Met receptor tyrosine kinase signals through a cortactin–Gab1 scaffold complex, to mediate invadopodia

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Met receptor tyrosine kinase signals through acortactin–Gab1 scaffold complex, to mediateinvadopodia

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, Montreal, Quebec H3A 1Y6, Canada2Rosalind and Morris Goodman Cancer Research Centre, McGill University, Montreal, Quebec H3A 1A3, Canada3Department of Medicine, McGill University, Montreal, Quebec H3A 1A1, Canada4Department of Oncology, McGill University, Montreal, Quebec H3A 1A1, Canada

*These authors contributed equally to this work`Author for correspondence ([email protected])

Accepted 23 January 2012Journal of Cell Science 125, 2940–2953� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.100834

SummaryInvasive carcinoma cells form actin-rich matrix-degrading protrusions called invadopodia. These structures resemble podosomesproduced by some normal cells and play a crucial role in extracellular matrix remodeling. In cancer, formation of invadopodia isstrongly 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 weshow that stimulation of breast cancer cells with the ligand for Met, hepatocyte growth factor, promotes invadopodia formation, and inaggressive 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-mediatedinvadopodia formation and cell invasion requires the scaffold protein Gab1. By a structure–function approach, we demonstrate that twoproline-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 theintegrity of the P4/5 region of Gab1. Both cortactin and Gab1 localize to invadopodia rosettes in Met-transformed cells and the specificuncoupling 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 insightsinto the molecular mechanisms of invadopodia formation and identify Gab1 as a scaffold protein involved in this process.

Key words: Invadopodia, Met RTK, Gab1, Cortactin, Matrix remodeling, Cell invasion

IntroductionMetastasis is the major cause of cancer-related mortality. During

the initial steps of metastatic dissemination, some cancer cells

acquire the ability to remodel extracellular matrix (ECM), invade

surrounding tissue locally, intravasate into lymphatic and blood

microvasculature by breaking basement membranes (BM) of the

vessels and extravasate at distant sites (Chaffer and Weinberg,

2011). Enhanced invasive capacity of many such cancer cells,

in particular carcinomas, is linked to their ability to form

invadopodia, specialized actin-rich membrane protrusions that

penetrate and remodel the ECM (Buccione et al., 2009; Gimona,

2008), and that are much like podosomes formed in macrophages

and osteoclasts (Linder, 2007). Consequently, these invasive

cancer cells can use invadopodia as functional structures to

perforate the basement membranes and guide the cell body into

blood vessels (Schoumacher et al., 2010).

Molecular mechanisms leading to invadopodia biogenesis are

only beginning to emerge. Invadopodia-like cellular structures

with the capacity to degrade ECM were originally identified in

chicken embryonic fibroblasts transformed by Rous sarcoma

virus (Chen, 1989), and were linked with constitutive activation

of the v-Src oncogene (Hauck et al., 2002). Since then, many

studies have established a role for increased Src kinase activity in

the formation of invadopodia in cancer cells and in invadopodia-

like structures of transformed fibroblasts, which are often

referred to as podosomes (Ayala et al., 2009; Bowden et al.,

2006; Webb et al., 2007; Oikawa et al., 2008; Balzer et al., 2010;

Kelley et al., 2010; Mader et al., 2011).

In addition to Src, other non-receptor tyrosine kinases, Abl

and Arg, localize to invadopodia, and are involved in biogenesis

of these cellular structures in MDA-MB-231 breast carcinoma

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

promotes invadopodia biogenesis (Eckert et al., 2011; Mader

et al., 2011). These findings raise the possibility that multiple

receptor tyrosine kinases, when deregulated in cancer, converge

This is an Open Access article distributed under the terms of the Creative Commons AttributionNon-Commercial Share Alike License (http://creativecommons.org/licenses/by-nc-sa/3.0),which permits unrestricted non-commercial use, distribution and reproduction in any mediumprovided that the original work is properly cited and all further distributions of the work oradaptation are subject to the same Creative Commons License terms.

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signals to promote invadopodia biogenesis contributing tometastatic progression.

The inner structure of invadopodia consists of an actin-rich core,

the formation of which is regulated by actin regulatory proteins,protein kinases, as well as regulators of lipid metabolism (Murphyand Courtneidge, 2011). During invadopodia formation in

response to EGF, actin polymerization is promoted followingcortactin tyrosine phosphorylation and localized release of theactin severing protein, cofilin (Oser et al., 2009). Src kinase also

promotes tyrosine phosphorylation of cortactin (Bowden et al.,2006), as well as the scaffold protein Tks5 (Blouw et al., 2008;Seals et al., 2005). Tks5 recruits the adaptor protein, Nck, to form a

trimeric complex (Stylli et al., 2009), which activates Wiscott–Aldrich syndrome protein (N-WASP) allowing recruitment ofArp2/3 to promote branched actin nucleation (Yamaguchi et al.,2005). Downstream from Src, a Tks5 protein complex is recruited

to phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2]-richmembrane regions through its phox homology (PX) domain andinitiates invadopodia biogenesis (Oikawa et al., 2008).

The Met RTK [also known as hepatocyte growth factor(HGF) receptor] is a proto-oncogene often implicated in cancer(Birchmeier et al., 2003). In normal tissues, Met and its ligand,

HGF, activate signals that induce epithelial cell dispersal,epithelial remodeling and invasive growth, which are importantduring development (Birchmeier et al., 2003). Met exerts aninvasive morphogenic program primarily through the scaffold

protein GRB2-associated-binding protein 1 (Gab1). Gab1 containsa pleckstrin homology (PH) domain, which tethers Gab1 tomembranes through interactions with PtdIns(3,4,5)P3 (Maroun

et al., 1999) and is recruited to and phosphorylated on multipletyrosine residues by an activated Met receptor (Birchmeier et al.,2003; Peschard et al., 2007). Upon phosphorylation, these residues

serve as docking sites for numerous SH2-domain-containingadaptor and signaling proteins, including Crk, Nck, P85 subunitof PI3K, Shp2 and PLCc (Abella et al., 2010; Schaeper et al., 2000;

Garcia-Guzman et al., 1999; Gual et al., 2000; Lamorte et al.,2000; Maroun et al., 1999; Maroun et al., 2000; Cunnick et al.,2001). Gab1 contains six proline rich motifs, two of which(proline-rich motifs four and five) are implicated in the constitutive

association with the adaptor protein growth factor receptor-boundprotein 2 (Grb2) through its C-terminal SH3 domain (Lock et al.,2002). Once recruited to a Met–Gab1 complex, these proteins

trigger activation of multiple signaling cascades, including PI3K–Akt (Maroun et al., 1999), Ras–MAPK (Maroun et al., 2000;Schaeper et al., 2000), Rac and Rap1 (Lamorte et al., 2000) and

Nck/N-WASP (Abella et al., 2010) that promote cell survival,actin cytoskeleton remodeling, as well as increased migration andinvasion (Benvenuti and Comoglio, 2007; Birchmeier et al., 2003;

Lai et al., 2009; Peschard and Park, 2007).

