Invasive matrix degradation at focal adhesions occurs via protease recruitment by a FAK-p130Cas complex.

Abstract

Tumor cell migration and the concomitant degradation of extracellular matrix (ECM) are two essential steps in the metastatic process. It is well established that focal adhesions (FAs) play an important role in regulating migration; however, whether these structures contribute to matrix degradation is not clear. In this study, we report that multiple cancer cell lines display degradation of ECM at FA sites that requires the targeted action of MT1-MMP. Importantly, we have found that this MT1-MMP targeting is dependent on an association with a FAK-p130Cas complex situated at FAs and is regulated by Src-mediated phosphorylation of Tyr 573 at the cytoplasmic tail of MT1. Disrupting the FAK-p130Cas-MT1 complex significantly impairs FA-mediated degradation and tumor cell invasion yet does not appear to affect invadopodia formation or function. These findings demonstrate a novel function for FAs and also provide molecular insights into MT1-MMP targeting and function.

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JCB: Article
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J. Cell Biol. Vol. 196 No. 3 375–385
www.jcb.org/cgi/doi/10.1083/jcb.201105153
JCB375
Correspondence to Mark A. McNiven: mcniven.mark@mayo.edu
Abbreviations used in this paper: FA, focal adhesion; FAK, focal adhesion
kinase; FAT, focal adhesion targeting; MMP, matrix metalloproteinase; PRR,
proline-rich region.
Introduction
It is well known that invading tumor cells are surrounded by
stroma and extracellular matrix (ECM) that is either displaced
or degraded during the metastatic process. This degradative
action is mediated by specialized organelles termed “invadopodia”
that are centrally situated, dot-shaped actin-rich membrane pro-
trusions that extend into the matrix-filled surroundings (Linder,
2007; Buccione et al., 2009; Caldieri et al., 2009). Active inva-
dopodia are known to markedly potentiate invasion and are
a hallmark of many types of cancer cells (Linder, 2007). Inva-
dopodia perform both secretory and endocytic processes by
mediating the targeted release of zinc-dependent matrix metal-
loproteinases (MMPs; Egeblad and Werb, 2002; Caldieri and
Buccione, 2010) at the cell base and the subsequent internal-
ization of the digested matrix for further processing by the lyso-
some (Coopman et al., 1996, 1998). Thus, cytoskeletal and
membrane dynamics play an essential role in supporting inva-
dopodia function (Gimona et al., 2008; Caldieri et al., 2009).
Both maturation and function of invadopodia are MMP
dependent (Artym et al., 2006). To date, 25 MMPs have been
identified in humans and are classified into either secreted
MMPs or membrane-type MMPs (MT-MMPs). These two fam-
ilies share many structural elements except that MT-MMPs
contain transmembrane or other membrane-tethering domains
(Egeblad and Werb, 2002), and both have been identified at
invadopodia. Among the known MMPs, MT1-MMP (MMP-14)
is the best characterized and is believed to be the most preva-
lent form (Sabeh et al., 2009). MT1-MMP contains a single
transmembrane domain, a 20-amino acid cytoplasmic tail,
and is up-regulated in many types of cancer (Egeblad and
Werb, 2002; Sato et al., 2005). Inhibition of MT1-MMP has
been shown to significantly impair tumor cell invasion; how-
ever, the spatial and temporal regulation of MT1-MMP is
not fully understood.
Although invadopodia are the primary site of action for
MT1-MMP, it is also believed to function at other cellular loca-
tions such as lamellipodia where it is recruited via an interaction
with CD44 (Mori et al., 2002). Moreover, Takino et al. (2006,
2007) have shown that in MT1-MMP–overexpressing Hela
cells, ECM degradation takes place at the leading edge and in
close proximity to focal adhesions (FAs). This is particularly
interesting as FAs and invadopodia are related organelles that
share many structural and regulatory components. In contrast to
the dot-shaped invadopodia, FAs are streak-like structures at the
basal membrane that bridge the actin cytoskeleton to the ECM,
T
that focal adhesions (FAs) play an important role in regu-
lating migration; however, whether these structures con-
tribute to matrix degradation is not clear. In this study, we
report that multiple cancer cell lines display degradation
of ECM at FA sites that requires the targeted action of
MT1-MMP. Importantly, we have found that this MT1-MMP
umor cell migration and the concomitant degrada-
tion of extracellular matrix (ECM) are two essential
steps in the metastatic process. It is well established
targeting is dependent on an association with a FAK–
p130Cas complex situated at FAs and is regulated by
Src-mediated phosphorylation of Tyr 573 at the cytoplas-
mic tail of MT1. Disrupting the FAK–p130Cas–MT1 com-
plex significantly impairs FA-mediated degradation and
tumor cell invasion yet does not appear to affect invado-
podia formation or function. These findings demonstrate a
novel function for FAs and also provide molecular insights
into MT1-MMP targeting and function.
