Figure 3. The cytoplasmic tail of the MT1 associ- ates with FAK. (a) Cartoon illustrating the domain structures of MT1-MMP that includes the Pro pep- tide, catalytic domain, hemopexin domain (Hpx), transmembrane domain (TM), and a cytoplasmic tail (CP) of only 20 amino acids. (b) To test if FAK interacts with the MT1 cytoplasmic tail, PANC-1 cells were transfected with either full-length MT1 or MT1 missing the cytoplasmic tail (CP), and subjected to a pull-down assay with GST-FRNK. Although a large amount of MT1 WT was pulled down by GST-FRNK, almost none of MT1CP ap- peared to associate. To test if MT1CP remains competent to degrade matrix at FAs, PANC-1 cells were plated and viewed as performed in Fig 1 f. The MT1CP-expressing cells failed to degrade any matrix (c) whereas cells expressing an MT1 CP fused with FAT domain, known to target pro- teins to FAs, exhibited excessive degradation at FA sites (d, arrow), suggesting that the MT1CP protein has defective targeting but not enzymatic activity. (e) The percentages of cells that degrade ECM at FAs were quantified. Values represent the mean of three independent experiments ± SD. Bars, 10 µm. FAK WT or a PRR mutant were expressed in 293T cells, followed by a FAK IP with S protein beads. As predicted, FAKPRR did not interact with MT1 (Fig. 2 e, lane 3). Within the FAK PRR domain the most characteristic features are two conserved PXXP motifs (second and third in the full- length protein; Mitra et al., 2005; Wu et al., 2005). Mutations of these two motifs (FAK P2/3A) did not fully abolish, but significantly impaired FAK’s interaction with MT1-MMP (Fig. 2 e, lane 4). Taken together, these data suggest that two PXXP motifs within the PPR domain of FAK are essential to interact with MT1-MMP. To test if FAK–MT1 association is important for matrix degradation at FA sites, HT-1080 cells were treated with FAK siRNA to knock down endogenous FAK (Fig. 2 f), then were plated on gelatin-coated coverslips for 16 h before IF analysis. Interestingly, degradation at FAs was significantly compro- mised in FAK knockdown cells (Fig. 2 g). To further confirm the role of FAK, FAK knockdown cells were rescued with siRNA-resistant rat-FAK. Surprisingly, WT FAK did not sig- nificantly increase degradation at FAs; however, expression of a FAK Y397F mutant rescued degradation to control levels (Fig. 2 h). As discussed later, this result is likely due to the fact that expression of WT FAK promotes FA turnover while the YF mutant is known to stabilize this structure (Hamadi et al., 2005; Wang et al., 2011). Accordingly, expression of a FAK double mutant, Y397F carrying the P2/3A mutation that re- duces MT1 binding, did not rescue matrix degradation at FAs (Fig. 2 i). Taken together, we conclude that FAK interacts with MT1-MMP through the PRR domain and this interaction is important for FA-mediated degradation. The cytoplasmic tail of MT1-MMP interacts with FAK indirectly through p130Cas MT1-MMP is a transmembrane protein with a 20-amino acid cytoplasmic tail (CP; Fig. 3 a) that is known to interact with a variety of cellular proteins such as MTCBP-1 and AP2 (Uekita et al., 2004). To test if FAK also interacts with this domain, we generated a MT1 mutant missing the cytoplasmic tail (MT1CP) and analyzed its interaction with FAK via GST pull-down assay. As predicted, removal of the CP domain completely abolished the FAK–MT1 interaction (Fig. 3 b), thus this mutant is likely defective in mediating degradation at FAs. Indeed, in contrast to MT1 WT (Fig. 1 f and Fig. S4 c), MT1 CP express- ing PANC-1 cells failed to generate any gelatin degradation at FAs (Fig. 3c). As this effect could result from defects in either MT1 targeting or enzymatic activity, we generated a chi- meric protein by replacing the MT1 cytoplasmic tail with the focal adhesion targeting domain of FAK (FAT). Thus, the cytoplasmic tail truncated protease could be constitutively targeted to FAs. As shown in Fig. 3 d, PANC-1 cells expressing MT1CP-FAT generated excessive degradation at FA sites (Fig. 3 e), indicating that MT1CP remains enzymatically active but cannot be targeted to FAs. Interaction between MT1-MMP and FAK is essential for FA-centric matrix degradation; however, whether this inter- action 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 de- tected (unpublished data), suggesting the participation of an adaptor. One candidate known to bind directly to both MT1 378 JCB • VOLUME 196 • NUMBER 3 • 2012