Although aberrant activation of Met is linked to increasedcancer cell invasion and is a hallmark of aggressive tumors withpoor prognosis, the ability of Met to coordinate invadopodia

formation has not been addressed (Camp et al., 1999; Cruz et al.,2003; Kammula et al., 2007; Lengyel et al., 2005; Okuda et al.,2008; Sawada et al., 2007; Tuynman et al., 2008; Wu et al., 1998;

Ponzo et al., 2010). Here we demonstrate that an activated MetRTK promotes invadopodia in carcinoma cells and that fibroblaststransformed with the constitutively active oncogenic variant of the

Met receptor, Tpr-Met, form invadopodia-like structures capableof remodeling ECM. We show that invadopodia induced by Metactivity are dependent on recruitment of Gab1 that localizes to

these structures and interacts directly with cortactin, a key

regulator of actin dynamics within invadopodia. We demonstratethat Met colocalizes with cortactin to invadopodia, and that Metactivity contributes to increased tyrosine phosphorylation of

cortactin independent of Src kinase. By structure–functionanalysis, we have established that a Gab1–cortactin interaction isrequired for assembly of functional invadopodia, in response tooncogenic Met signals.

ResultsTpr-Met induces formation of invadopodia rosettes infibroblasts

Recent studies have suggested that invasive and metastaticpotential of cancer cells and malignantly transformed fibroblastsis tightly linked with the ability of these cells to produce

invadopodia (Gimona, 2008; Buccione et al., 2009; Schoumacheret al., 2010). Therefore it is possible that malignant phenotypes,tumorigenicity and metastatic potential of cells transformed byoncogenic variants of the Met receptor are at least in part due to

the acquired ability of these cells to produce invadopodia orsimilar actin-rich proteolytically active membrane protrusionsthat enable remodeling of ECM. To investigate this possibility,

we used Fischer rat 3T3 (FR3T3) fibroblasts transformed with theoncogenic variant of the Met receptor, Tpr-Met. Upon Tpr-Met-mediated transformation, FR3T3 fibroblasts acquire many

features of malignantly transformed cancer cells, including theability to invade through the ECM, as well as to develop tumorsand metastases in nude mice (Fixman et al., 1996; Fixman et al.,

1997; Saucier et al., 2002). In line with our previous findings,control FR3T3 cells used in this study spread and formeda contact-inhibited monolayer in culture, whereas FR3T3fibroblasts transformed with Tpr-Met developed a distinct

elongated cell morphology, formed foci, lost contact inhibitionand acquired increased migratory and invasive capacity (Fig. 1)(Fixman et al., 1995; Saucier et al., 2002).

To establish whether introduction of the Tpr-Met oncogenecould induce the formation of invadopodia and remodel theextracellular matrix, we examined the ability of control and Tpr-Met-transformed FR3T3 fibroblasts to produce ventral actin-rich

protrusions when plated on fluorescent gelatin. In this assay, weobserved that control FR3T3 fibroblasts formed extensive actinstress fibers (as defined by phalloidin staining), and were unable to

remodel gelatin matrix (Fig. 1A). At the same time, FR3T3fibroblasts transformed with Tpr-Met formed few stress fibers, butproduced prominent ventral rosettes of actin filaments associated

with underlying areas of degraded fluorescent gelatin matrix,typical of invadopodia (Fig. 1A,D; supplementary material Movie1). To confirm that the observed structures were indeedinvadopodia, we stained these cells with invadopodia markers

Tks5 and cortactin and showed their colocalization with the actinrosettes (Fig. 1E; supplementary material Fig. S1). These cellularstructures, which we call invadopodia rosettes, were found in 50%

of the cells (usually one rosette per cell), and penetrated throughthe gelatin matrix in 35% of cells at steady state (Fig. 1B). Toexclude the possibility that the observed formation of invadopodia

rosettes by Tpr-Met-transformed FR3T3 fibroblasts was due toclonal effects, we repeated these experiments using independentlyderived clones of Tpr-Met-transformed FR3T3 fibroblasts

and obtained similar results (Fig. 1A–C). Hence, our findingsdemonstrate that upon transformation with the Tpr-Met oncogene,FR3T3 fibroblasts acquire the ability to produce invadopodia

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rosettes, similar to those produced by cancer cells and fibroblasts

transformed with the Src oncogene (Murphy and Courtneidge,2011).

Met activity is required for invadopodia formation incancer cells

Increased activity of receptor tyrosine kinases, including Met, isoften observed in malignancies and is associated with invasive

capacity of cancer cells (Birchmeier et al., 2003; Corso et al.,2005; Lai et al., 2009). The roles of different RTKs in biogenesisof invadopodia and similar proteolytically active protrusive

cellular structures, of invasive cancer cells, are poorlyunderstood. Given our observation that a constitutively activeoncogenic form of Met, Tpr-Met, could induce formation of

invadopodia rosettes in fibroblasts, and a number of previousreports that aberrant signaling downstream from Met can promotecancer invasion and metastasis (Lai et al., 2009), we sought to

verify whether activation of Met in cancer cells could promoteinvadopodia biogenesis. For this purpose, we used two differentcancer cell lines: an invasive basal breast carcinoma cell line

MDA-MB-231, in which Met activation is HGF dependent and a

gastric carcinoma cell line MKN45, in which Met is amplified

and is constitutively active (Fushida et al., 1993). MDA-MB-231

cells increase invadopodia formation in response to activation of

the epidermal growth factor receptor (EGFR) tyrosine kinase

(Nam et al., 2007; Pichot et al., 2010) but also express the Met

receptor (Fig. 2B). Stimulation of MDA-MB-231 cells plated on

fluorescent gelatin in the presence of HGF, led to enhanced

activation of Met receptor as visualized using a phosphorylation-

specific antibody that recognizes the active receptor (Fig. 2B),

but also increased the number of invadopodia by approximately

twofold, compared with non-stimulated cells (Fig. 2A,C,D).

Approximately 30% of MKN45 cells form invadopodia in the

absence of HGF stimulation (Fig. 2E–H). Strikingly, specific

inhibition of the Met RTK using a small molecule inhibitor,

PHA665752 (Christensen et al., 2003), resulted in a profound

change in cell morphology, and abrogated the ability of MKN45

cells to form invadopodia and remodel the gelatin matrix

(Fig. 2E–H). To confirm that this is dependent on Met activity,

we employed knockdown of Met expression using a specific

Fig. 1. Tpr-Met-transformed FR3T3 fibroblasts form invadopodia. (A) FR3T3 cells or FR3T3 cells stably overexpressing Tpr-Met (Tpr-Met 3 and Tpr-Met

4) were cultured for 24 hours on glass coverslips coated with Oregon-Green-conjugated gelatin (gelatin matrix). Cells were stained with phalloidin and confocal

images were taken at the ventral plane of the cells. Representative images are shown. (B) Quantification of the ability of FR3T3 cells to form actin rosettes or

active invadopodia in response to Tpr-Met. Values are the means of three independent experiments. Active invadopodia are defined as actin-rich rosettes

overlaying matrix remodeling. (C) SDS-PAGE was performed on cell lysates from FR3T3 cells or FR3T3 cells stably overexpressing Tpr-Met and probed for

Met-P (pMet), Met and actin. (D) Confocal Z-sections were collected, deconvolved using IMARIS software and volume rendered to reconstruct the 3D image.