Invasive matrix degradation at focal
adhesions occurs via protease recruitment
by a FAK–p130Cas complex
Yu Wang and Mark A. McNiven
Department of Biochemistry and Molecular Biology, and the Center for Basic Research in Digestive Diseases, Mayo Clinic and Graduate School, Rochester, MN 55905
© 2012 Wang and McNiven This article is distributed under the terms of an Attribution–
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T H E J O U R N A L O F C E L L B I O L O G Y

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JCB • VOLUME 196 • NUMBER 3 • 2012 376
MMP, not a secreted form, was responsible for degradation at
FAs. MT1-MMP is the most prominent member of MT-MMPs
and has been shown by many others to support ECM degradation
(Sato et al., 2005; Itoh, 2006; Strongin, 2006). As shown in
Fig. 1, c–c’, knocking down MT1 in HT-1080 cells (dashed
line) significantly impaired ECM degradation, including the
and the dynamic turnover of FAs is essential for the motility
of both normal and tumor cells (Mitra et al., 2005; Gimona
and Buccione, 2006). Despite distinct morphologies, FAs
and invadopodia share many components including integrins,
talin, paxillin, actin, cortactin, and dynamin, to name just a few
(Linder and Aepfelbacher, 2003). A central component of FAs
that provides both a scaffolding and signaling function is focal
adhesion kinase (FAK). Although not observed at invadopodia
(Bowden et al., 2006), FAK has been implicated in the regu-
lation of invadopodia formation by modulating downstream
signaling through Src and p130Cas (Hsia et al., 2003; Chan
et al., 2009). Despite these molecular similarities, FAs are gen-
erally perceived to mediate cell migration but are not involved
in matrix degradation.
Recently we have observed that a variety of cancer cell
lines degrade matrix not only at invadopodia, but also at nu-
merous peripheral sites that resemble FAs. Here we have
confirmed that this peripheral ECM degradation represents
bona fide FA sites, and have further revealed that this FA-type
degradation is MT1-MMP dependent. Moreover, we find that
this protease is targeted to FAs by a physical interaction
between MT1-MMP and a FAK–p130Cas complex in response
to the activation of Src. Formation of this FAK–p130Cas–MT1
complex is essential for the FA-centric ECM degradation
as well as invasive migration of tumor cells through Matrigel.
Results
MT1-MMP mediates matrix degradation
at focal adhesions
Matrix degradation by a wide variety of different tumor cell
lines appears to occur at linear, peripherally located FA-like sites
as well as the well-characterized, centrally situated and dot-
shaped invadopodia. To determine if this pattern represents the
action of FAs, human fibrosarcoma cells (HT-1080) were plated
on fluorescent gelatin-coated coverslips for 6 h, and stained
for the FA marker vinculin. As shown in Fig. 1 a, many of the
peripheral, linear degradation sites colocalized with vinculin.
The enlarged images reveal that mature FAs (Fig. 1 a’) gen-
erated a solid, dark pattern of degradation whereas nascent
FAs (Fig. 1 a’’) appeared to be in the process of degrading
matrix. A similar result was obtained from several cell lines
(Fig. S1). As many actin stress fibers terminate in FAs, we colo-
calized actin with a FA marker, and sites of matrix degradation
as just one feature of many used to distinguish FAs from other
adhesive structures (Fig. S1, c and d). It is well known that cells
exert physical tension on the ECM at FA sites that can result
in matrix tearing. To distinguish enzymatic activity from physical
effects, HT-1080 cells were treated with 10 µM of the MMP
inhibitor BB94 (Davies et al., 1993) as the cells were plated on
gelatin. As shown in Fig. S2 (a–a’), overall degradation includ-
ing the ones at FAs were completely inhibited, while no sig-
nificant change in FA morphology was observed. From these
observations we conclude that ECM degradation at FAs occurs
by the proteolytic activity of MMPs.
