Post on 12-Oct-2020
Page 1 of 29
Regulation of sarcoma cell migration, invasion and invadopodia
formation by AFAP1L1 through a phosphotyrosine-dependent pathway
Sien R. TIE1,4, David J. McCARTHY1,4, Tulene S. KENDRICK1, Alison LOUW1,2, Cindy
LE1, Jiulia SATIAPUTRA1, Nicole KUCERA1, Michael PHILLIPS3, and Evan INGLEY1*
1 Cell Signalling Group, Harry Perkins Institute of Medical Research and
Centre for Medical Research, The University of Western Australia, Nedlands, WA
6009, Australia.
2 Royal Perth Hospital Medical Research Foundation, Royal Perth Hospital,
Perth, WA 6000, Australia.
3 Harry Perkins Institute of Medical Research and Centre for Medical
Research, The University of Western Australia, Perth, WA 6000, Australia.
4 Contributed equally to this study.
* Corresponding Author:
Associate Professor Evan Ingley
Harry Perkins Institute of Medical Research
Level 7, QQ Block, QEII Medical Centre
6 Verdun Street
Nedlands, WESTERN AUSTRALIA 6009
Telephone: 61-08-6151-0738
Facsimile: 61-08-6151-0701
Email: evan.ingley@uwa.edu.au
Running title: AFAP1L1 binds Nck2 and Vav2 at sarcoma invadopodia
Word count: 4,468
Page 2 of 29
ABSTRACT
Invasion and metastasis are controlled by the invadopodia, which delivers matrix-
degrading enzymes to the invasion interface permitting cancer cell penetration and spread
into healthy tissue. We have identified a novel pathway that directs Lyn/Src family tyrosine
kinase signals to the invadopodia to regulate sarcoma cell invasion via the molecule
AFAP1L1, a new member of the AFAP (Actin Filament Associated Protein) family. We
show that AFAP1L1 can transform cells, promote migration, and co-expression with active
Lyn profoundly influences cell morphology and movement. AFAP1L1 intersects several
invadopodia pathway components through its multiple domains and motifs, including; (i)
pleckstrin homology domains that bind phospho-lipids generated at the plasma membrane
by PI-3 kinase, (ii) a direct filamentous-actin binding domain, and (iii) phospho-tyrosine
motifs (pY136 and pY566) that specifically bind Vav2 and Nck2 SH2 domains,
respectively. These phosphotyrosine motifs are essential for AFAP1L1-mediated
cytoskeleton regulation. Through its interaction with Vav2, AFAP1L1 regulates Rac activity
and down-stream control of PAK1/2/3 (p21-activated kinases) phosphorylation of myosin
light chain kinase (MLCK) and myosin light chain-2 (MLC2). AFAP1L1 interaction with
Nck2 recruits actin-nucleating complexes. Significantly, in osteosarcoma cell lines
knockdown of AFAP1L1 inhibits phosphorylated MLC2 recruitment to filamentous-actin
structures, disrupts invadopodia formation, cell attachment, migration and invasion. These
data define a novel pathway that directs Lyn/SFK tyrosine kinase signals to sarcoma cell
invadopodia through specific recruitment of Vav2 and Nck2 to phosphorylated AFAP1L1 to
control cell migration and invasion.
Key words: Sarcoma, migration, Invadopodia, Nck2, Vav2, AFAP1L1
Page 3 of 29
INTRODUCTION
The invasive cell phenotype is mediated by invadopodia, which are actin-rich
matrix-degrading protrusive subcellular structures that form on the ventral cell surface and
are critically controlled by Src family tyrosine kinases (SFK).8, 34, 39, 46, 54 Through
invadopodia-mediated delivery of metalloprotease (MMP)-containing vesicles via the
vesicle-tethering exocyst complex,38 they facilitate basement membrane and extracellular
matrix degradation 43. Several actin-binding adaptor proteins are utilized in the initiation,
assembly and maturation of invadopdia, including the recently characterized actin filament
associated protein (AFAP) family.17, 45 As with the actin related protein complex-2/3
(Arp2/3) and cortactin, the neuronal Wiskott-Aldrich syndrome protein (N-WASp) binding
adaptor,37 AFAP family members are SFK substrates that localize to invadopodia.14, 45 SFK
regulate invadopodia initiation and assembly down-stream of receptor tyrosine kinases
and integrins by stimulating GTPases such as Rac1 and cdc42,35 as well as via
phosphorylating substrates/scaffolds such as tyrosine kinase substrate-5 (Tks5).30 In
addition, cortactin brings together actin nucleation regulators including Arp2/3, N-WASp,
WASp interacting protein (WIP) and cofilin.37, 44
AFAP family proteins also intersect protein kinase-C (PKC) mediated invadopodia
regulation.18 However, the molecular pathways modulated by SFK phosphorylation of
AFAP proteins and their consequences have not been delineated. To address this
important question, we analysed the interactions, pathways and biological consequences
of SFK tyrosine phosphorylation of AFAP-1-Like-1 (AFAP1L1) in sarcoma cell
invadopodia. The SFK member Lyn binds and phosphorylates AFAP1L1 at two tyrosine
residues, Y136 and Y566, mediating specific interaction with Vav2, a Rac-guanidine
nucleotide exchange factor, and Nck2, the N-WASp/WIP/cortactin binding adaptor,
respectively. AFAP1L1 localizes to invadopodia in sarcoma cells and mutation of its Vav2
and Nck2 binding motifs (Y136, Y566) mitigates its ability to regulate cytoskeletal changes
through Nck2 and Vav2. Knockdown of AFAP1L1 in sarcoma cells inhibits invadopodia re-
formation, cell attachment, migration and invasion. Conversely, ectopic expression of
AFAP1L1 alters cell movement and promotes mitogen gradient-directed cell migration and
an invasive phenotype. These data illustrate a novel pathway that directs Lyn/SFK tyrosine
kinase signals to the invadopodia in sarcoma cells through specific pY-motif mediated
interaction of AFAP1L1 with Vav2 and Nck2 to regulate cell migration and invasion.
Page 4 of 29
RESULTS
AFAP1L1 localizes to invadopodia in sarcoma cells and binds F-actin.
We utilized an AFAP1L1 specific antibody (Supplementary Figure S1) to assess
AFAP1L1 expression in human osteosarcoma cell lines. Immunoblot analysis detected
robust expression of AFAP1L1 in the invadopodia forming U2OS and MG-63
osteosarcoma cells lines (Figure 1a). Intriguingly, endogenous AFAP1L1 localized to the
F-actin rich sub-nuclear ventral invadopodia structures in U2OS cell (Figure 1b). To
facilitate live cell imaging and manipulation of AFAP1L1 in sarcoma cells (U2OS, MG-63)
we generated stable lines expressing eGFP-tagged AFAP1L1, selecting cells that
expressed levels of the tagged AFAP1L1 similar to endogenous levels. Immunoflourescent
analysis of these lines showed that the tagged AFAP1L1 also localized to the F-actin rich
sub/proximal-nuclear ventral invadopodia structures in these sarcoma cells (Figure 1c, 1d,
Movie S1, Movie S2). Further, when grown in 3D solid extracellular matrix cultures
AFAP1L1 localized to the typical comet-like F-actin rich invadopodia structures that
projected deep into the extracellular matrix (Figure 1e). AFAP1L1 also strongly co-
localized with the invadopodia marker cortactin in sarcoma cells U2OS (Figure 1f) and
MG-63 (Supplemental Figure S2).
