JCB: Article Y G O L O I B L L E C F O L A N R U O J E H T Invasive matrix degradation at focal adhesions occurs via protease recruitment by a FAK–p130Cas complex Yu Wang and Mark A. McNiven Department of Biochemistry and Molecular Biology, and the Center for Basic Research in Digestive Diseases, Mayo Clinic and Graduate School, Rochester, MN 55905 T tion of extracellular matrix (ECM) are two essential umor cell migration and the concomitant degrada- steps in the metastatic process. It is well established that focal adhesions (FAs) play an important role in regu- lating migration; however, whether these structures con- tribute to matrix degradation is not clear. In this study, we report that multiple cancer cell lines display degradation of ECM at FA sites that requires the targeted action of MT1-MMP. Importantly, we have found that this MT1-MMP targeting is dependent on an association with a FAK– p130Cas complex situated at FAs and is regulated by Src-mediated phosphorylation of Tyr 573 at the cytoplas- mic tail of MT1. Disrupting the FAK–p130Cas–MT1 com- plex significantly impairs FA-mediated degradation and tumor cell invasion yet does not appear to affect invado- podia formation or function. These findings demonstrate a novel function for FAs and also provide molecular insights into MT1-MMP targeting and function. Introduction It is well known that invading tumor cells are surrounded by stroma and extracellular matrix (ECM) that is either displaced or degraded during the metastatic process. This degradative action is mediated by specialized organelles termed “invadopodia” that are centrally situated, dot-shaped actin-rich membrane pro- trusions that extend into the matrix-filled surroundings (Linder, 2007; Buccione et al., 2009; Caldieri et al., 2009). Active inva- dopodia are known to markedly potentiate invasion and are a hallmark of many types of cancer cells (Linder, 2007). Inva- dopodia perform both secretory and endocytic processes by mediating the targeted release of zinc-dependent matrix metal- loproteinases (MMPs; Egeblad and Werb, 2002; Caldieri and Buccione, 2010) at the cell base and the subsequent internal- ization of the digested matrix for further processing by the lyso- some (Coopman et al., 1996, 1998). Thus, cytoskeletal and membrane dynamics play an essential role in supporting inva- dopodia function (Gimona et al., 2008; Caldieri et al., 2009). Both maturation and function of invadopodia are MMP dependent (Artym et al., 2006). To date, 25 MMPs have been identified in humans and are classified into either secreted MMPs or membrane-type MMPs (MT-MMPs). These two fam- ilies share many structural elements except that MT-MMPs contain transmembrane or other membrane-tethering domains (Egeblad and Werb, 2002), and both have been identified at invadopodia. Among the known MMPs, MT1-MMP (MMP-14) is the best characterized and is believed to be the most preva- lent form (Sabeh et al., 2009). MT1-MMP contains a single transmembrane domain, a 20-amino acid cytoplasmic tail, and is up-regulated in many types of cancer (Egeblad and Werb, 2002; Sato et al., 2005). Inhibition of MT1-MMP has been shown to significantly impair tumor cell invasion; how- ever, the spatial and temporal regulation of MT1-MMP is not fully understood. Although invadopodia are the primary site of action for MT1-MMP, it is also believed to function at other cellular loca- tions such as lamellipodia where it is recruited via an interaction with CD44 (Mori et al., 2002). Moreover, Takino et al. (2006, 2007) have shown that in MT1-MMP–overexpressing Hela cells, ECM degradation takes place at the leading edge and in close proximity to focal adhesions (FAs). This is particularly interesting as FAs and invadopodia are related organelles that share many structural and regulatory components. In contrast to the dot-shaped invadopodia, FAs are streak-like structures at the basal membrane that bridge the actin cytoskeleton to the ECM, Correspondence to Mark A. McNiven: mcniven.mark@mayo.edu Abbreviations used in this paper: FA, focal adhesion; FAK, focal adhesion kinase; FAT, focal adhesion targeting; MMP, matrix metalloproteinase; PRR, proline-rich region. © 2012 Wang and McNiven This article is distributed under the terms of an Attribution– Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). The Rockefeller University Press $30.00 J. Cell Biol. Vol. 196 No. 3 375–385 www.jcb.org/cgi/doi/10.1083/jcb.201105153 JCB 375