Figure 4. Interaction between MT1 and FAK is mediated by p130Cas. (a) To test if p130Cas adapts MT1 to FAK, 293T cells were transfected with MT1-GFP alone or together with HA-p130Cas, and the homogenates were incubated with the GST-PRR domain of FAK. Although GST-PRR exhibited modest interaction with MT1, this was markedly increased when p130Cas was coexpressed. (b) To further test p130Cas as an adaptor between MT1 and FAK, HT-1080 cells were treated with control or p130Cas siRNA, then lysed and IP with a FAK antibody. Although both p130Cas and MT1 were coprecipitated in control cells, MT1 coIP was significantly reduced in p130Cas knockdown cells, suggesting p130Cas links MT1 to FAK. (c) Illustration of the domains of full-length Cas and the truncation mutants used to study MT1 interaction. SP, SH3 and proline rich domain; SD, substrate domain; SC, serine-rich region and C terminus. (d and e) MT1-GFP was coexpressed with the indicated SFB-tagged Cas mutants in 293T cells, and IP with S-beads. The substrate domain alone is sufficient to interact with MT1 (d), whereas deletion of this domain abolished Cas–MT1 interaction (e). To test if p130Cas is required for ECM degrada- tion at FAs, HT-1080 cells were treated with p130Cas siRNA (f) and plated on gelatin for 16 h. p130Cas knockdown significantly reduced degradation at FAs (g). Re-expression of Cas WT (h) but not the MT1-binding mutant (i) rescued degradation at FAs. (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. (Gingras et al., 2008; Gonzalo et al., 2010) and to the second and third PXXP motifs of FAK is p130Cas (Harte et al., 1996; Mitra et al., 2005). However, whether these three proteins form a functional complex is not clear. To test this possibility, we transfected 293T cells with either MT1 alone or together with p130Cas and then performed pull-down assays with the puri- fied GST-PRR of FAK. As shown in Fig. 4 a, p130Cas strongly binds to GST-PRR, and overexpression of p130Cas signifi- cantly enhances the amount of MT1-MMP in the pulldown, suggesting that p130Cas facilitates MT1–FAK interaction. Selective recruitment of MT1-MMP to focal adhesions • Wang and McNiven 379