In COS7 cells, which do not readily form invadopodia, AFAP1L1 co-localization with
F-actin structures (Figure 1g). Bioinformatic analysis of the AFAP1L1 protein sequence
identified a potential F-actin binding region in the carboxyl leucine-rich/alpha-helical 620-
777 amino acids. To directly test this region for F-actin binding, a co-sedimentation assay
was performed using polymerized F-actin and a purified GST-fusion of the 620-777aa
region of AFAP1L1. This region of AFAP1L1 did indeed show strong direct binding to F-
actin (Figure 1h). Interestingly, this region of AFAP1L1 also showed the capacity to
multimerize when tested by yeast two-hybrid analysis (Figure 1i). We then assessed the
ability of this region to direct AFAP1L1 localization to invadopodia in U2OS sarcoma cells
(Figure 1j). Importantly, deletion of the C-terminal region (630-777aa) of AFAP1L1
completely eliminated co-localization with all F-actin structures and resulted in localization
of AFAP1L1 to the nucleus in U2OS sarcoma cells (Figure 1j; (i), (ii)). Expression of the C-
terminal 620-777aa region alone showed that this region directed a strong co-localization
with F-actin (Figure 1j; (iii)). However, compared to the full-length molecule little
localization to invadopodia could be detected, indicating that while this region promotes
strong F-actin localization other regions are required for localization to the invadopodia F-
actin fraction. Deletion of the individual C-terminal homology domains, the coiled-coil (iv),
leucine-rich (v), and alpha-helical region (vi) showed that removal of the coiled coil region
Page 5 of 29
disrupted localization to invadopodia, although it could still co-localize with other F-actin
structures. However, deletion of the leucine-rich or alpha-helical domains fully disrupted
localization to all F-actin structures and the mutated AFAP1L1 now localized to nuclear
puncta. Further, the coiled-coil mutant of AFAP1L1 also localized to non-F-actin containing
punctate structures close to the F-actin containing inadopodia that may be sites of
invadopodia formation prior to localized F-actin assembly.
Down-regulation of AFAP1L1 in sarcoma cells inhibits invadopodia formation,
cell attachment, migration, and invasion.
To define the importance of AFAP1L1 to sarcoma cells and invadopodia we
undertook RNAi mediated knockdown of AFAP1L1 in U2OS and MG-63 sarcoma cells and
analysed the biological consequences. Two independent siRNA oligonucleotides (S3, S4)
could efficiently mediate strong reduction in AFAP1L1 protein in both U2OS and MG-63
sarcoma cells (Figure 2a). Knockdown of AFAP1L1 did not significantly affect proliferation
of U2OS cells (Supplementary Figure S3). The ability of U2OS cells to attach and spread
on tissue culture dishes in the absence of serum was greatly diminished with knock-down
of AFAP1L1 as measured by cell foot print area (Figure 2b) and cellular impedance
(xCELLigence E-plates) (Figure 2c). Knockdown of AFAP1L1 also reduced the ability of
these cells to migrate in response to a serum gradient (on serum coated surfaces to
mitigate their spreading defect in the absence of serum) as measured by Boyden chamber
assays (Figure 2d), and their ability to form invadopodia (Figure 2e). Additionally, in MG-63
cells there were also significant effects on the cytoskeleton, in particular their ability to
reform invadopodia with vanadate stimulation after serum starvation (Figure 2f).
Importantly, AFAP1L1 was required for efficient invasion through extracellular matrix
coated with serum for U2OS cells as measured in Matrigel coated Boyden chamber
assays (Figure 2g).
As AFAP1L1 knockdown in U2OS cells impeded invadopodia formation we tested if
re-expression of wild-type AFAP1L1 or mutants of AFAP1L1 could rescue this phenotype.
Indeed U2OS cells treated with the S3 RNAi and transfected with mouse AFAP1L1
reformed invadopodia structures (Supplemental Figure S4(i-ii)). However, expression of
the C-terminal deleted mutant (AA 1-630) or the C-terminal region alone (AA 620-777)
failed to re-form invadopodia structures (Supplemental Figure S4(iii-iv)).
Page 6 of 29
AFAP1L1 interacts with and is phosphorylated by the SFK Lyn, and
stimulation of tyrosine phosphorylation regulates total AFAP1L1 localization to
invadopodia.
Through a yeast two-hybrid (Y2H) screen, AFAP1L1 was isolated as a binding
partner of the SFK member Lyn (Figure 3a). This assay showed that Lyn bound to
AFAP1L1 through its SH3 domain (Figure 3a). This interaction was supported through co-
immunoprecipitation and co-localization of Lyn and AFAP1L1 when ectopically expressed
in COS-7 cells (Figure 3b, c). Interestingly, co-expression with Lyn increased the level of
tyrosine phosphorylation of AFAP1L1, suggesting that Lyn might phosphorylate AFAP1L1
(Figure 3b). This possibility was tested directly with kinase assays using
immunoprecipitates of inactive (Y397F) or hyperactive (Y508F) Lyn and a purified GST-
fusion of AFAP1L1. Significantly, these experiments demonstrate that active Lyn could
interact with and directly phosphorylate AFAP1L1 (Figure 3d).
When U2OS (expressing eGFP-AFAP1L1) sarcoma cells were starved overnight
and then stimulated with vanadate and visualized by live cell imaging, AFAP1L1 localized
to invadopodia within 5min of stimulation and continued to accumulate in invadopodia over
a 30min time course (Figure 3e, Movie S3, Movie S4).
Heterologous expression of AFAP1L1 promotes anchorage-independent
growth, serum-directed migration and an invasive phenotype.
Stable ectopic expression of AFAP1L1 was undertaken in NIH3T3 cells as they did
not express any detectable endogenous AFAP1L1 and are commonly used as a non-
transformed cell line that are sensitive to oncogenic transformation, especially by SFK
pathway oncogenes. This showed enforced expression of AFAP1L1 resulted in
anchorage-independent growth (cellular transformation) as demonstrated by markedly
enhanced colony formation in soft-agar assays (Figure 4a). This observation indicates that
raising the level of AFAP1L1 may promote oncogenic transformation. In NIH3T3 cells
expressing AFAP1L1 increasing Lyn activity via wild-type Lyn expression resulted in
altered cell morphology. This was accentuated further with hyperactive Lyn (LynY508F)
(Figure 4b). Interestingly, these cells also displayed a more “transformed” spindle-shaped
phenotype with reduced spreading on tissue culture dishes (Figure 4b). In scratch/wound-
healing assays elevated expression of AFAP1L1 led to a decrease in migration rate of
NIH3T3 cells (Figure 4c).
