2940 Research Article Met receptor tyrosine kinase signals through a cortactin–Gab1 scaffold complex, to mediate invadopodia Charles V. Rajadurai1,2, Serhiy Havrylov2,3,*, Kossay Zaoui2,3,*, Richard Vaillancourt1,2, Matthew Stuible1,2, Monica Naujokas2, Dongmei Zuo2, Michel L. Tremblay1,2 and Morag Park1,2,3,4,` 1Department of Biochemistry, McGill University, Montre´al, Que´bec H3A 1Y6, Canada 2Rosalind and Morris Goodman Cancer Research Centre, McGill University, Montre´al, Que´bec H3A 1A3, Canada 3Department of Medicine, McGill University, Montre´al, Que´bec H3A 1A1, Canada 4Department of Oncology, McGill University, Montre´al, Que´bec H3A 1A1, Canada *These authors contributed equally to this work `Author for correspondence ([email protected]) Accepted 23 January 2012 Journal of Cell Science 125, 2940–2953 ß 2012. Published by The Company of Biologists Ltd doi: 10.1242/jcs.100834 Summary Invasive carcinoma cells form actin-rich matrix-degrading protrusions called invadopodia. These structures resemble podosomes produced by some normal cells and play a crucial role in extracellular matrix remodeling. In cancer, formation of invadopodia is strongly associated with invasive potential. Although deregulated signals from the receptor tyrosine kinase Met (also known as hepatocyte growth factor are linked to cancer metastasis and poor prognosis, its role in invadopodia formation is not known. Here we show that stimulation of breast cancer cells with the ligand for Met, hepatocyte growth factor, promotes invadopodia formation, and in aggressive gastric tumor cells where Met is amplified, invadopodia formation is dependent on Met activity. Using both GRB2- associated-binding protein 1 (Gab1)-null fibroblasts and specific knockdown of Gab1 in tumor cells we show that Met-mediated invadopodia formation and cell invasion requires the scaffold protein Gab1. By a structure–function approach, we demonstrate that two proline-rich motifs (P4/5) within Gab1 are essential for invadopodia formation. We identify the actin regulatory protein, cortactin, as a direct interaction partner for Gab1 and show that a Gab1–cortactin interaction is dependent on the SH3 domain of cortactin and the integrity of the P4/5 region of Gab1. Both cortactin and Gab1 localize to invadopodia rosettes in Met-transformed cells and the specific uncoupling of cortactin from Gab1 abrogates invadopodia biogenesis and cell invasion downstream from the Met receptor tyrosine kinase. Met localizes to invadopodia along with cortactin and promotes phosphorylation of cortactin. These findings provide insights into the molecular mechanisms of invadopodia formation and identify Gab1 as a scaffold protein involved in this process. Journal of Cell Science Key words: Invadopodia, Met RTK, Gab1, Cortactin, Matrix remodeling, Cell invasion Introduction Molecular mechanisms leading to invadopodia biogenesis are Metastasis is the major cause of cancer-related mortality. During only beginning to emerge. Invadopodia-like cellular structures the initial steps of metastatic dissemination, some cancer cells with the capacity to degrade ECM were originally identified in acquire the ability to remodel extracellular matrix (ECM), invade chicken embryonic fibroblasts transformed by Rous sarcoma surrounding tissue locally, intravasate into lymphatic and blood virus (Chen, 1989), and were linked with constitutive activation microvasculature by breaking basement membranes (BM) of the of the v-Src oncogene (Hauck et al., 2002). Since then, many vessels and extravasate at distant sites (Chaffer and Weinberg, studies have established a role for increased Src kinase activity in 2011). Enhanced invasive capacity of many such cancer cells, the formation of invadopodia in cancer cells and in invadopodia- in particular carcinomas, is linked to their ability to form like structures of transformed fibroblasts, which are often invadopodia, specialized actin-rich membrane protrusions that referred to as podosomes (Ayala et al., 2009; Bowden et al., penetrate and remodel the ECM (Buccione et al., 2009; Gimona, 2006; Webb et al., 2007; Oikawa et al., 2008; Balzer et al., 2010; 2008), and that are much like podosomes formed in macrophages Kelley et al., 2010; Mader et al., 2011). and osteoclasts (Linder, 2007). Consequently, these invasive In addition to Src, other non-receptor tyrosine kinases, Abl cancer cells can use invadopodia as functional structures to and Arg, localize to invadopodia, and are involved in biogenesis perforate the basement membranes and guide the cell body into of these cellular structures in MDA-MB-231 breast carcinoma blood vessels (Schoumacher et al., 2010). cells (Mader et al., 2011; Smith-Pearson et al., 2010). Activation of the epidermal growth