View of invadopodia from above (arrows) and below (arrowheads) the gelatin matrix. (E) FR3T3 cells expressing Tpr-Met were plated on glass coverslips coated

with unlabeled collagen for 24 hours and immunostained for invadopodia markers Tks5 and cortactin to identify invadopodia rosettes. The boxed region in the

third image is shown at higher magnification in the fourth panel. Scale bars: 10 mm.

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Fig. 2. Invasive breast cancer cells, MDA-MB-231, and gastric cancer cells, MKN45, form invadopodia in response to Met RTK signaling. (A) MDA-MB-

231 cells were cultured on gelatin matrix for 3 hours and stimulated with 0.5 nM HGF for an additional 3 hours. Cells were stained for the invadopodia markers

actin (phalloidin) or cortactin. (B) SDS-PAGE was performed on cell lysates of MDA-MB-231 cells stimulated with 0.5 nM HGF and non-stimulated cells.

(C,D) The ability of MDA-MB-231 cells to form invadopodia in response to HGF stimulation was quantified. Values are the means of three independent

experiments. (E) MKN45 cells were cultured on gelatin matrix in the presence of 0.1 mM Met inhibitor PHA665752 or DMSO for 24 hours. MKN45 cells were

treated with 50 nM siRNA targeting Met or control siRNA. Cells were trypsinized 48 hours after treatment and plated on gelatin matrix and cultured for an

additional 24 hours. Cells were stained for markers of invadopodia, actin (phalloidin) or cortactin and confocal images were acquired at the ventral plane of the

cells. DIC images of cells stained with actin (red) and DAPI (blue) taken at a lower magnification (636) are shown on the right. Representative images are shown.

(F,G) The loss of invadopodia formation in MKN45 cells in response to treatment with Met inhibitor PHA665752 or siRNA-mediated knockdown of Met.

(H) SDS-PAGE was performed on cell lysates of MKN45 cells treated with 0.1 mM Met inhibitor or 50 nM siRNA to Met or the respective vehicles (DMSO)

or control siRNA and probed for Met-P (pMet), Met and tubulin. Scale bars: 10 mm.

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siRNA and observed the same phenotype: the ability of MKN45cells to form invadopodia upon inactivation of Met signal is

decreased by half (Fig. 2E–H). Together, these data demonstratethat in invasive cancer cells, activation of Met, both dependentand independent of stimulation with HGF, elicits signals that

result in increased invadopodia biogenesis and extracellularmatrix remodeling.

Ability of fibroblasts to form functional invadopodiarosettes is determined by an intact multiprotein dockingsite of Tpr-Met

Constitutive activation of the Tpr-Met oncogene is accompaniedby auto-phosphorylation at two key tyrosine residues of the C-terminal region (Y482 and Y489, corresponding to Y1349 and

Y1356 residues of the wild-type Met receptor). These residuescreate a multiprotein docking site for phosphorylation-dependentrecruitment of the adaptor proteins, Grb2 and Shc, and the scaffold

protein Gab1 (Fixman et al., 1997; Fournier et al., 1996; Nguyenet al., 1997; Ponzetto et al., 1994) and are essential for thebiological and transforming activity of the Tpr-Met oncogene andMet RTK (Fixman et al., 1995; Fixman et al., 1997; Lock et al.,

2003; Ponzetto et al., 1996). Whereas interaction between Grb2and Tpr-Met requires direct binding of the Grb2 SH2 domain to aphosphorylated Y1356 residue of Tpr-Met (Nguyen et al., 1997),

Gab1 can be recruited to Tpr-Met both directly, through interactionof the Gab1 Met-binding domain (MBD) to a phosphorylatedY1349 residue (Weidner et al., 1996), and indirectly, through Grb2

(Lock et al., 2003; Lock et al., 2000; Nguyen et al., 1997).

To assess whether signals from the multiprotein docking site ofTpr-Met also determine the ability of FR3T3 fibroblasts to form

functional invadopodia rosettes, we established stable cell linesexpressing mutants of the Tpr-Met oncogene, carrying Y1349Fand/or Y1356F substitutions, and quantified their ability to produce

proteolytically active invadopodia rosettes on fluorescent gelatin.In comparison with Tpr-Met-transformed FR3T3 fibroblasts,substitution of Y1349 or Y1356 residues of Tpr-Met withphenylalanine led to slight decreases in the number of actin

rosettes (,20%) as well as proteolytically active invadopodiarosettes (,30%; Fig. 3A,B). However, FR3T3 cells expressingTpr-Met mutants with both Y1349 and Y1356 replaced by

phenylalanine [Tpr-Met Y1349, Y1356F], produced considerablyfewer actin rosettes (,20% that of cells expressing WT Tpr-Met)and were unable to remodel the gelatin matrix (,5% that of cells

expressing WT Tpr-Met; Fig. 3A,B), even though this mutant Tpr-Met is catalytically active (Fixman et al., 1997; Nguyen et al.,1997). In a similar manner to parental FR3T3 fibroblasts, these cells

also formed enhanced actin stress fibers (Fig. 3A,B), indicative ofthe reversal of the transformed phenotype.

Gab1 is required for invadopodia formation induced byMet in fibroblasts and cancer cells

Because both tyrosine resides, 1349 and 1356, of Tpr-Met are

required for recruitment of the Gab1 scaffold (Fixman et al., 1997;Nguyen et al., 1997), we tested the requirement for Gab1 ininvadopodia formation. Tpr-Met was stably expressed in

immortalized mouse embryonic fibroblasts with knockout ofGab1 (Gab12/2 MEFs) (Holgado-Madruga and Wong, 2003) andthe ability of these cells to form invadopodia rosettes when

cultured on fluorescent gelatin matrix was examined. In theabsence of Gab1, expression of Tpr-Met failed to initiate theformation of actin rosettes or matrix remodeling (Fig. 4A–C;

supplementary material Movie 2). However, when expression of

Gab1 was rescued by introduction of a GFP-fused variant of this

scaffold protein, Gab12/2 MEFs expressing Tpr-Met acquired the

ability to form proteolytically active actin rosettes, in a manner

similar to Tpr-Met-transformed FR3T3 fibroblasts (Fig. 4A–C;

supplementary material Movie 3, Fig. S2C,D). In addition, upon

rescue of Gab1 expression in Gab12/2 MEFs, Tpr-Met signals

lead to increased peripheral actin ruffles and reduced actin stress

fibers (Fig. 4A).

To establish whether Gab1 expression is also crucial for Met-

dependent invadopodia formation in cancer cells, we performed

siRNA-mediated knockdown of Gab1 expression in MKN45

gastric cancer cells. Decreased Gab1 expression, led to a fourfold

decrease in invadopodia formation (Fig. 4D–F). Together these

data support that Gab1 plays an essential role in the formation of

invadopodia, both in Tpr-Met transformed fibroblasts and in Met-

dependent gastric cancer cells.