We also tested the effect of overexpression of AFAP1L1 in HEK293 cells, which
express low levels of AFAP1L1 but are also non-transformed similar to NIH3T3 cells. In
Page 7 of 29
these cells overexpression of AFAP1L1 did not significantly influence proliferation of the
cells (Supplemental Figure S5). We found significant influence of AFAP1L1 expression on
cell attachment, spreading, movement, migration and response to extracellular matrix
(Figure 4d-f, Supplemental Figure S6a, b). Elevated AFAP1L1 reduced the ability of
HEK293 cells to attach and spread (Figure 4d). Further, tracking the cell movement in low
confluence cultures showed that AFAP1L1 over-expressing cells had a significantly
reduced propensity for stochastic movement (Figure 5e), similar to the reduced migration
(in response to release from contact inhibition) of NIH3T3 cells in the scratch/wound-
healing assay (Figure 4c). Interestingly, these cells also had a dramatic response to
extracellular matrix – elevated AFAP1L1 induced an invasive phenotype where cells grew
into the solid matrix (Figure 4f). Further, HEK293 cells expressing AFAP1L1 grew to larger
and more pleomorphic colonies in liquid cultures supplemented with extracellular matrix
(Supplemental Figure S6a, b). Elevated expression of AFAP1L1 significantly increased
migration in response to a serum gradient in Boyden chamber assays (Figure 4g, h).
Phosphorylation of AFAP1L1 by Lyn/SFK links directly to Vav2 and Nck2, and
the AFAP1L1 PH domains mediate PIP binding.
We utilized our phosphotyrosine (pY) Y2H system24 using AFAP1L1 as the bait
during co-expression with the active Lyn kinase domain, and tested against a Y2H cDNA
library to uncover what pathways phosphorylated AFAP1L1 intersected. This screen
revealed that the SH2 domains of Nck2 and Vav2 could bind to AFAP1L1 in a
phosphotyrosine dependent manner (Figure 5a, b). Clones encoding Vav2 from the pY-
Y2H screen encompassed both its C-terminal SH3 and SH2 domains. When tested
independently both domains bound to AFAP1L1, with the SH3 domain of Vav2 binding a
classical PxPxxP motif within AFAP1L1 close to the tyrosine motif (Y136) responsible for
specific binding to the SH2 domain of Vav2 (Figure 5a(i)-(ii)). Importantly, this interaction
of Vav2 and AFAP1L1 was recapitulated in co-immunoprecipitation and co-localization
experiments in COS7 cells expressing Vav2 and AFAP1L1 (Figure 5a(iii), (iv)). Further, a
purified NusA fusion of the SH2 domain of Vav2 (tagged with a fluorescent label) bound to
the AFAP1L1 band in lysates from COS7 cells co-expressing AFAP1L1 and hyperactive
Lyn (Y508F), whereas the Y136F mutant of AFAP1L1 did not show any binding (Figure
5a(v)).
Clones encoding Nck2 from the pY-Y2H screen only encompassed its C-terminal
SH2 domain, and mutagenesis revealed that this binding was dependent upon tyrosine
566 of AFAP1L1 (Figure 5b(i)) and this interaction was confirmed in co-
Page 8 of 29
immunoprecipitation and co-localization experiments in COS7 cells (Figure 5b(ii), (iii)).
Additionally, a purified NusA fusion of the SH2 domain of Nck2 (tagged with a fluorescent
label) also bound to the AFAP1L1 band in lysates from COS7 cells co-expressing
AFAP1L1 and hyperactive Lyn (Y508F), but not with the Y566F mutant of AFAP1L1
(Figure 5b(iv)). Collectively, these data demonstrate that Nck2 can bind AFAP1L1 via the
C-terminal SH2 domain interacting specifically with the pY566 motif of AFAP1L1.
Interestingly, COS7 cells showed major alterations to their morphology when
AFAP1L1 and hyperactive Lyn were co-expressed - the cells displayed a small rounded
appearance, which was not observed when kinase inactive Lyn was co-expressed with
AFAP1L1 (Figure 5c(i), (ii)). Significantly, only when both sites (Y136 and Y566) were
mutated did the COS7 cell morphology revert to their normal appearance (Figure 5c(iii)-
(v)). Interestingly, in COS7 cells, the related AFAP1L1 molecule AFAP1 mediated the
formation of peripheral podosomes when co-expressed with hyperactive Lyn (Figure
5c(vi)).
Having established a molecular pathway regulated by phosphorylation of AFAP1L1
by Lyn/SFK through specific recruitment of Vav2 and Nck2 utilizing heterologous systems,
we now sought to determine if this pathway held true in sarcoma cells that endogenously
express AFAP1L1. Interestingly, eGFP-tagged AFAP1L1 in sarcoma cells showed highly
dynamic recruitment into invadopodia and movement along stress fibers (Figure 3e, Movie
S3, Movie S4). Further, vanadate stimulation promoted tyrosine phosphorylation of
endogenous AFAP1L1 in U2OS cells (Figure 5d). This phosphorylation was sensitive to
SFK inhibition by the inhibitor PP2, utilizing an antibody that specifically recognizes the
pY136 site of AFAP1L1 (Supplemental Figure S7). Consequently, we then looked to see if
Vav2 and Nck2 associated with AFAP1L1 after vanadate stimulation, and found that this
was indeed the case with both Vav2 and Nck2 co-immunoprecipitating with AFAP1L1 in
U2OS and MG-63 sarcoma cells (Figure 5f, g). Importantly, growth factor/integrin
stimulation of U2OS cells with EGF and fibronectin (FN) also induced Vav2 co-
immunoprecipitation with AFAP1L1 (Figure 5e). Further, we also observed significant co-
localization of both Vav2 and Nck2 with AFAP1L1 in stimulated U2OS cells (Figure 5h(i-
ii)). The importance of the Y136 and Y566 sites for AFAP1L1 involvement in invadopodia
formation was tested on U2OS cells treated with the S3 RNAi to knockdown endogenous
AFAP1L1 and then transfected with mouse AFAP1L1 with the Y136 and Y566 sites
mutated to phenylalanine. This showed that the Y136F/Y566F mutant of AFAP1L1 was
unable to re-form invadopodia structures unlike the wild-type AFAP1L1 protein
(Supplemental Figure S4(i), (vi)). These data provide support for a novel pathway of
Page 9 of 29
AFAP1L1 leading to Vav2 and Nck2 being present in U2OS and MG-63 sarcoma cells,
localizing to invadopodia, which is activated when cells are stimulated through vanadate or
growth factor/integrin ligation.
AFAP1L1 contains two pleckstrin homology (PH) domains (Figure-1i) that could
each directly bind to phospholipids when assayed using PIP-strips (Echelon)
(Supplemental Figure S8a). Moreover, the PH domains could mediate plasma membrane
localization in PDGF stimulated NIH3T3 cells, demonstrating that they are functionally
capable of binding phosphatidyl inositol lipids (PIPs) generated by PI3 kinase
(Supplemental Figure S8b). In addition, expression of mouse AFAP1L1 with its PH
domains deleted (ΔPH1/2) in U2OS cells that had endogenous AFAP1L1 knocked down
with the S3 RNAi were unable to re-form invadopodia structures unlike cells transfected
with wild-type AFAP1L1 (Supplemental Figure S4(i), (v)). Together, these results show
AFAP1L1 can utilize its PH domains to mediate localization to the plasma membrane of
cells and are an important component of its signaling to invadopodia.
AFAP1L1 signals to Rac and PAK/MLCK pathways.
