1-Y573F mutant that cannot bind p130Cas (d). To test if a reduction of FAK and p130Cas exerts an inhibitory effect, HT-1080 cells were transfected with siRNA twice, and then rescued with either WT or mutant proteins (b and c). Although knockdown of either FAK or p130Cas significantly impaired invasion (e), this could be reversed by expression of WT proteins. Mutant proteins that cannot reconstitute the FAK–p130Cas–MT1 complex did not provide a functional rescue (e). The graphed data represent results from three independent experiments ± SD, and were normalized to the average of control cells (as 1). (f) Illustration depicting the central theme of this study: a FAK–p130Cas complex mediates the docking of MT1-MMP at FAs. MT1-MMP is trafficked to the surface via exocytotic vesicles for fusion with the membrane to release MT1 that can be recruited by the FAK–p130Cas complex at FAs. In addition, MT1 is recruited to lamellipodia by CD44 that may diffuse along the membrane for recruitment to FAs. Src kinase activity at FAs mediates MT1 phosphorylation at Y573 that facilitates an interaction with the FAK–p130Cas complex at FAs.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC3275373&req=5

fig7: FA-mediated matrix degradation promotes tumor cell invasion. PANC-1 cells were transfected with MT1-MMP WT or mutants (a), and invasive potential was analyzed by the Boyden chamber assay in which filters were coated with 1 mg/ml Matrigel. Constructs that facilitate FA-type degradation (MT1-WT, ΔCP-FAT) significantly promoted invasion compared with the GFP vector or the MT1-Y573F mutant that cannot bind p130Cas (d). To test if a reduction of FAK and p130Cas exerts an inhibitory effect, HT-1080 cells were transfected with siRNA twice, and then rescued with either WT or mutant proteins (b and c). Although knockdown of either FAK or p130Cas significantly impaired invasion (e), this could be reversed by expression of WT proteins. Mutant proteins that cannot reconstitute the FAK–p130Cas–MT1 complex did not provide a functional rescue (e). The graphed data represent results from three independent experiments ± SD, and were normalized to the average of control cells (as 1). (f) Illustration depicting the central theme of this study: a FAK–p130Cas complex mediates the docking of MT1-MMP at FAs. MT1-MMP is trafficked to the surface via exocytotic vesicles for fusion with the membrane to release MT1 that can be recruited by the FAK–p130Cas complex at FAs. In addition, MT1 is recruited to lamellipodia by CD44 that may diffuse along the membrane for recruitment to FAs. Src kinase activity at FAs mediates MT1 phosphorylation at Y573 that facilitates an interaction with the FAK–p130Cas complex at FAs.
Mentions: The observations described above have implicated a novel role for a FAK–p130Cas–MT1 complex in the degradation of ECM at FAs. To test if this FA-centric protein complex contributes to tumor cell invasion, an in vitro Boyden chamber invasion assay was implemented. As PANC-1 cells do not express endogenous MT1-MMP, cells were transfected to express MT1 WT, Y573F, or MT1ΔCP-FAT (Fig. 7 a). Cells were suspended in serum-free medium and plated in the upper chamber 24 h after transfection, then challenged to invade through a Matrigel-coated filter into the lower chamber containing full serum (10% FBS) medium. After a 36-h period, cells were fixed and the number of invaded cells per field was quantified. Importantly, the protein components that facilitate ECM degradation at FAs (MT1 WT and MT1ΔCP-FAT, Fig. 3 e) also significantly enhanced invasion through the filter compared with MT1 Y573F or GFP vector (Fig. 7 d). As a reciprocal approach, HT-1080 cells were transfected twice with siRNA against FAK or p130Cas, and subsequently rescued with either WT or mutant constructs (Fig. 7, b and c). Interestingly, cells with reduced levels of FAK or p130Cas exhibited significantly reduced invasion compared with siRNA control cells, which is consistent with previous reports (Ueda et al., 2003; Chan et al., 2009; Cunningham-Edmondson and Hanks, 2009). Moreover, reexpression of the respective WT proteins rescued invasion whereas expression of the mutants FAK P2/3A and CasΔSD that perturb formation of the trimeric complex did not (Fig. 7 e). Taken together, these data suggest that the targeting of MT1-MMP to FAs by the FAK–p130Cas complex is important for cancer cell invasion.

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