Proline-rich motifs of Gab1 are required for formation of

functional invadopodia rosettes and invasion of Tpr-Met-

transformed fibroblasts

Upon binding to a phosphorylated Met receptor, or Tpr-Met

oncogene, Gab1 recruits many proteins that trigger diverse

downstream signaling cascades (Birchmeier et al., 2003; Lai

Fig. 3. A multi-substrate docking site on Tpr-Met is required for

functional invadopodia formation. (A) FR3T3 cells stably overexpressing

various mutants of Tpr-Met with phenylalanine substituted for tyrosine, were

cultured on gelatin-matrix-coated coverslips for 24 hours. Cells were stained

with phalloidin and confocal images were acquired. Arrows indicate areas of

matrix remodeling. Representative images are shown. (B) The ability of Tpr-

Met mutants to promote formation of actin rosettes or active invadopodia was

quantified. Values are the means of three independent experiments. SDS-

PAGE was performed on lysates from cells expressing Tpr-Met mutants and

probed for Met and tubulin. Scale bars: 10 mm.

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et al., 2009). Having established that Gab1 is crucial for

biogenesis of invadopodia rosettes in Tpr-Met-transformed

fibroblasts, and invadopodia in gastric carcinoma MKN45 cells,

we sought to identify downstream interaction partners of Gab1

implicated in these events using a structure–function approach.

For this, we rescued Gab1 expression in Gab12/2 MEFs stably

expressing Tpr-Met (2B-Tpr-Met-1) using various deletion or

point mutants of the Gab1 scaffold protein. In contrast to wild-

type (WT) Gab1, two of the Gab1 deletion mutants, Gab1DMBD,

lacking the Met binding domain, and Gab1DP4/5, missing the

fourth and fifth proline-rich motifs of Gab1, failed to appreciably

rescue the formation of actin rosettes in Gab12/2 MEFs

expressing Tpr-Met (Fig. 5A,B,D). Instead, cells expressing

Gab1DMBD and Gab1DP4/5 mutants to similar levels as WT

Gab1, formed prominent actin stress fibers, and their ability to

form actin rosettes were decreased by 75%, compared with cells

expressing WT Gab1 (Fig. 5B,D,F).

Although Gab12/2 MEFs expressing Tpr-Met (2B-Tpr-Met-1)

could invade reconstituted extracellular matrix (Matrigel) at a

basal level, rescue of WT Gab1 expression increased the invasive

capacity of these cells by fourfold (Fig. 5C,E). Furthermore,

consistent with the reduced capacity of Gab12/2 Tpr-Met MEFs

rescued using Gab1DMBD or Gab1DP4/5 deletion mutants to

form actin rosettes, there was a more than 50% reduction in their

invasive capacity, when compared with cells expressing WT

Gab1 (Fig. 5C,E).

The MBD domain of Gab1 binds directly to a tyrosine-

phosphorylated Met receptor, and Gab1 proline-rich motifs 4 and

5 are implicated in indirect recruitment to Met, mediated through

the Grb2 adaptor protein (Lock et al., 2003). To determine whether

the failure of the Gab1DP4/5 or Gab1DMBD deletion mutants to

support formation of actin rosettes and rescue invasiveness of

Gab12/2 Tpr-Met MEFs is due to their inability to bind Tpr-Met,

we performed reciprocal immunoprecipitations of Tpr-Met or Gab1

mutants in HEK 293 cells, and examined co-immunoprecipitates

for the presence of Gab1 and Tpr-Met, respectively. Whereas

Gab1DMBD failed to bind Tpr-Met, Gab1DP4/5 was recruited,

although its tyrosine phosphorylation was reduced by 20%

(Fig. 6A–E). Notably, FR3T3 fibroblasts, transformed by a Tpr-

Met mutant (N1358H), specifically uncoupled from Grb2, formed

actin rosettes to a similar level as cells expressing WT Tpr-Met,

indicating that Grb2-dependent recruitment of Gab1 to the Met

receptor is dispensable for actin rosette formation (Fig. 6H–J).

Given that Gab1DP4/5 recruitment to Met is not impaired, we

examined the ability of the Gab1DP4/5 mutant to localize to

invadopodia. For this, we performed both confocal analysis on

fixed cells and time-lapse video microscopy analysis of

FR3T3-Tpr-Met cells that form actin rosettes and examined the

Fig. 4. Gab1 is required for Met-induced invadopodia formation. (A) Gab1 null cells stably overexpressing Tpr-Met (Gab1 null Tpr-Met) and rescued with

GFP–Gab1 were cultured on gelatin-matrix-coated coverslips for 24 hours. Cells were stained with phalloidin and confocal images were acquired. (B) Proteins

from cell extracts of Gab1 null Tpr-Met cells as well as GFP–Gab1-rescued cells were resolved by SDS-PAGE and immunoblotted for GFP, Met-P (pMet),

Met and actin. (C) Gab1 null Tpr-Met cells or cells rescued with GFP–Gab1 were transiently transfected with RFP–actin and subjected to time-lapse video

microscopy. One frame from each video is shown. (D) MKN45 cells were treated with 100 nM control siRNA or smartpool siRNA against Gab1 for 48 hours.

Cells were trypsinized and plated on gelatin matrix for 24 hours, stained with phalloidin and confocal images were acquired. (E,F) The ability of MKN45 cells to

form invadopodia in response to treatment with siRNA against Gab1 or control siRNA was quantified. An immunoblot showing the knockdown of Gab1 in

MKN45 cells treated with siRNA against Gab1 but not in control siRNA-treated cells is shown. Scale bars: 10 mm.

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ability GFP-Gab1 WT or GFP-Gab1DP4/5 to localize to these

structures. We observed that GFP-Gab1DP4/5 localized to the

actin rosettes in a similar manner to GFP–Gab1WT (Fig. 6F,G;

supplementary material Movies 4, 5). Together, these results

substantiate that the formation of actin rosettes in Tpr-Met-

transformed fibroblasts is dependent on assembly of multiprotein

complexes that involve Tpr-Met, Gab1, and possibly other

protein(s) recruited through the fourth and/or fifth proline-rich

motifs of Gab1.

Proline-rich motifs of Gab1 are involved in interaction

with cortactin

Previous studies have identified the adaptor protein cortactin as

a key component of invadopodia, both in cancer cells and

oncogenically transformed fibroblasts (Bowden et al., 1999;

Bowden et al., 2006; Clark and Weaver, 2008; Clark et al., 2007;

Cortesio et al., 2008; Cosen-Binker and Kapus, 2006; DesMarais

et al., 2009; Oser et al., 2009). Cortactin is thought to promote

invadopodia formation by association with actin regulatory

proteins Nck, N-WASP and Arp2/3, which initiate actin

polymerization (DesMarais et al., 2009; Murphy and

Courtneidge, 2011; Yamaguchi et al., 2005).

The fourth and fifth proline-rich motifs of Gab1, which are

crucial for formation of actin rosettes in Tpr-Met transformed

fibroblasts, are similar to xPPxPxKPx consensus sequences

recognized by the SH3 domain of cortactin (Rubini et al., 2010;

Sparks et al., 1996). This raises the possibility that Gab1 serves as a

scaffold to recruit cortactin to sites of emerging actin rosette or

vice versa. To test this hypothesis, we performed reciprocal

immunoprecipitation of Gab1 and cortactin and detected co-

immunoprecipitation between the two proteins when either

transiently expressed in HEK 293 cells (Fig. 7A,B) or between

endogenous Gab1 and cortactin in BT549 cells (Fig. 7H).

