Invasive matrix degradation at focal adhesions occurs via protease recruitment by a 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.
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
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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.
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.