Figure 5. Src regulates formation of a FAK–p130Cas–MT1 complex. (a) To confirm MT1-MMP as a Src substrate, MT1-GFP was cotransfected with SrcY530F in 293T cells, IP with a GFP antibody, and blotted with a phosphor-Tyr antibody (pY20). MT1 was strongly phosphorylated by Src. (SE, short exposure; LE, long exposure). (b) To test if phosphorylation of Y573 at the MT1 cytoplasmic tail is important for binding to p130Cas, HA-Cas was cotrans- fected with MT1 WT, CP, or Y573F, followed by IP with an HA antibody. MT1CP did not interact with p130Cas, whereas MT1 Y573F significantly reduced the interaction. (c) To further test a role for Src in regulating MT1–p130Cas interactions, HT-1080 cells were treated with DMSO or 10 µM PP2 for 3 h before IP with p130Cas antibody, and PP2 treatment significantly reduced MT1–p130Cas binding. (d–e’) HT-1080 cells were plated on gelatin in the presence of DMSO or PP2 for 6 h. PP2 significantly attenuated FA-mediated degradation compared with DMSO control. (f) Percentages of PP2- or DMSO- treated cells that degraded matrix at FAs. Data represent results from three independent experiments ± SD. MT1-MMP is recruited to FAs and increased upon elevated Src activity. HT1080 cells were transfected with paxillin-GFP and stained for endogenous MT1 (g–j), or transfected with MT1-mCherry and plated onto gelatin for 6 h and stained for paxillin (h). Certain colocalization between MT1 and FA marker (g and h, arrows and inserts) or sites of degrada- tion (h) was observed. Same treatment was applied to cells coexpressing either MT1-GFP (i) or zyxin-dsRed (j) together with a constitutively active SrcY530F mutant. Importantly, SrcY530F markedly enhanced MT1’s recruitment to FAs (I and j, arrows and inserts). Bars, 10 µm. To test if these three proteins interact endogenously, FAK was immunoprecipitated from HT-1080 cells, and both p130Cas and MT1 were detected in the Co-IP (Fig. 4 b). Importantly, the FAK–MT1 interaction was attenuated in p130Cas knockdown cells (Fig. 4 b), suggesting that p130Cas mediates the inter- action between MT1-MMP and FAK. Although it is known that p130Cas binds to FAK via the N-terminal SH3 domain (Harte et al., 1996), and FAK recruits p130Cas to FAs (Fig. S3, a–b’), it remained unclear how p130Cas might interact with MT1. To address this question, a series of SFB-p130Cas truncation mutants were generated (Fig. 4 c) and coexpressed with MT1-GFP in 293T cells fol- lowed by Co-IP analysis. As shown in Fig. 4 d, the substrate domain (SD) of p130 Cas was sufficient to bind to MT1, and the deletion of this domain abolished the interaction (Fig. 4 e). Although p130Cas is an established signaling molecule at FA sites (O’Neill et al., 2000), its function during matrix degra- dation remains elusive. To test this premise, HT-1080 cells were treated with p130Cas siRNA for 72 h to knock down the endogenous protein (Fig. 4 f), and then were plated on gelatin- coated coverslips. As shown in Fig. 4 g, p130Cas knockdown significantly reduced FA-mediated degradation, while FA morphology remained unchanged (Fig. S3, c–c’). To further test p130Cas’s effect, p130Cas WT or SD (Fig. 4, h and i) were reexpressed and tested for matrix degradation. Importantly, p130Cas WT but not the MT1-binding mutant (SD) rescued ECM degradation at FAs. These data suggest that ECM degra- dation at FAs is regulated by a novel mechanism involving a FAK–p130Cas–MT1 complex. Src regulates FAK–p130Cas–MT1 complex formation and ECM degradation at focal adhesions Src kinase is a central signaling molecule known to promote ECM degradation (Yeatman, 2004; Courtneidge et al., 2005; Kelley et al., 2010) by phosphorylating a variety of substrates including Tyr573 on the cytoplasmic tail of MT1-MMP (Nyalendo et al., 2007). Consistent with this premise, we observed a marked 380 JCB • VOLUME 196 • NUMBER 3 • 2012