By undertaking a structure–function analysis in HEK 293 cells,

we established that Gab1-cortactin interaction is dependent on

the SH3 domain of cortactin (Fig. 7E,C). Notably, WT cortactin

was efficiently co-immunoprecipitated with Gab1, whereas both

a cortactin mutant lacking the SH3 domain (cortactinDSH3) and

cortactin with a point mutation rendering the SH3 domain unable

to bind consensus proline-rich sequences (cortactin W525K),

failed to bind Gab1 (Fig. 7E,C). Additionally, in reciprocal

experiments, cortactin co-immunoprecipitated efficiently with

WT Gab1, and could interact, although to a lesser extent, with

either Gab1DP4 or Gab1DP5 mutants alone, but failed to interact

with the Gab1DP4/5 mutant (Fig. 7D). Gab1 was efficiently

pulled down from cell lysates by a GST fusion protein containing

the SH3 domain of cortactin, but not by a fusion protein

expressing the cortactin SH3 W525K mutant (Fig. 7I), indicating

that an intact SH3 domain of cortactin is both necessary and

sufficient for interaction with Gab1. Although the interaction

between cortactin and Gab1 requires the SH3 domain of cortactin

and the proline-rich motifs of Gab1, it does not exclude the

possibility that this interaction is mediated by an intermediate. To

confirm that this interaction was indeed direct, we performed far-

western blotting of Gab1, and a known cortactin SH3 binding

partner dynamin as positive control, with the GST-fused cortactin

SH3 or SH3 W525K mutant. As expected dynamin interacted

with GST–cortactin SH3 domain fusion protein but failed to

Fig. 5. Met-RTK-driven

invadopodia biogenesis is

dependent on two PxxP motifs in

Gab1. (A) Schematic diagram of

Gab1, indicating Met binding domain

(MBD) and a proline-rich region 4/5

(P4/5) and sites of recruitment for

downstream signaling proteins. PH;

pleckstrin homology domain.

(B) Gab1 null Tpr-Met cells rescued

with either WT Gab1, Gab1DMBD or

Gab1DP4/5 were cultured on gelatin

matrix for 24 hours, fixed, stained

with phalloidin and confocal images

acquired. Arrows indicate rosettes

and arrowheads indicate matrix

remodeling. (C) Cells rescued with

either WT Gab1 or the indicated

Gab1 mutants were subjected to

Boyden chamber invasion assays.

Representative images are shown.

(D,E) Quantification of invadopodia

response (D) and Boyden chamber

invasion response (E) is shown for

three clones for each mutant and WT

Gab1-rescued cells. Values are the

means of three independent

experiments. (F) SDS-PAGE was

performed on lysates from the

corresponding rescue cells and

probed for GFP, Met-P (pMet), Met

and tubulin. Scale bars: 10 mm.

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interact with GST–cortactin SH3-W525K mutant fusion protein

(Fig. 7J). In a similar manner, Gab1 bound to the intact SH3

domain of cortactin but failed to interact with the SH3 W525K

mutant, indicating that the interaction between the proline-rich

motifs of Gab1 and SH3 domain of cortactin is direct (Fig. 7C,J).

Cortactin colocalizes with Gab1 in Tpr-Met-transformed

fibroblasts and is required for formation of invadopodia

rosettes

Recently we demonstrated that in the process of circular dorsal

ruffle formation in response to Met activation, Gab1 served as a

scaffold protein linking activated Met to nucleation of branched

actin filaments, by recruiting the adaptor protein Nck and actin

nucleation promoting factor, N-WASP (Abella et al., 2010). In

order to understand whether a Gab1–cortactin complex could

play a similar role within invadopodia rosettes, we examined the

colocalization of Gab1 and cortactin in Tpr-Met-transformed

fibroblasts. Both Gab1 and cortactin were found to colocalize

at actin rosettes, representative of proteolytically active

invadopodia rosettes (Fig. 7F).

The ability of Tpr-Met-transformed fibroblasts to produce

proteolytically active invadopodia rosettes is strongly linked with

Fig. 6. Gab1DP4/5 is recruited to Tpr-Met and localizes to invadopodia but fails to initiate actin rosette formation. (A,D) HEK 293 cells were transiently

transfected with the indicated constructs; proteins from lysates were immunoprecipitated with Met 147 antibody (A) or HA antibody (D) and probed as indicated

using the Odessey detection system (Li-Cor). (B,E) Densitometric analysis of western blots was performed using Odessey software and tyrosine phosphorylation of

Gab1 mutants. Their ability to be recruited to Met is depicted as a percentage of WT Gab1. Values are the means of three independent experiments. (C) Proteins from

lysates of Gab-null Tpr-Met cells rescued with GFP–Gab1 (WT) or GFP–Gab1DP4/5 were immunoprecipitated with GFP antibody and probed as indicated.

(F) FR3T3 Tpr-Met cells were transiently transfected with RFP-actin and GFP-Gab1DP4/5 and subjected to time-lapse video microscopy. One frame depicting

Gab1DP4/5 localization to an actin rosette is shown. (G) FR3T3 Tpr-Met cells were transiently transfected with GFP-Gab1DP4/5, plated on gelatin matrix for 24

hours and stained for cortactin and GFP–Gab1. (H) FR3T3 cells stably overexpressing a Tpr-Met mutant that is specifically uncoupled from Grb2 (N1358H) were

plated on gelatin matrix for 24 hours and stained for invadopodia markers actin (phalloidin) and cortactin. (I) Ability of Tpr-Met N1358H mutants to induce actin

rosette was assessed. Values are the means of two independent clones (FR3T3 Tpr-Met-N1358H-1 and FR3T3 Tpr-Met-N1358H-2). Levels of Tpr-Met were assessed

by western blotting. (J) Tpr-Met was immunoprecipitated from lysates prepared from FR3T3 Tpr-Met-N1358H-1 and FR3T3 Tpr-Met-N1358H-2 clones as well as

from FR3T3 Tpr-Met 3 cells using Met 147 antibody. SDS-PAGE was performed and proteins immunoblotted as indicated. Scale bars: 10 mm.

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the potential to produce ventral actin rosettes (Fig. 1A,B). Hence

both proteolytically active and non-degrading actin rosettes

probably represent a similar cellular structure at different stages

of formation. Using this advantage, we further validated the

importance of cortactin for assembly of Tpr-Met-dependent

invadopodia rosettes. Knockdown of cortactin expression in Tpr-

Met-transformed FR3T3 cells, using two different siRNA

duplexes, led to more prominent actin stress fibers and a 50%

decrease in the formation of actin rosettes, as compared with

control, Tpr-Met-transformed FR3T3 cells transfected with non-

targeting siRNA duplexes (supplementary material Fig. S3A,B).

Furthermore, in each case, the remaining actin rosettes observed

in Tpr-Met-transformed FR3T3 fibroblasts were only partially

formed, compared with cells treated with non-targeting siRNAs

(supplementary material Fig. S3A). In summary, these

experiments substantiate Gab1 and cortactin as components of

a protein complex that determines the capacity of Tpr-Met-

transformed fibroblasts to form invadopodia rosettes and remodel

ECM.