The interaction of AFAP1L1 with Vav2, which has Rho family guanine nucleotide
exchange activity, suggested AFAP1L1 might regulate down-stream pathways from Vav2,
including Rac1 and p21-activated kinase (PAK) networks to mediate its effects on the
cytoskeleton. Significantly, ectopic expression of AFAP1L1 in COS7 cells increased the
levels of active Rac1, but not RhoA in PAK1-PBD and Rhotekin-PBD pull-down assays
(Figure 6a). Further, upon extended vanadate stimulation (1h) or co-expression with
hyperactive Lyn (Y508F), a greatly diminished pool of active Rac1 was observed that was
not mediated by vanadate stimulation or LynY508F alone. This observation suggests that
high levels of phospho-AFAP1L1 can result in feedback inhibition and consequently down-
regulation of active Rac1.
Down-stream of Rac1 are the p21-activated kinases (PAK1/2/3).26 Therefore, we
assayed their activation status in COS7 cells expressing AFAP1L1 during a time course of
vanadate stimulation. Here we found that AFAP1L1 expression led to significant activation
of PAK1/3 (with modest PAK2 activation), which was further enhanced by vanadate
stimulation up to 30min, and then subsequently down regulated by 1h of treatment (Figure
6b). In contrast, cells not ectopically expressing AFAP1L1 only showed minimal PAK1/3
activation but pronounced PAK2 activation (Figure 6b). The transient PAK activation (with
subsequent down-regulation at 1h) of vanadate stimulated AFAP1L1 expressing cells
correlates with the observed down-regulation of Rac1 activity at this time point (Figure 6a).
Page 10 of 29
Downstream of the p21-activated kinases are several pathways, including the
phosphorylation and inhibition of myosin light chain kinase (MLCK, which phosphorylates
myosin light chain 2, MLC2), which is directed towards cytoskeletal reorganization.42
Consequently, we assessed the levels of MLC2 phosphoryaltion in cells expressing
AFAP1L1 and found that indeed the increased PAK1/3 levels correlated with decreased
phospho-MLC2 levels, and that while vanadate stimulated an increase of pMLC2 in control
cells, those ectopically expressing AFAP1L1 showed significant inhibition of this response
(Figure 6b).
With the significant effects of AFAP1L1 on cytoskeletal function and that ectopic
expression of AFAP1L1 affects PAK/MLC2 we then looked and the effects of knockdown
of AFAP1L1 on the signaling to PAK/MLC2. Importantly, knockdown of AFAP1L1 reduced
the activation of PAK1/3, while increasing that of PAK2, when U2OS cells were stimulated
with vanadate (Figure 6c, d). Commensurate with these changes in PAK isoform activation
were also significant alterations to the level of phosphorylation and subcellular distribution
of MLC2, with knock-down of AFAP1L1 causing less pMLC2 and total MLC2 being
recruited to the F-actin compartment, resulting in more pMLC2/MLC2 remaining in the
cytoplasm (Figure 6d). These data illustrate that AFAP1L1 provides a major signaling
mechanism to regulate major cytoskeletal remodeling in sarcoma cells, especially through
modulating PAK activity and MLC2 loading onto F-actin, potentially primarily through its
interaction with Vav2.
Page 11 of 29
DISCUSSION
We report that the scaffolding protein AFAP1L1 directly links Vav2 and Nck2
pathways to regulate invadopodia formation and function in sarcoma cells. This is through
our novel findings that two specific pY motifs phosphorylated by Lyn/SFKs in AFAP1L1,
pY136 and pY566, bind to the SH2 domains of Vav2 and Nck2, respectively. This provides
a regulatory mechanism for linking the down-stream pathways of Vav2 through Rac1-
PAK1/3, and Nck2 regulation of F-actin via the WIP/cortactin/Tks5//N-WASP/Arp2/3
complex,5, 6, 19, 29, 33, 47, 55 within sarcoma cell invadopodia (summarized in Figure 7). These
results provide an important mechanistic explanation for the regulation of invadopodia by
AFAP1L1 in sarcoma cells. Our novel findings also significantly expand the recent findings
on AFAP1L1 interacting with vinculin at invadopodia and its regulation of disease
progression in colorectal cancers48 and provide an impetus to delineate if the Vav2/Nck2
connection to AFAP1L1 also mediates its links to colorectal cancer progression.
AFAP1L1 contains a direct F-actin binding and multimerization region in its C-
terminus and two functional central PH domains that bind PIPs generated by growth
factor-stimulated PI3-kinase, providing direct links to both the F-actin cytoskeleton
(including the ability to cross-link the cytoskeletal network) and tethering to growth factor
activated plasma membrane loci. In addition, as AFAP1L1 is able to form dimers (and
potentially higher order multimers), and through its proline rich motifs that interact with
several SH3 domains, i.e. Lyn, Vav2 and cortactin,17 this could facilitate simultaneous co-
localization of multiple distinct AFAP1L1 complexes within the same tethered space.
AFAP1L1 could also be directly interacting with Tks5 and p190RhoGAP within
invadopodia via their SH3 domains through binding to PXP motifs within AFAP1L1, similar
to that reported for AFAP1.10 These observations together with the ability of AFAP1L1 to
directly bind the invadopodia/podosome marker cortactin17, associate with vinculin (found
at focal adhesions as well as invadopodia)48 and the fact that AFAP1L1 encompasses a
homologous site to that which is phosphorylated by PKC in AFAP1 (which regulates
podosome, and potentially invadopodia, lifespan),14 we have now significant evidence for
placing AFAP1L1 as a crucial regulator of invadpodia in sarcoma cells and that it directly
intersects multiple critical invadopodia regulatory networks. The strongest evidence for the
importance of AFAP1L1 for invadopodia regulation, from other investigators17, 45, 48 and as
we report here, relates to the U2OS cell line. This suggests there are important cell type
specific ancillary components that may be direct interacting partners of AFAP1L1, which
help mediate this cell-specific phenotype.
Page 12 of 29
The regulation of invasion and metastasis by the invadopodia34, 36, 46, 54 is complex
and involves many players that are also involved in other cytoskeletal functions, e.g.
attachment and migration,2, 3, 9, 27, 32, 34, 35, 38, 43, 46, 54 and this includes the AFAP1 family
(encompassing AFAP1, AFAP1L1 and AFAP1L2/XB130) which are scaffold forming
proteins providing direct network connections that facilitate cytoskeletal regulation.10, 14, 31,
45, 56 The initiation of invadopodia assembly involves cdc42 activity to facilitate targeting
and activation of actin nucleation complexes (e.g. Arp2/3, N-WASp, WIP) and tyrosine
phosphorylation of substrates by SFKs (e.g. cortactin, N-WASp, paxillin, Tks5) to enhance
invadosome assembly.8, 34, 36, 46, 49, 54 Placing AFAP1L1, with its direct binding to Nck2 and
Vav2 when phosphorylated by SFKs(Lyn), within the invadpodia provides further links
between their other known direct partners (i.e. for Nck2; N-WASp, WIP, cortactin, Tks5)
and the Rac1/PAK pathway via Vav2 within the invadosome.1, 9, 13, 19, 33, 37, 47, 55 While Nck
and its partners are central players in formation and maintenance of invadopodia,5, 40, 47
the involvement of Rac1 activators such as Vav2 has been less well characterized and
indeed a recently identified Trio-Rac1-Pak1 pathway appears strongly associated with
disassembly of invadopodia.33 Intriguingly, there is a link between Rac1 and Nck in
promoting actin nucleation via causing disassociation of the WASP-family verprolin
homologous protein (WAVE1) complex,15 and potentially through AFAP1L1 bringing
together the Nck and Rac1 (via Vav2) complexes it could also regulate the actin
polymerization dynamics within invadopodia. The phosphorylation status of AFAP1L1, and
thus its recruitment/regulation of Nck2 and Vav2 pathways, may also regulate the type of
cell-matrix complexes that a cell forms, i.e. focal adhesions or invadopodia/podosomes, as
several of its binding partners (e.g. vinculin, F-actin, SFKs) can be found in both types of
structures. This may also facilitate the much higher temporal dynamics of
invadopodia/pososomes compared to more stabilized actin structures as occur in focal
adhesions.
