Figure 2. An interaction between FAK and MT1 is required for matrix degradation at focal adhe- sions. (a) To test for FA proteins that might interact with MT1, PANC-1 cells were transfected with vec- tors encoding either GFP alone or MT1-GFP then lysed and IP with a GFP antibody. The pellet was then subjected to Western blot analysis using anti- bodies against several major FA proteins. FAK is the most prominent MT1-interacting protein of those tested. (b) Illustration of FAK and the dele- tion constructs used to map FAK-MT1 binding. (c and d) Western blot analysis of homogenates from PANC-1 cells expressing MT1-GFP and incu- bated with different GST-FAK fragments for 2 h. Importantly, only portions of FAK that contain the PRR domain were able to interact with MT1. (e) To further define the importance of the PRR domain in MT1 binding, MT1-GFP was coexpressed with SFB-tagged FAK WT, the PRR domain deletion mutant (PRR), or point mutations that disrupt the second and third PXXP motif (P2/3A), followed by IP with S-protein beads. Deletion or mutation of the PRR domain significantly impaired FAK–MT1 interactions. To test if FAK is required for degrada- tion at FAs, HT-1080 cells were treated with FAK siRNA (f), then plated on gelatin for 16 h before IF analysis. FAK knockdown significantly reduced degradations at FAs (g). Re-expression of FAK Y397F rescued FA-type degradation (h), whereas FAK Y397F carrying P2/3A mutation (i) had only a minimal effect. (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. ones at FAs (Fig. 1 d). To further test the role of MT1-MMP in ECM degradation, we used a pancreatic cancer cell line (PANC-1) that does not express endogenous MT1 (Fig. 1 e). Although gelatin degradation was not observed by these cells even 24 h after plating (Fig. S2 b-b’), some FA-type degradations were observed in cells transfected with MT1-GFP just 2 h after plat- ing on gelatin (Fig. 1 f). The same findings were obtained by cells expressing either an internal mCherry-tagged (see Fig. 5 h) or a nontagged MT1, indicating that this protease is essential for FA-mediated degradation. A physical interaction between MT1-MMP and FAK is required for matrix degradation at focal adhesions As MT1-MMP mediates ECM degradation at FAs, it is likely that a FA component acts to bind and recruit this protease to these sites. To test this possibility, MT1-GFP was over- expressed in PANC-1 cells, then immunoprecipitated (IP) with a GFP antibody and blotted for several core FA proteins, in- cluding FAK, paxillin, vinculin, talin, and 1-integrin. Of all these proteins, FAK was by far the most prominent interacting partner (Fig. 2 a). As FAK localizes to FAs and is generally not considered an invadopodial component (Bowden et al., 2006; Chan et al., 2009), this FAK–MT1 interaction was pre- dicted to occur at FAs. To define this novel interaction, we generated a series of FAK truncation mutants as illustrated in Fig. 2 b. PANC-1 cells expressing MT1-GFP were lysed and incubated with different GST-tagged FAK domains to test for interaction. As shown in Fig. 2, c and d, the proline-rich region (PRR) of FAK is sufficient to pull down MT1. To confirm this finding, MT1-GFP and SFB-tagged (SFB: S pro- tein, FLAG, and streptavidin-binding peptide triple tagged) Selective recruitment of MT1-MMP to focal adhesions • Wang and McNiven 377