met receptor tyrosine kinase signals through a cortactin ... · protein grb2-associated-binding...
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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