Met RTK localizes to invadopodia and promotes tyrosinephosphorylation of cortactin

So far, only non-receptor tyrosine kinases, Src, Arg and Abl, have

been shown to localize to invadopodia. Given that Met was found

at other structurally related actin-rich structures, such as circular

dorsal ruffles, we sought to explore the possibility that Met could

Fig. 7. Cortactin interacts with Gab1 through its SH3 domain with proline-rich regions on Gab1. HEK 293 cells were transiently transfected with the

indicated constructs; proteins from lysates were immunoprecipitated with HA antibody (A,D,E) or cortactin antibody (B) and immunoblotted as indicated.

(C) Schematic diagram depicting a potential interaction of the cortactin SH3 domain and Gab1 proline-rich consensus motifs. (F,G) FR3T3 cells expressing Tpr-

Met were transiently transfected with GFP-Gab1 and then plated on gelatin matrix for 24 hours. Cells were stained for GFP, cortactin and phalloidin. (F) X–Z and

Y–Z projection of a 40 Z-stack showing the localization of Gab1 and cortactin to membrane protrusions (arrows). (H) Endogenous Gab1 was immunoprecipitated

from BT549 cells and probed for cortactin. (I) GST–cortactin SH3 or GST–cortactin SH3-W525K mutant fusion proteins were coupled to GST beads and used to

pull down proteins from lysates of HEK 293 cells transiently expressing HA–Gab1. (J) Proteins from HEK 293 cells, transfected with GFP-Gab1 or HA-dynamin,

were immunoprecipitated with either HA or GFP antibody. The immune complex was separated by SDS-PAGE and transferred to a nitrocellulose membrane.

Nitrocellulose membranes were incubated with fusion proteins of either cortactin GST–SH3 domain or GST–SH3-W525K and immunoblotted as indicated. Scale

bars: 10 mm.

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localize to invadopodia. Indeed, in MKN45 cells, which showconstitutive Met activity and produce cortactin-positive actin-rich

protrusions that remodel the underlying matrix, Met localized

to actively degrading invadopodia protrusions, as defined by

colocalization with cortactin and degraded fluorescent gelatin

areas (Fig. 8A). Given the observed colocalization of Met

Fig. 8. Met colocalizes with cortactin to invadopodia, and cortactin tyrosine phosphorylation is highly dependent on Met kinase activity. (A) MKN45 cells

were plated on gelatin matrix for 24 hours and stained for cortactin and Met. The boxed regions are shown enlarged in the insets. (B) HEK 293 cells were transfected

with the indicated constructs and the cell extracts were immunoprecipitated with cortactin antibody, 4F11. Immune complexes were separated by SDS-PAGE and

probed as indicated. (C) MKN45 cells were treated with 10 mM PP2, 10 mM SU6656, 10 mM Imatinib, 0.1 mM PHA665752 or vehicle (DMSO). Cell extracts were

separated by SDS-PAGE and probed as indicated or immunoprecipitated using anti-cortactin (4F11) or anti-Crk antibodies. Immune complexes were separated by

SDS-PAGE and probed as indicated. (D) MKN45 cells were plated on gelatin matrix in the presence of 10 mM PP2, 10 mM SU6656, 10 mM Imatinib, 0.1 mM

PHA665752 or vehicle (DMSO) for 24 hours and stained with phalloidin. Representative images are shown. (E,F) The ability of MKN45 cells to form invadopodia in

the presence of PP2, SU6656, Imatinib or PHA665752 was quantified. Values are the mean of three independent experiments. Scale bars: 10 mm.

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receptor and cortactin at invadopodia and the role of tyrosine

phosphorylation of cortactin in regulation of invadopodiadynamics, we examined whether Met could also promotephosphorylation of cortactin on tyrosine residues. When

expressed in HEK 293 cells without additional stimuli,cortactin was not detectably tyrosine phosphorylated; however,co-expression with Tpr-Met was sufficient to trigger its strongtyrosine phosphorylation of cortactin (Fig. 8B). In a similar

fashion, in MKN45 cells, where Met is amplified andconstitutively active, cortactin was tyrosine phosphorylated(Fig. 8C), which was abolished upon treatment of the cells

with the Met inhibitor PHA665752, suggesting that tyrosinephosphorylation of cortactin in these cancer cells is dependent onMet kinase activity (Fig. 8C). Intriguingly, it has been shown

previously that cortactin is a substrate for the non-receptortyrosine kinase Src, and that cortactin phosphorylation ontyrosine is triggered by Src activity. However, in MKN45 cells,

inhibition of Met RTK does not detectably alter Src activity(Fig. 8C). Moreover, treatment of MKN45 cells with Srcinhibitors PP2 or SU6656 or the Abl inhibitor Imatinib, or incombinations, had little to no effect on cortactin tyrosine

phosphorylation (Fig. 8C) indicating that in MKN45 gastriccancer cells Src or Abl kinase activity is not essential to triggertyrosine phosphorylation of cortactin, whereas Met activation is

required. Additionally, tyrosine phosphorylation of cortactin inMKN45 cells treated with inhibitors of Src, Abl or Met, correlatewith the ability of treated cells to form invadopodia, with Met

inhibition exerting the strongest impact (Fig. 8D–F). Takentogether, these data support the proposal that Met can promotetyrosine phosphorylation of cortactin and invadopodia biogenesis

independently of Src.

DiscussionIt is becoming increasingly evident that the invasive capacity ofcancer cells is often determined by their ability to assemble

invadopodia, actin-rich protrusive cellular structures that mediatefocal ECM remodeling (Gimona, 2008; Buccione et al., 2009;Murphy and Courtneidge, 2011). Invadopodia are produced

predominantly by cancer cells, and with some exceptions, areabsent in normal cells, and hence might serve as a feasiblespecific target for first-line anti-metastatic therapies. Despite this

attractive therapeutic possibility, molecular mechanisms that leadto invadopodia biogenesis in cancer remain largely undefined.

Research performed so far has attributed induction ofinvadopodia and invadopodia-like structures mostly to

oncogenic activation of the non-receptor tyrosine kinase Src infibroblasts (Hauck et al., 2002; Bowden et al., 2006; Webb et al.,2007; Oikawa et al., 2008; Balzer et al., 2010; Kelley et al., 2010;

Murphy and Courtneidge, 2011), as well as Abl and Arg kinases(Smith-Pearson et al., 2010; Mader et al., 2011), and growthfactor receptor tyrosine kinases, including EGFR (Kimura et al.,

2010; Mader et al., 2011) and PDGFRa (Eckert et al., 2011).

Here we report for the first time that signals from an activatedMet RTK, which is implicated in cancer invasiveness(Birchmeier et al., 2003; Peschard and Park, 2007), can induce

invadopodia rosettes in fibroblasts, and increase invadopodiabiogenesis in human cancer cells. We demonstrate that upontransformation with a constitutively active oncogenic variant of

the Met receptor, Tpr-Met, fibroblasts acquire the ability to formventral proteolytically active actin rosettes (Fig. 1). Inaccordance with a recently proposed nomenclature (Murphy

and Courtneidge, 2011), we refer to this structure as an‘invadopodia rosette’. Importantly, we also show that activation

of Met, either through engagement with the ligand, HGF, or as aresult of genomic amplification of the MET locus, increasesinvadopodia biogenesis in basal-like breast cancer and gastriccarcinoma cells (Fig. 2). These observations are physiologically

relevant. Increase in plasma HGF levels, as well as elevatedlevels of Met expression and activity, including constitutiveactivation resulting from gene amplification, are linked with

increased cancer invasiveness and metastasis, and correlate withpoor prognosis in many types of cancer, including basal-likebreast cancers (Camp et al., 1999; Lengyel et al., 2005; Ponzo

et al., 2009) and gastric carcinomas (Kammula et al., 2007;Tuynman et al., 2008; Wu et al., 1998).