Interestingly, while over-expression of AFAP1L1 in HEK293 cells led to overall
increases in Rac1 and PAK1/3 activity, after sustained stimulation (1hr vanadate, co-
expression dominant active LynY508F, Figure-4a, b) Rac1 and PAK1/3 activity was
decreased, showing that depending on duration of SFK activity AFAP1L1 can up and
down-regulate Rac1/PAK1/3 activity. Manipulating AFAP1L1 levels also had profound
effects on the differential activation of PAK isoforms, PAK1/3 and PAK2, and differential
compartmentalization of the PAK substrate MLC2 (cytosolic and F-action subcellular
fractions). Potentially, AFAP1L1 could be both promoting assembly through Nck2
pathways and regulating disassembly through Vav2/Rac1 pathways within invadopodia,
Page 13 of 29
providing dynamic invadopodia turnover, which is important for invasion of cancer cells as
increased invadopodia turnover through Rac1 enhances this process.4
AFAP1L1 also had significant enhancing effects on mitogen gradient-directed
migration, which is also intimately controlled by Vav2/Rac1/Pak1.11, 12, 20, 29 Consequently,
AFAP1L1 could be providing an integration of both migration/locomotion control and
invasion/invadopodia regulation, thus linking these two important aspects of cancer cell
invasion and metastasis. Indeed, the pathways feeding into AFAP1L1 complex formation
and localization, i.e. SFKs and PI3-kinase, are both intimately linked to promoting
migration as well as invasion.8, 16, 51, 57
Additionally, our data show that signaling from AFAP1L1 is modulated by a cells
contact with extracellular matrix, which could explain the differential effects on cell growth
in 2D monolayer cultures (which show no major effects on cell proliferation) compared to
3D and in vivo cultures (which show significant effects on proliferation/survival) of cells
over/under expressing AFAP1L117, 48.
While high expression of total AFAP1L1 is associated with sarcomas (and
colorectal cancer progression)17, 48 it will be important to determine if the activation status
(i.e. phosphorylation of Y136 and Y566 sites, for which we have generated specific
antibodies) also correlate with disease status and clinical outcomes. Important clinical
applications of the molecular understanding we now have of the AFAP1L1 pathway need
to be addressed through the use of both advanced cancer mouse models with altered
AFAP1L1 (e.g. sarcomas bearing wild-type or Y136/566F mutant AFAP1L1), and
identifying therapeutic avenues to disrupt the AFAP1L1 pathway in sarcoma, and
potentially other (e.g. colorectal cancer) patients.
Page 14 of 29
MATERIALS AND METHODS
Cell culture, stable cell line generation and in vivo assays
U2OS, MG-63, A549, PANC-1, HEK293, NIH3T3 and COS7 cells (sourced from ATCC
and mycoplasma free) were maintained in Dulbecco's Modified Eagle's medium (DMEM)
supplemented with penicillin and streptomycin (Invitrogen), 10% fetal bovine serum (FBS)
(Gibco) and cultured at 37°C. Stable cell lines expressing eGFP tagged mouse AFAP1L1
were generated by selecting eGFP positive cells after 7 days of culture by fluorescence
activated cell sorting (FACS) (BD FACS Aria II flow cytometer, Beckman-Coulter). NIH3T3
cell lines expressing different Lyn mutants were generated by retroviral infection
(pMSCV2.2neo-Lyn; wild-type, kinase inactive Y397F, or dominant active Y508F Lyn) and
selecting lines using neomycin (1mg/ml) for 10 days. Egg whites were used as a source of
extracellular matrix for 3D cultures of cells on coverslips, as described.25 For transient
expression of plasmids cells were transfected using Lipofectamine2000 as per the
manufacturer’s instructions (Life Technologies). Cell proliferation assays were performed
using the IncuCyte Zoom (Essen Biosciences Inc.) system, the cell screen (Innovatis Inc.)
system or the xCELLigence (ACEA Biosciences Inc.) system, according to the
manufacturers protocol.
Soft agar cultures of NIH3T3 clones stably expressing untagged mouse AFAP1L1
or control vector (pMSCV2.2neo) were performed essentially as described.7
Spontaneous cell movement in sub-confluent cultures was analysed on timed series
(1min intervals) phase-contrast images of live cell cultures using Gradientech Tracking
Tool (v1.07, Gradientech).
Wound-healing/scratch assays were performed on NIH3T3 cells by growing cells to
confluence in 6-well trays and then dragging a 200µl pipette tip across the monolayer to
remove a track of cells.28 Phase-contrast images were then taken at time points over a 16h
period and the degree of scratch closure quantified.
Cell attachment and spreading analysis was performed by plating cells on untreated
tissue-culture plastic in serum-free media or media supplemented with 10%FBS and
phase-contrast images captured at 4h post plating and the percentage of cells attaching
and spreading quantified.
Modified Boyden chamber cell migration and invasion assays were performed in 24-
well plates (8µm pore size, BD Biosciences) coated with FBS before adding cells (2.0 X
104) in the top chamber in serum-free media. For invasion assays chambers were coated
Page 15 of 29
with a thin layer of Matrigel (BD Biosciences), 10µl of Matrigel (1mg/ml) was layered and
allowed to set on the filter chamber membrane, then air-dried overnight. The lower
chamber was filled with media containing 10% FBS and cells that had migrated after 16h
to the underside of the filter were fixed, visualized and enumerated.
Real-time cell attachment, spreading, proliferation and migration assays were also
performed using an xCELLigence RTCA DP system with E-plate-16 (for
attachment/spreading assays) or CIM-plate-16 (for migration assays, 8µm pore size)
according to the manufacturer’s instructions (ACEA Biosciences Inc.) and analysed using
RTCS Software (v1.2, ACEA Biosciences Inc.).
Plasmid construction and in vitro assays
All plasmid constructs were generated by subcloning and/or site directed
mutagenesis using oligonucleotides, and were confirmed by Sanger sequencing.