factor (EGF) as well as platelet derived growth factor (PDGF) receptor tyrosine kinases (RTK) also This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Share Alike License (http://creativecommons.org/licenses/by-nc-sa/3.0), promotes invadopodia biogenesis (Eckert et al., 2011; Mader which permits unrestricted non-commercial use, distribution and reproduction in any medium et al., 2011). These findings raise the possibility that multiple provided that the original work is properly cited and all further distributions of the work or adaptation are subject to the same Creative Commons License terms. receptor tyrosine kinases, when deregulated in cancer, converge Met RTK induces invadopodia through Gab1 2941 signals to promote invadopodia biogenesis contributing to these structures and interacts directly with cortactin, a key metastatic progression. regulator of actin dynamics within invadopodia. We demonstrate The inner structure of invadopodia consists of an actin-rich core, that Met colocalizes with cortactin to invadopodia, and that Met the formation of which is regulated by actin regulatory proteins, activity contributes to increased tyrosine phosphorylation of protein kinases, as well as regulators of lipid metabolism (Murphy cortactin independent of Src kinase. By structure–function and Courtneidge, 2011). During invadopodia formation in analysis, we have established that a Gab1–cortactin interaction is response to EGF, actin polymerization is promoted following required for assembly of functional invadopodia, in response to cortactin tyrosine phosphorylation and localized release of the oncogenic Met signals. actin severing protein, cofilin (Oser et al., 2009). Src kinase also promotes tyrosine phosphorylation of cortactin (Bowden et al., Results 2006), as well as the scaffold protein Tks5 (Blouw et al., 2008; Tpr-Met induces formation of invadopodia rosettes in Seals et al., 2005). Tks5 recruits the adaptor protein, Nck, to form a fibroblasts trimeric complex (Stylli et al., 2009), which activates Wiscott– Recent studies have suggested that invasive and metastatic Aldrich syndrome protein (N-WASP) allowing recruitment of potential of cancer cells and malignantly transformed fibroblasts Arp2/3 to promote branched actin nucleation (Yamaguchi et al., is tightly linked with the ability of these cells to produce 2005). Downstream from Src, a Tks5 protein complex is recruited invadopodia (Gimona, 2008; Buccione et al., 2009; Schoumacher to phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2]-rich et al., 2010). Therefore it is possible that malignant phenotypes, membrane regions through its phox homology (PX) domain and tumorigenicity and metastatic potential of cells transformed by initiates invadopodia biogenesis (Oikawa et al., 2008). oncogenic variants of the Met receptor are at least in part due to The Met RTK [also known as hepatocyte growth factor the acquired ability of these cells to produce invadopodia or (HGF) receptor] is a proto-oncogene often implicated in cancer similar actin-rich proteolytically active membrane protrusions (Birchmeier et al., 2003). In normal tissues, Met and its ligand, that enable remodeling of ECM. To investigate this possibility, HGF, activate signals that induce epithelial cell dispersal, we used Fischer rat 3T3 (FR3T3) fibroblasts transformed with the epithelial remodeling and invasive growth, which are important oncogenic variant of the Met receptor, Tpr-Met. Upon Tpr-Met- during development (Birchmeier et al., 2003). Met exerts an mediated transformation, FR3T3 fibroblasts acquire many invasive morphogenic program primarily through the scaffold features of malignantly transformed cancer cells, including the protein GRB2-associated-binding protein 1 (Gab1). Gab1 contains ability to invade through the ECM, as well as to develop tumors a pleckstrin homology (PH) domain, which tethers Gab1 to and metastases in nude mice (Fixman et al., 1996; Fixman et al., membranes through interactions with PtdIns(3,4,5)P3 (Maroun 1997; Saucier et al., 2002). In line with our previous findings, et al., 1999) and is recruited to and phosphorylated on multiple control FR3T3 cells used in this study spread and formed tyrosine residues by an activated Met receptor (Birchmeier et al., a contact-inhibited monolayer in culture, whereas FR3T3 2003; Peschard et al., 2007). Upon phosphorylation, these residues fibroblasts transformed with Tpr-Met developed a distinct serve as docking sites for numerous SH2-domain-containing elongated cell morphology, formed foci, lost contact inhibition adaptor and signaling proteins, including Crk, Nck, P85 subunit and acquired increased migratory and invasive capacity (Fig. 1) Journal of Cell Science of PI3K, Shp2 and PLCc (Abella et