Invadopodia observed in basal breast cancer MDA-MB-231 cellsupon HGF stimulation (Fig. 2) morphologically resemble those

formed in response to epidermal growth factor (EGF) activation ofthe EGFR (Oser et al., 2009; Mader et al., 2011), suggesting that asimilar molecular machinery promoting invadopodia formation is

be driven by multiple upstream signals. By contrast, in gastriccarcinoma MKN45 cells, which carry genomic amplification ofMET, formation of invadopodia is abolished by Met inhibition

or substantially decreased following Met knockdown (Fig. 2)indicating that in Met-addicted cancer cells, the Met signal is themajor upstream driver of invadopodia biogenesis.

Sequential signals are involved in assembly of functional

invadopodia (Oikawa et al., 2008). These involve initial signalsthat trigger the establishment of precursor actin-rich membraneprotrusions, followed by signals that regulate targeted secretion

of metalloproteases for ECM remodeling known as invadopodiamaturation (Artym et al., 2006; Murphy and Courtneidge, 2011).In many cell types the formation of invadopodia in response to

Met signaling is dependent on the scaffold protein Gab1 (Fig. 4).In Gab12/2 fibroblasts, Tpr-Met fails to induce formation ofinvadopodia rosettes and this phenotype is rescued by expression

of Gab1 (Fig. 4; supplementary material Movies 2,3). SiRNA-mediated knockdown of Gab1 also leads to decreased Met-dependent invadopodia biogenesis in MKN45 gastric carcinomacells (Fig. 4). Following Gab1 knockdown in MKN45 cells or in

fibroblasts expressing a Tpr-Met mutant unable to recruit Gab1Y1349F/Y1356FY, the inability to produce invadopodia and/oractin rosettes (Figs 3, 4) is due to decreased assembly of actin-

rich core structures (supplementary material Movies 2,3),consistent with a role for Met-Gab1 signaling in the regulationof an early step in invadopodia formation. This supports previous

reports that Gab1 plays a role in Met-dependent regulation ofother actin-rich cellular structures, such as lamellipodia (Frigaultet al., 2008) and circular dorsal ruffles (Abella et al., 2010).

Hence, depending on the cellular context, Gab1 provides acommon link between Met signals and assembly of actin-richstructures at the cell periphery.

Using a structure–function approach we have identified the

requirement of two proline-rich motifs of Gab1 (P4/5) for actinrosette formation and have shown that these provide directbinding sites for the SH3 domain of cortactin, a protein involved

in actin dynamics (Figs 5, 7). The P4/5 motifs also bind the Grb2adaptor protein, which indirectly recruits Gab1 to Met (Locket al., 2002). Given that direct recruitment of Grb2 to Met is

dispensable for actin rosette formation (Fig. 6), whereas theGab1DP4/5 mutant itself is still recruited to and phosphorylatedby Tpr-Met (Fig. 6), but fails to rescue assembly of actin rosettes

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in Gab12/2 fibroblasts, we propose that a Gab1–cortactininteraction mediated through proline-rich motifs of Gab1 isrequired for Met-dependent invadopodia formation (Fig. 5).

Cortactin recruitment is essential for invadopodia formation

downstream from multiple signals. In one model, invadopodiaassembly in response to EGF is initiated at early precursors thatcontain cortactin, N-WASP and Arp2/3 (Oser et al., 2009; Artym

et al., 2006). Recruitment of cortactin to the early precursors isthought to be mediated through the Arp2/3 complex (Uruno et al.,2001) and subsequent phosphorylation of cortactin on tyrosineresidues promotes release and activation of cofilin, resulting in

actin severing and increased barbed end formation (Oser et al.,2009). A second model proposes that the podosome, in Srctransformed fibroblasts, is initiated by the accumulation of

PtdIns(3,4)P2 in the vicinity of existing focal adhesions, and bysubsequent recruitment of Tks5 scaffold protein (Oikawa et al.,2008; Murphy and Courtneidge, 2011). Clustering of N-WASP

on Tks5 (Oikawa et al., 2008; Oikawa et al., 2009) andrecruitment of cortactin (Crimaldi et al., 2009) is responsiblefor actin nucleation during initiation of actin rosettes.

Interestingly, Gab1 interacts with N-WASP and cortactin andlocalizes to actin-rich circular dorsal ruffles (Abella et al., 2010)as well as invadopodia in response to Met activation and mightthus function in a similar manner to Tks5 to promote the

assembly of actin rosette.

Met promotes robust tyrosine phosphorylation of cortactin (Fig. 8)(Crostella et al., 2001). Hence one possible role for a Gab1–cortactincomplex at invadopodia precursors is to promote localized Met–

dependent tyrosine phosphorylation of cortactin, leading to increasedbranched actin nucleation and actin rosette assembly. In support ofthis, Gab1 and Met localize to cortactin-positive matrix remodeling

invadopodia (Fig. 8). To date Met is the only RTK shown to localizeto invadopodia. Hence Met could promote actin nucleation, not onlythrough phosphorylation-dependent regulation of cortactin, but alsoby influencing activity of other invadopodia-associated proteins. In

support of this, Src activity is not essential for invadopodiaformation in Tpr-Met-transformed fibroblasts or Met-addictedMKN45 carcinoma cells (supplementary material Fig. S4; Fig. 8).

Interestingly, overexpression of cortactin in human non-small celllung cancer (HNSCLC) cells is associated with acquired resistance totreatment with EGFR inhibitors, a phenomenon that is often

observed in HNSCLC and other cancer cells as a result ofgenomic amplification of MET and increased Met activation(Kosaka et al., 2011; Lai et al., 2009). Overexpression of cortactin

in HNSCLC cells has also been linked to attenuated Met receptordownregulation and augmented Met-mediated biological responses(Timpson et al., 2007), further supporting a physiological role forMet–cortactin functional interaction.

In summary, in this study we have identified a Met signaling

axis as an important determinant of invadopodia biogenesis. Wehave demonstrated that Gab1, one of the main scaffold proteinsrecruited to active Met, is crucial for the formation of Met-

dependent invadopodia, by regulating assembly of their actinrosettes. Our study highlights Met–Gab1 signaling as analternative target for therapeutic disruption of these structures

in invasive cancer cells.