Expression constructs for Lyn and the standard and phospho-tyrosine-specific yeast two-
hybrid assays were as previously described.21-24, 41, 50, 52, 53
The SH2 domains of Nck2 and Vav2 were expressed as NusA fusions and purified
using specific affinity columns (GE Healthcare) and gel filtration using Profinia (Bio-rad)
and BioLogic DuoFlow chromatography (Bio-rad) instruments. The NusA-fusions were
labelled with fluorescent probes for use as direct immunoblotting reagents using the
IRDye-800CW protein labelling kit (LI-COR Biosciences). The purified C-terminal GST
fusion of AFAP1L1 was also used in F-actin co-sedimentation assays using the Actin
Binding Protein Biochem Kit (BK013, Cytoskeleton).
Kinase assays were performed using Lyn immunoprecipitated from COS7 cells
transfected with inactive Lyn (LynY397F) or hyper-active Lyn (LynY508F) using anti-Lyn
antibodies (sc-15, Santa Cruz Biotechnology) and subsequent incubation with [γ-32P]-ATP
in the presence or absence of substrate (GST or GST-AFAP1L1) essentially as described
previously.50
Rac1 and RhoA activation status was measured using the Rac1 and RhoA
activation assays kits (BK035, BK036, Cytoskeleton) according to the manufacture’s
instructions on COS7 cells transiently transfected with combinations of the HA-tagged
AFAP1L1, inactive Lyn (LynY397F) and hyperactive Lyn (LynY508F) mammalian
expression plasmids at 48h post transfections, either with or without vanadate stimulation
for 1h.
Page 16 of 29
Antibodies and reagents
Polyclonal rabbit antibodies were raised against AFAP1L1 using the purified C-terminal
region (aa 627-777) of murine AFAP1L1 fused to GST as the antigen, and high titer serum
was purified on protein-A beads (antibody specificity is detailed in Supplemental Figure
S1). Additional antibodies used for immunoblotting and/or immunoprecipitation were anti-
pY(PY100; 9411), anti-PAK1/2/3(2604), anti-MLC2(3672), anti-pMLC2(pT18/pS19, 3674),
pPAK1/2(pS144/pS141, 2606) (Cell Signaling Technology, Danvers, MA); anti-βactin(AC-
15, ab6276), anti-Nck2(ab109239), anti-Vav2(ab52640) (Abcam, Cambridge, United
Kingdom); anti-Lyn(sc-15), anti-AFAP1L1(sc-162497), anti-cortactin (H-191, sc-11408),
anti-GFP(FL, sc-8834) (Santa Cruz Biotechnology); and anti-pY-HRP(4G10), anti-Myc-tag
(clone 4A6), anti-HA-tag (HA.11 clone) (Merck-Millipore, Billerica, MA). TRITC-labeled
phalloidin (P1591, Sigma-Aldrich) was used to visualize filamentous actin (F-actin). RNAi
knockdown of AFAP1L1 was achieved using individual siGENOME oligonucleotides
(S43843 and S43844, Dharmacon, Thermoscientific).
Cell lysis, fractionation, immunoblotting, and immunoprecipitation
Cells were generally lysed in raft buffer as previously described.24 For
cytosolic/cytoskeletal fractionation studies cells were lysed as previously described.41 The
protein concentration of lysates was measured by the Bradford method (Bio-rad). For
immunoprecipitation, 0.5 to 2 mg of total protein from clarified cell lysates were incubated
with specific antibodies (0.5 to 5µg) for 2h at 4°C, collected with protein G-Sepharose
beads (Sigma-Aldrich) for 16h (4°C) before washing in lysis buffer, SDS-PAGE and
analysis by immunoblotting. After probing with specific primary antibodies, proteins were
revealed using either secondary antibody coupled to horseradish peroxidase (GE
Healthcare or Cell Signaling Technology,) and detection by enhanced chemiluminescence
(GE Healthcare), or with fluorescently labeled secondary antibodies and an Odyssey
scanner (LI-COR Biosciences, Lincoln, NE).
Microscopy and immunofluorescence
Glass coverslips (No. 1.5H, Marienfeld) were either uncoated or coated with either poly-L-
lysine (Sigma-Aldrich) or serum before seeding cells. After specific experimentation
treatments/time points, coverslips were gently washed with PBS (37°C) then fixed in 4%
paraformaldehyde/PBS for 15 min at 37°C, followed by permeabilization with 0.5% triton
X-100 in PBS (5 min), then blocked for 2h at 25°C in 3% BSA/PBS/Tween-20(0.1%).
Coverslips were incubated with primary antibody diluted in blocking buffer at 4°C for 16h,
Page 17 of 29
then washed 3 times in PBS/Tween-20 (15 min) before incubation with fluorophore-
conjugated (Alexa-Fluor-labeled-488/594, Life-Technologies) secondary antibodies (with
Hoechst 33342 at 0.01µg/ml) for 1h at 25°C, then washed again in PBS/Tween-20 before
mounting in Vectashield (H-1000, Vector Labs). Fluorescent microscopy was performed
using an x60 plan-Apo 1.40 oil objective on an inverted Elcips-Ti microscope (Nikon,
Japan), fitted with a CoolSNAP HQ2 CCD camera (Photometrics). Images were captured,
then z-stacks deconvolved and analysed using NIS Elements 4.x (Nikon), or on a
DeltaVision Elite system and analysed with softWoRx suite2.0 (GE Healthcare).
Statistical analysis
For general statistical analysis experiments were repeated as three biological replicates
and analysed by students t-test or ANOVA with two-tailed analysis, with orthogonal
comparisons.
Page 18 of 29
AUTHOR CONTRIBUTION
Contribution: David J. McCarthy and Sien Ron Tie contributed equally to the manuscript
and designed, supported and performed experiments, and analyzed data. Tulene S.
Kendrick, Alison Louw, Cindy Le, Jiulia Satiaputra, and Nicole Kucera designed and
performed experiments, and analysed data. Michael Phillips designed and analysed
experiments and contributed to writing the manuscript. Evan Ingley designed and
supported the research, designed and undertook experiments, analyzed data and
contributed to writing the manuscript.
ACKNOWLEDGEMENTS
We thank Janice Lam, Matt Lee, Rebecca Shapiro, Morgane Davies, Irma Larma and
Kevin Li for technical assistance.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
FUNDING
This work was supported by grants from the National Health and Medical Research
Council (513714 and 634352), the Medical Research Foundation of Royal Perth Hospital,
and the Cancer Council of Western Australia. Evan Ingley received support from the
Cancer Council of Western Australia, The Harry Perkins Institute of Medical Research,
Sock-it-to-Sarcoma and the Hollywood Private Hospital Research Foundation.
ABBREVIATIONS USED:
SFK, Src family kinases.
Page 19 of 29
REFERENCES
1 Abe K, Rossman KL, Liu B, Ritola KD, Chiang D, Campbell SL et al. Vav2 is an activator of Cdc42, Rac1, and RhoA. J Biol Chem 2000; 275: 10141-10149.
2 Albiges-Rizo C, Destaing O, Fourcade B, Planus E, Block MR. Actin machinery and
mechanosensitivity in invadopodia, podosomes and focal adhesions. J Cell Sci 2009; 122: 3037-3049.
3 Badowski C, Pawlak G, Grichine A, Chabadel A, Oddou C, Jurdic P et al. Paxillin
phosphorylation controls invadopodia/podosomes spatiotemporal organization. Mol Biol Cell 2008; 19: 633-645.
