Figure 6. ECM degradation at FAs and in- vadopodia are differentially regulated. (a–c) To test if MT1-MMP, FAK, and p130Cas are required for invadopodia formation and func- tion, BxPC-3 cells were treated with siRNA against these proteins for 72 h, followed by plating on gelatin for 16 h. Cells were stained for cortactin as an invadopodia marker. (a) MT1-MMP knockdown (dashed line) signifi- cantly impaired invadopodia formation and ECM degradation. Importantly, in contrast to FA-based degradation, FAK (b) or p130Cas (c) knockdown had no effect on invadopodia function. (d and e) To confirm that Src kinase regulates invadopodia formation and function, BxPC-3 cells were plated on gelatin in the presence of DMSO or 10 µM PP2 for 16 h. Cells were then stained for actin as an inva- dopodia marker. PP2 completely abolished invadopodia formation and degradation. (f) The percentages of cells that degrade ECM at invadopodia were quantified. Data rep- resent results from three independent experi- ments ± SD. Bars, 10 µm. tyrosine phosphorylation of MT1-MMP when coexpressed with active Src (Y530F; Fig. 5 a). As expression of an MT1-Y573F mutant did not eliminate the phospho-tyrosine signal (un- published data), we assume that other tyrosine substrates exist on MT1-MMP. Importantly, however, phosphorylation of Y573 appears to facilitate the MT1–p130Cas interaction as the MT1 Y573F mutant exhibited a significantly reduced affinity to p130Cas compared with the MT1 WT in a Co-IP assay (Fig. 5 b). To confirm a role for Src, HT-1080 cells were treated with the Src family kinase inhibitor PP2 (10 µM, 3 h) before IP with p130Cas antibody. As shown in Fig. 5 c, PP2 treatment significantly reduced p130Cas–MT1 interaction compared with the DMSO control. Because p130Cas adapts MT1-MMP to FAK, we predicted that a Y573F mutant should also reduce the MT1–FAK interaction. This was indeed the case, as confirmed by both Co-IP and GST pull-down assay (Fig. S4, a and b). To test if Src activity is important for ECM degradation at FAs, HT-1080 cells were plated on gelatin in the presence of 10 µM PP2. As expected, PP2 treatment significantly inhibited ECM degradation at FAs (Fig. 5, e–e’). Consistent with this result, PANC-1 cells expressing MT1 Y573F showed a reduced FA-type degradation compared with MT1 WT–expressing cells (Fig. S4, c–d’). Taken together, these data suggest that Src regulates matrix degradation at FAs by phos- phorylating MT1-MMP on Y573 that promotes MT1’s inter- action with the FAK–p130Cas complex. ECM degradation at FAs and invadopodia are differentially regulated The observations described above suggest that Src-mediated formation of a FAK–p130Cas–MT1 complex regulates ECM degradation at FAs. It is intriguing to test if invadopodia are regulated by the same mechanism. It is well established that Src kinase is a central regulator of invadopodia (Courtneidge et al., 2005). Surprisingly the numerous, centrally located degradation sites generated by the HT-1080 cells were not attenuated by PP2 treatment (Fig. 5 e). Importantly, these central sites did not co-stain for invadopodial markers such as actin and cortactin (Fig. S5, a and c), suggesting that these are distinct structures. In contrast, BxPC-3 cells, which do form numerous classical Selective recruitment of MT1-MMP to focal adhesions • Wang and McNiven 381