Because the FA-like degradation was sharply focused rather
than diffuse in nature, we hypothesized that a membrane-type
Figure 1. MT1-MMP mediates ECM degradation at focal adhesions. (a) To
test if extracellular matrix degradation occurs at focal adhesions, HT-1080
cells were plated on fluorescent gelatin (green)-coated coverslips for 6 h
before fixation and staining with a vinculin antibody (red). Matrix degrada-
tion occurred at vinculin-positive FA sites. Boxed regions provide a higher
magnification showing the correlation between vinculin and matrix degra-
dation in the gelatin matrix (dark spots in a’ and a’’). (b–c’) To define if
MT1-MMP is required for the FA-centered degradation, HT-1080 cells
were treated with MT1 siRNA for 72 h to knock down endogenous
MT1-MMP (b), and then were plated on gelatin-coated coverslips (c and
c’). MT1 knockdown cells (dashed lines) exhibit a significant reduction in
matrix degradation. (d) Quantification of the percentage of cells (in c) that
degraded ECM at FAs indicates that MT1 is required for this type of degra-
dation. Values represent the mean of three independent experiments ± SD.
(e and f) In reciprocal experiments MT1-MMP was overexpressed in a
human pancreatic tumor cell line (PANC-1) that does not express endogenous
MTI-MMP (e) or degrade matrix (Fig. S2). Just 2 h after plating on gelatin
the transfected PANC-1 cells exhibited substantial matrix degradation at
FA sites (f–f’’). Additional examples of pancreatic tumor cells degrading
matrix at FAs are provided in Fig. S1. Bars, 10 µm.

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377 Selective recruitment of MT1-MMP to focal adhesions • Wang and McNiven
to these sites. To test this possibility, MT1-GFP was over-
expressed in PANC-1 cells, then immunoprecipitated (IP) with
a GFP antibody and blotted for several core FA proteins, in-
cluding FAK, paxillin, vinculin, talin, and 1-integrin. Of all
these proteins, FAK was by far the most prominent interacting
partner (Fig. 2 a). As FAK localizes to FAs and is generally
not considered an invadopodial component (Bowden et al.,
2006; Chan et al., 2009), this FAK–MT1 interaction was pre-
dicted to occur at FAs. To define this novel interaction, we
generated a series of FAK truncation mutants as illustrated in
Fig. 2 b. PANC-1 cells expressing MT1-GFP were lysed and
incubated with different GST-tagged FAK domains to test
for interaction. As shown in Fig. 2, c and d, the proline-rich
region (PRR) of FAK is sufficient to pull down MT1. To
confirm this finding, MT1-GFP and SFB-tagged (SFB: S pro-
tein, FLAG, and streptavidin-binding peptide triple tagged)
ones at FAs (Fig. 1 d). To further test the role of MT1-MMP in
ECM degradation, we used a pancreatic cancer cell line (PANC-1)
that does not express endogenous MT1 (Fig. 1 e). Although
gelatin degradation was not observed by these cells even 24 h
after plating (Fig. S2 b-b’), some FA-type degradations were
observed in cells transfected with MT1-GFP just 2 h after plat-
ing on gelatin (Fig. 1 f). The same findings were obtained by
cells expressing either an internal mCherry-tagged (see Fig. 5 h)
or a nontagged MT1, indicating that this protease is essential
for FA-mediated degradation.
A physical interaction between MT1-MMP
and FAK is required for matrix degradation
at focal adhesions
As MT1-MMP mediates ECM degradation at FAs, it is likely
that a FA component acts to bind and recruit this protease
Figure 2. An interaction between FAK and MT1
is required for matrix degradation at focal adhe-
sions. (a) To test for FA proteins that might interact
with MT1, PANC-1 cells were transfected with vec-
tors encoding either GFP alone or MT1-GFP then
lysed and IP with a GFP antibody. The pellet was
then subjected to Western blot analysis using anti-
bodies against several major FA proteins. FAK
is the most prominent MT1-interacting protein of
those tested. (b) Illustration of FAK and the dele-
tion constructs used to map FAK-MT1 binding.