4 Barrio-Real L, Kazanietz MG. Rho GEFs and cancer: linking gene expression and
metastatic dissemination. Sci Signal 2012; 5: pe43. 5 Buday L, Wunderlich L, Tamas P. The Nck family of adapter proteins: Regulators of
actin cytoskeleton. Cell Signal 2002; 14: 723-731. 6 Bustelo XR. Vav proteins, adaptors and cell signaling. Oncogene 2001; 20: 6372-
6381. 7 Chauhan AK, Li YS, Deuel TF. Pleiotrophin transforms NIH 3T3 cells and induces
tumors in nude mice. Proc Natl Acad Sci USA 1993; 90: 679-682. 8 Chen WT, Chen JM, Parsons SJ, Parsons JT. Local degradation of fibronectin at
sites of expression of the transforming gene product pp60src. Nature 1985; 316: 156-158.
9 Courtneidge SA, Azucena EF, Pass I, Seals DF, Tesfay L. The SRC substrate
Tks5, podosomes (invadopodia), and cancer cell invasion. Cold Spring Harb Symp Quant Biol 2005; 70: 167-171.
10 Crimaldi L, Courtneidge SA, Gimona M. Tks5 recruits AFAP-110, p190RhoGAP,
and cortactin for podosome formation. Exp Cell Res 2009; 315: 2581-2592. 11 Dang I, Gorelik R, Sousa-Blin C, Derivery E, Guerin C, Linkner J et al. Inhibitory
signalling to the Arp2/3 complex steers cell migration. Nature 2013; 503: 281-284. 12 Delorme-Walker VD, Peterson JR, Chernoff J, Waterman CM, Danuser G,
DerMardirossian C et al. Pak1 regulates focal adhesion strength, myosin IIA distribution, and actin dynamics to optimize cell migration. J Cell Biol 2011; 193: 1289-1303.
13 Desmarais V, Yamaguchi H, Oser M, Soon L, Mouneimne G, Sarmiento C et al. N-
WASP and cortactin are involved in invadopodium-dependent chemotaxis to EGF in breast tumor cells. Cell Motil Cytoskeleton 2009; 66: 303-316.
14 Dorfleutner A, Cho Y, Vincent D, Cunnick J, Lin H, Weed SA et al. Phosphorylation
of AFAP-110 affects podosome lifespan in A7r5 cells. J Cell Sci 2008; 121: 2394-2405.
Page 20 of 29
15 Eden S, Rohatgi R, Podtelejnikov AV, Mann M, Kirschner MW. Mechanism of regulation of WAVE1-induced actin nucleation by Rac1 and Nck. Nature 2002; 418: 790-793.
16 Frame MC. Newest findings on the oldest oncogene; how activated src does it. J
Cell Sci 2004; 117: 989-998. 17 Furu M, Kajita Y, Nagayama S, Ishibe T, Shima Y, Nishijo K et al. Identification of
AFAP1L1 as a prognostic marker for spindle cell sarcomas. Oncogene 2011; 30: 4015-4025.
18 Gatesman A, Walker VG, Baisden JM, Weed SA, Flynn DC. Protein kinase Calpha
activates c-Src and induces podosome formation via AFAP-110. Mol Cel Biol 2004; 24: 7578-7597.
19 Heo J, Thapar R, Campbell SL. Recognition and activation of Rho GTPases by
Vav1 and Vav2 guanine nucleotide exchange factors. Biochemistry 2005; 44: 6573-6585.
20 Huang CH, Tang M, Shi C, Iglesias PA, Devreotes PN. An excitable signal
integrator couples to an idling cytoskeletal oscillator to drive cell migration. Nat Cell Biol 2013; 15: 1307-1316.
21 Ingley E, Williams JH, Walker CE, Tsai S, Colley S, Sayer MS et al. A novel ADP-
ribosylation like factor (ARL-6), interacts with the protein-conducting channel SEC61beta subunit. FEBS Lett 1999; 459: 69-74.
22 Ingley E, Sarna MK, Beaumont JG, Tilbrook PA, Tsai S, Takemoto Y et al. HS1
interacts with Lyn and is critical for erythropoietin-induced differentiation of erythroid cells. J Biol Chem 2000; 275: 7887-7893.
23 Ingley E, Chappell D, Poon SY, Sarna MK, Beaumont JG, Williams JH et al. Thyroid
hormone receptor-interacting protein 1 modulates cytokine and nuclear hormone signaling in erythroid cells. J Biol Chem 2001; 276: 43428-43434.
24 Ingley E, Schneider JR, Payne CJ, McCarthy DJ, Harder KW, Hibbs ML et al. Csk-
binding protein mediates sequential enzymatic down-regulation and degradation of Lyn in erythropoietin-stimulated cells. J Biol Chem 2006; 281: 31920-31929.
25 Kaipparettu BA, Kuiatse I, Tak-Yee Chan B, Benny Kaipparettu M, Lee AV,
Oesterreich S. Novel egg white-based 3-D cell culture system. Biotechniques 2008; 45: 165-168, 170-161.
26 Knaus UG, Bokoch GM. The p21Rac/Cdc42-activated kinases (PAKs). Int J
Biochem Cell Biol 1998; 30: 857-862. 27 Li A, Dawson JC, Forero-Vargas M, Spence HJ, Yu X, Konig I et al. The actin-
bundling protein fascin stabilizes actin in invadopodia and potentiates protrusive invasion. Curr Biol 2010; 20: 339-345.
28 Liang CC, Park AY, Guan JL. In vitro scratch assay: a convenient and inexpensive
method for analysis of cell migration in vitro. Nat Protoc 2007; 2: 329-333.
Page 21 of 29
29 Liu BP, Burridge K. Vav2 activates Rac1, Cdc42, and RhoA downstream from
growth factor receptors but not beta1 integrins. Mol Cell Biol 2000; 20: 7160-7169. 30 Lock P, Abram CL, Gibson T, Courtneidge SA. A new method for isolating tyrosine
kinase substrates used to identify fish, an SH3 and PX domain-containing protein, and Src substrate. EMBO J 1998; 17: 4346-4357.
31 Lodyga M, Bai XH, Kapus A, Liu M. Adaptor protein XB130 is a Rac-controlled
component of lamellipodia that regulates cell motility and invasion. J Cell Sci 2010; 123: 4156-4169.
32 Mader CC, Oser M, Magalhaes MA, Bravo-Cordero JJ, Condeelis J, Koleske AJ et
al. An EGFR-Src-Arg-cortactin pathway mediates functional maturation of invadopodia and breast cancer cell invasion. Cancer Res 2011; 71: 1730-1741.
33 Moshfegh Y, Bravo-Cordero JJ, Miskolci V, Condeelis J, Hodgson L. A Trio-Rac1-
Pak1 signalling axis drives invadopodia disassembly. Nat Cell Biol 2014; 16: 574-586.
34 Murphy DA, Courtneidge SA. The 'ins' and 'outs' of podosomes and invadopodia:
characteristics, formation and function. Nat Rev Mol Cell Biol 2011; 12: 413-426. 35 Nakahara H, Otani T, Sasaki T, Miura Y, Takai Y, Kogo M. Involvement of Cdc42
and Rac small G proteins in invadopodia formation of RPMI7951 cells. Genes Cells 2003; 8: 1019-1027.