Materials and MethodsCell culture and cDNA transfections

FR3T3, HEK 293, MEFs and MDA231 cells were maintained in Dulbecco’smodified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS).MKN45 cells were maintained in RPMI containing 10% FBS. The generation of

FR3T3 cell lines stably overexpressing the WT Tpr-Met and Tpr-Met mutants has

been previously described by Saucier et al. and Fixman et al. GFP-tagged WT andmutant Gab1 were generated as described in Abella et al. (Saucier et al., 2002;

Fixman et al., 1997; Abella et al., 2010). Tpr-Met was cloned into pLXSH vectorand stably expressed in MEFs by retroviral infection. Cell lines were selected in

0.5 mg/ml hygromysin or 2 mg/ml of puromysin. pCDNA-cortactin WT and themutants were described previously (Stuible et al., 2008). DsRed–cortactin was a

kind gift from Mark A. McNiven (Mayo Clinic, Rochester, MN). For transienttransfection assays, HEK 293 cells were transfected with Lipofectamine Plus

reagent and MEFs and FR3T3 cells were transfected with Lipo2000 according tothe manufacturer’s instructions (Invitrogen, Carlsbad, CA). Cells were used for

biochemical assays 24 hours post-transfection.

siRNA knockdown

Human Gab1- and Met-specific siRNAs were purchased from Dhamacon Inc.(Lafayette, CO) as a smartpool containing four different oligonucleotides:

siGENOME, SMARTpool M-003553, Human Gab1 and siGENOMESMARTpool L-003156, Human Met; siRNAs were transfected using Hiperfect

transfection reagent at 100 nM and 50 nM concentrations, respectively. After48 hours, cells were trypsinized and plated for biological assays or for western

blotting for an additional 24 hours. Rat cortactin-specific siRNA sequences,

S102732177 and S100169386, were purchased from Qiagen (Valencia, CA) andtransfected at 50 nM concentration using Hiperfect transfection reagent. After 48

hours, cells were trypsinized and plated for biological assays or for western blotanalysis for an additional 24 hours.

Antibodies and reagents

Antibody 147 was raised against a C-terminal peptide of the human Met protein

(Maroun et al., 1999; Rodrigues et al., 1991). Tks5 antibody (1736) was a kind giftfrom Sara Courtneidge (Sanford-Burnham Medical Research Institute, CA).

Commercial antibodies: Gab1 and cortactin 4F11 were from UpstateBiotechnology (Lake Placid, NY), pan phospho tyrosine (pTYR-100) and

pMet1234/35 (phosphorylated Met;Met-p) were from Cell SignalingTechnologies (Danvers, MA), actin was from Santa Cruz Biotechnology, Inc.

(Santa Cruz, CA), Tubulin was from Sigma (St Louis, MO), GFP antibody,Phalloidin Alexa Fluor 488, 546 and 647, and Alexa-Fluor-488-, 555- and 647-

conjugated secondary antibodies were from Molecular Probes (Eugene, OR),HA.11 monoclonal antibody was from Covance (Berkeley, CA). Met inhibitor

PHA665752 was a kind gift from Pfizer Inc. (New York, NY). Src inhibitors PP2and Su6656 were purchased from EMD Chemicals (Gibbstown, NJ) and Sigma-

Aldrich, respectively. HGF was a generous gift from Genentech (San Francisco,

USA).

Immunoprecipitation and western blotting

Cells were harvested in T&D lysis buffer (150 mM NaCl, 20 nM Tris-HCl, 1 mMEDTA, 1 mM EGTA, 1% Triton X-100, 1% deoxycholate, pH 7.4). All lysis

buffers were supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF),1 mM sodium vanadate, 1 mM sodium fluoride, 10 mg/ml aprotinin and 10 mg/ml

leupeptin. For immunoprecipitation, lysates (1 mg) were incubated with theindicated antibodies for 2 hours at 4 C with gentle rotation. To collect immune

complexes, 25 ml of 50% slurry of either protein-A– or protein-G–Sepharose wasadded for an additional hour. The immune complex was washed three times in the

lysis buffer and resolved by SDS-PAGE and transferred to a nitrocellulosemembrane. Membranes were blocked in 3% BSA in TBST (10 mM Tris pH 8.0,

150 mM NaCl, 2.5 mM EDTA, 0.1% Tween 20) for 1 hour, incubated withprimary and secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.,

West Grove, PA) in TBST for 2 hours and 1 hour, respectively. After four washeswith TBST, bound proteins were visualized with an ECL detection kit (Amersham

Biosciences) or Odessey detection system (Li-Cor, Lincoln, NE). Densitometricanalysis of western blots was performed using Odyssey software.

Fluorescent gelatin degradation assay

Coverslips were coated with poly-D lysine for 20 minutes, washed three times withPBS, incubated for 20 minutes in 0.4% glutaraldehyde (Sigma-Aldrich) and

washed three times with PBS. Oregan-Green-conjugated gelatin (Invitrogen) wasdiluted to 20 mg/ml in 0.1% unconjugated gelatin (Stem Cell Technologies,

Vancouver, BC, Canada) and incubated on the coverslips at 37 C for 1 hour,washed three times with PBS and quenched with 70% ethanol for 20 minutes.

Finally Oregan-Green-conjugated gelatin-coated coverslips were washed withDMEM, and 50,000 cells were plated and incubated at 37 C in 5% CO2 for

24 hours unless specified otherwise. Cells were fixed with 4% paraformaldehyde

and continued with regular immunofluorescence protocol, as described previously(Abella et al., 2010). Samples were mounted using Immunomount from Thermo

Scientific (Pittsburgh, PA) and images were acquired using a 1006objective on aconfocal microscope, unless mentioned otherwise.

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Live cell imaging

Cells grown on gelatin-coated glass coverslips (35 mm) were positioned on themotorized stage on the Axiovert 200 M (Carl Zeiss, Inc.) inverted microscope,equipped with 1006 plan Apochromat NA 1.4 objective and an AxioCam HRMdigital camera, and equipped with a small transparent environmental chamber,Climabox (Carl Zeiss, Inc.) with 5% (v/v) CO2 in air at 37 C. The microscope wasdriven by AxioVision LE software (Carl Zeiss, Inc.). The motorized stageadvanced to pre-programmed locations and photographs were collected for 30minutes.

Far-western blotting

HEK 293 cells were transiently transfected with the indicated constructs,immunoprecipitated and separated by SDS-PAGE and transferred tonitrocellulose membranes. The membranes were incubated with either cortactinGST–SH3 or GST–SH3–W525K fusion proteins in lysis buffer A (20 mM HepespH 7.5, 120 mM NaCl, 2 mM EDTA, 10% glycerol, 1 mM PMSF, 10 mg/mlaprotinin and 10 mg/ml leupeptin) and bound GST-fused proteins were detectedusing anti-GST antibodies.

AcknowledgementsWe thank members of the Park lab for their helpful comments on themanuscript. We thank Claire Brown and Aleksandrs Spurmanis fortheir help with deconvolution of confocal Z-stacks using IMARISsoftware. We thank Ken McDonald for his help with FACS. Wewould like to thank Genentech Inc. for HGF and Marina Holgado-Madruga for Gab1-null cells.

FundingThis work was supported by a fellowship from Canadian Institutes ofHealth Research [grant number CGD-96470 to C.V.R.]; the USDepartment of Defense Breast Cancer Research Initiative [grantnumber XWH-09-1-00 to R.V.]; Canadian Institutes of HealthResearch/Fonds de la Recherche en Sante du Quebec training grantin cancer research from the McGill Integrated Cancer ResearchTraining Program [grant number FRN53888 to S.H. and K.Z.]; andby an operating grant from the Canadian Institutes of HealthResearch [grant number MOP-106635 to M.P.]. M.P. holds the Dianeand Sal Guerrera Chair in Cancer Genetics. Deposited in PMC forimmediate release.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.100834/-/DC1

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Met RTK induces invadopodia through Gab1 2953