(c and d) Western blot analysis of homogenates
from PANC-1 cells expressing MT1-GFP and incu-
bated with different GST-FAK fragments for 2 h.
Importantly, only portions of FAK that contain the
PRR domain were able to interact with MT1. (e) To
further define the importance of the PRR domain
in MT1 binding, MT1-GFP was coexpressed with
SFB-tagged FAK WT, the PRR domain deletion
mutant (PRR), or point mutations that disrupt the
second and third PXXP motif (P2/3A), followed by
IP with S-protein beads. Deletion or mutation of
the PRR domain significantly impaired FAK–MT1
interactions. To test if FAK is required for degrada-
tion at FAs, HT-1080 cells were treated with FAK
siRNA (f), then plated on gelatin for 16 h before
IF analysis. FAK knockdown significantly reduced
degradations at FAs (g). Re-expression of FAK
Y397F rescued FA-type degradation (h), whereas
FAK Y397F carrying P2/3A mutation (i) had only
a minimal effect. (j) The percentages of cells (g–i)
that degraded ECM at FAs were quantified.
Values represent the mean of three independent
experiments ± SD. Bars, 10 µm.

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JCB • VOLUME 196 • NUMBER 3 • 2012 378
The cytoplasmic tail of MT1-MMP
interacts with FAK indirectly
through p130Cas
MT1-MMP is a transmembrane protein with a 20-amino acid
cytoplasmic tail (CP; Fig. 3 a) that is known to interact with a
variety of cellular proteins such as MTCBP-1 and AP2 (Uekita
et al., 2004). To test if FAK also interacts with this domain, we
generated a MT1 mutant missing the cytoplasmic tail (MT1CP)
and analyzed its interaction with FAK via GST pull-down
assay. As predicted, removal of the CP domain completely
abolished the FAK–MT1 interaction (Fig. 3 b), thus this mutant
is likely defective in mediating degradation at FAs. Indeed, in
contrast to MT1 WT (Fig. 1 f and Fig. S4 c), MT1 CP express-
ing PANC-1 cells failed to generate any gelatin degradation
at FAs (Fig. 3c). As this effect could result from defects in
either MT1 targeting or enzymatic activity, we generated a chi-
meric protein by replacing the MT1 cytoplasmic tail with
the focal adhesion targeting domain of FAK (FAT). Thus, the
cytoplasmic tail truncated protease could be constitutively
targeted to FAs. As shown in Fig. 3 d, PANC-1 cells expressing
MT1CP-FAT generated excessive degradation at FA sites
(Fig. 3 e), indicating that MT1CP remains enzymatically
active but cannot be targeted to FAs.
Interaction between MT1-MMP and FAK is essential for
FA-centric matrix degradation; however, whether this inter-
action is direct was unclear. To test this, we purified His-MT1
protein from Escherichia coli, and performed direct binding
analysis with GST-PRR of FAK. No direct interaction was de-
tected (unpublished data), suggesting the participation of an
adaptor. One candidate known to bind directly to both MT1
FAK WT or a PRR mutant were expressed in 293T cells,
followed by a FAK IP with S protein beads. As predicted,
FAKPRR did not interact with MT1 (Fig. 2 e, lane 3). Within
the FAK PRR domain the most characteristic features are
two conserved PXXP motifs (second and third in the full-
length protein; Mitra et al., 2005; Wu et al., 2005). Mutations
of these two motifs (FAK P2/3A) did not fully abolish, but
significantly impaired FAK’s interaction with MT1-MMP
(Fig. 2 e, lane 4). Taken together, these data suggest that two
PXXP motifs within the PPR domain of FAK are essential
to interact with MT1-MMP.
To test if FAK–MT1 association is important for matrix
degradation at FA sites, HT-1080 cells were treated with FAK
siRNA to knock down endogenous FAK (Fig. 2 f), then were
plated on gelatin-coated coverslips for 16 h before IF analysis.
Interestingly, degradation at FAs was significantly compro-
mised in FAK knockdown cells (Fig. 2 g). To further confirm
the role of FAK, FAK knockdown cells were rescued with
siRNA-resistant rat-FAK. Surprisingly, WT FAK did not sig-
nificantly increase degradation at FAs; however, expression
of a FAK Y397F mutant rescued degradation to control levels
(Fig. 2 h). As discussed later, this result is likely due to the fact
that expression of WT FAK promotes FA turnover while the
YF mutant is known to stabilize this structure (Hamadi et al.,
2005; Wang et al., 2011). Accordingly, expression of a FAK
double mutant, Y397F carrying the P2/3A mutation that re-
duces MT1 binding, did not rescue matrix degradation at FAs
(Fig. 2 i). Taken together, we conclude that FAK interacts with
MT1-MMP through the PRR domain and this interaction is
important for FA-mediated degradation.
Figure 3. The cytoplasmic tail of the MT1 associ-
ates with FAK. (a) Cartoon illustrating the domain
structures of MT1-MMP that includes the Pro pep-
tide, catalytic domain, hemopexin domain (Hpx),
transmembrane domain (TM), and a cytoplasmic
tail (CP) of only 20 amino acids. (b) To test if FAK
interacts with the MT1 cytoplasmic tail, PANC-1
cells were transfected with either full-length MT1
or MT1 missing the cytoplasmic tail (CP), and
subjected to a pull-down assay with GST-FRNK.
Although a large amount of MT1 WT was pulled
down by GST-FRNK, almost none of MT1CP ap-
peared to associate. To test if MT1CP remains
competent to degrade matrix at FAs, PANC-1 cells
were plated and viewed as performed in Fig 1 f.
The MT1CP-expressing cells failed to degrade
any matrix (c) whereas cells expressing an MT1
CP fused with FAT domain, known to target pro-
teins to FAs, exhibited excessive degradation at
FA sites (d, arrow), suggesting that the MT1CP
protein has defective targeting but not enzymatic
activity. (e) The percentages of cells that degrade
ECM at FAs were quantified. Values represent the
mean of three independent experiments ± SD.
Bars, 10 µm.

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379 Selective recruitment of MT1-MMP to focal adhesions • Wang and McNiven
p130Cas and then performed pull-down assays with the puri-
fied GST-PRR of FAK. As shown in Fig. 4 a, p130Cas strongly
binds to GST-PRR, and overexpression of p130Cas signifi-
cantly enhances the amount of MT1-MMP in the pulldown,
suggesting that p130Cas facilitates MT1–FAK interaction.
(Gingras et al., 2008; Gonzalo et al., 2010) and to the second
and third PXXP motifs of FAK is p130Cas (Harte et al., 1996;
Mitra et al., 2005). However, whether these three proteins form
a functional complex is not clear. To test this possibility, we
transfected 293T cells with either MT1 alone or together with
Figure 4. Interaction between MT1 and FAK is mediated by p130Cas. (a) To test if p130Cas adapts MT1 to FAK, 293T cells were transfected with MT1-GFP
alone or together with HA-p130Cas, and the homogenates were incubated with the GST-PRR domain of FAK. Although GST-PRR exhibited modest
interaction with MT1, this was markedly increased when p130Cas was coexpressed. (b) To further test p130Cas as an adaptor between MT1 and FAK,
HT-1080 cells were treated with control or p130Cas siRNA, then lysed and IP with a FAK antibody. Although both p130Cas and MT1 were coprecipitated
in control cells, MT1 coIP was significantly reduced in p130Cas knockdown cells, suggesting p130Cas links MT1 to FAK. (c) Illustration of the domains of
full-length Cas and the truncation mutants used to study MT1 interaction. SP, SH3 and proline rich domain; SD, substrate domain; SC, serine-rich region and
C terminus. (d and e) MT1-GFP was coexpressed with the indicated SFB-tagged Cas mutants in 293T cells, and IP with S-beads. The substrate domain alone
is sufficient to interact with MT1 (d), whereas deletion of this domain abolished Cas–MT1 interaction (e). To test if p130Cas is required for ECM degrada-
tion at FAs, HT-1080 cells were treated with p130Cas siRNA (f) and plated on gelatin for 16 h. p130Cas knockdown significantly reduced degradation at
FAs (g). Re-expression of Cas WT (h) but not the MT1-binding mutant (i) rescued degradation at FAs. (j) The percentages of cells (g–i) that degraded ECM
at FAs were quantified. Values represent the mean of three independent experiments ± SD. Bars, 10 µm.