36 Nurnberg A, Kitzing T, Grosse R. Nucleating actin for invasion. Nat Rev Cancer
2011; 11: 177-187. 37 Oser M, Yamaguchi H, Mader CC, Bravo-Cordero JJ, Arias M, Chen X et al.
Cortactin regulates cofilin and N-WASp activities to control the stages of invadopodium assembly and maturation. J Cell Biol 2009; 186: 571-587.
38 Poincloux R, Lizarraga F, Chavrier P. Matrix invasion by tumour cells: a focus on
MT1-MMP trafficking to invadopodia. J Cell Sci 2009; 122: 3015-3024. 39 Ridley AJ. Life at the leading edge. Cell 2011; 145: 1012-1022. 40 Rivera GM, Antoku S, Gelkop S, Shin NY, Hanks SK, Pawson T et al. Requirement
of Nck adaptors for actin dynamics and cell migration stimulated by platelet-derived growth factor B. Proc Natl Acad Sci USA 2006; 103: 9536-9541.
41 Samuels AL, Klinken SP, Ingley E. Liar, a novel Lyn-binding nuclear/cytoplasmic
shuttling protein that influences erythropoietin-induced differentiation. Blood 2009; 113: 3845-3856.
42 Sanders LC, Matsumura F, Bokoch GM, de Lanerolle P. Inhibition of myosin light
chain kinase by p21-activated kinase. Science 1999; 283: 2083-2085.
Page 22 of 29
43 Schoumacher M, Goldman RD, Louvard D, Vignjevic DM. Actin, microtubules, and vimentin intermediate filaments cooperate for elongation of invadopodia. J Cell Biol 2010; 189: 541-556.
44 Seals DF, Azucena EF, Jr., Pass I, Tesfay L, Gordon R, Woodrow M et al. The
adaptor protein Tks5/Fish is required for podosome formation and function, and for the protease-driven invasion of cancer cells. Cancer Cell 2005; 7: 155-165.
45 Snyder BN, Cho Y, Qian Y, Coad JE, Flynn DC, Cunnick JM. AFAP1L1 is a novel
adaptor protein of the AFAP family that interacts with cortactin and localizes to invadosomes. Eur J Cell Biol 2011; 90: 376-389.
46 Stylli SS, Kaye AH, Lock P. Invadopodia: at the cutting edge of tumour invasion. J
Clin Neurosci 2008; 15: 725-737. 47 Stylli SS, Stacey TT, Verhagen AM, Xu SS, Pass I, Courtneidge SA et al. Nck
adaptor proteins link Tks5 to invadopodia actin regulation and ECM degradation. J Cell Sci 2009; 122: 2727-2740.
48 Takahashi R, Nagayama S, Furu M, Kajita Y, Jin Y, Kato T et al. AFAP1L1, a novel
associating partner with vinculin, modulates cellular morphology and motility, and promotes the progression of colorectal cancers. Cancer Med 2014; 3: 759-774.
49 Tehrani S, Tomasevic N, Weed S, Sakowicz R, Cooper JA. Src phosphorylation of
cortactin enhances actin assembly. Proc Natl Acad Sci USA 2007; 104: 11933-11938.
50 Tilbrook PA, Ingley E, Williams JH, Hibbs ML, Klinken SP. Lyn tyrosine kinase is
essential for erythropoietin-induced differentiation of J2E erythroid cells. EMBO J 1997; 16: 1610-1619.
51 Timpson P, Jones GE, Frame MC, Brunton VG. Coordination of cell polarization
and migration by the Rho family GTPases requires Src tyrosine kinase activity. Current Biol 2001; 11: 1836-1846.
52 Tsai S, Bartelmez S, Sitnicka E, Collins S. Lymphohematopoietic progenitors
immortalized by a retroviral vector harboring a dominant-negative retinoic acid receptor can recapitulate lymphoid, myeloid, and erythroid development. Genes Dev 1994; 8: 2831-2841.
53 Vojtek AB, Hollenberg SM, Cooper JA. Mammalian Ras interacts directly with the
serine/threonine kinase Raf. Cell 1993; 74: 205-214. 54 Weaver AM. Invadopodia: specialized cell structures for cancer invasion. Clin Exp
Metastasis 2006; 23: 97-105. 55 Wells CM, Bhavsar PJ, Evans IR, Vigorito E, Turner M, Tybulewicz V et al. Vav1
and Vav2 play different roles in macrophage migration and cytoskeletal organization. Exp Cell Res 2005; 310: 303-310.
56 Xu J, Bai XH, Lodyga M, Han B, Xiao H, Keshavjee S et al. XB130, a novel adaptor
protein for signal transduction. J Biol Chem 2007; 282: 16401-16412.
Page 23 of 29
57 Yamaguchi H, Yoshida S, Muroi E, Yoshida N, Kawamura M, Kouchi Z et al.
Phosphoinositide 3-kinase signaling pathway mediated by p110alpha regulates invadopodia formation. J Cell Biol 2011; 193: 1275-1288.
Page 24 of 29
FIGURE LEGENDS
Figure 1. AFAP1L1 localizes to invadopodia in sarcoma cells, which requires its F-
actin binding/dimerization domain. (a) Immunoblot analysis of AFAP1L1 expression in
osteosarcoma (U2OS and MG-63), pancreatic carcinoma (PANC-1), and lung carcinoma
(A549) cell lines, relative to β-actin levels. (b) Subcellular localization of AFAP1L1 in U2OS
osteosarcoma cells detected with an AFAP1L1 specific antibody. Co-localization of
AFAP1L1 (green) and F-actin (red) rich sub-nuclear invadopodia is indicated (arrow head),
nuclei stained with Hoechst 33342 (blue). Maximum image projection in XY plane and
reconstructed image slices at point of strongest co-localization (arrow head) in YZ and XZ
planes. Scale bar=10µm. (c) Subcellular localization of AFAP1L1 in U2OS osteosarcoma
cells stably expressing eGFP-tagged AFAP1L1. Co-localization of eGFP-AFAP1L1 (green)
and F-actin (red) rich sub-nuclear invadopodia is indicated (arrow head), nuclei stained
with Hoechst 33342 (blue). Maximum image projection in XY plane and reconstructed
image slices at point of strongest co-localization (arrow head) in YZ and XZ planes. Scale
bar=10µm. (d) Subcellular localization of AFAP1L1 in MG-63 osteosarcoma cells stably
expressing eGFP-tagged AFAP1L1. Co-localization of eGFP-AFAP1L1 (green) and F-
actin (red) rich sub-nuclear invadopodia is indicated (arrow head), nuclei stained with
Hoechst 33342 (blue). Maximum image projection in XY plane and reconstructed image
slices at point of strongest co-localization (arrow head) in YZ and XZ planes. Scale
bar=10µm. (e) Subcellular localization of AFAP1L1 in U2OS osteosarcoma cells stably
expressing eGFP-tagged AFAP1L1 cultured in 3D extracellular matrix (egg white). Co-
localization of eGFP-AFAP1L1 (green) and F-actin (red) rich sub-nuclear invadopodia is
indicated (arrow heads), nuclei stained with Hoechst 33342 (blue). Maximum image
projection in XY plane and reconstructed image slices at point of strongest co-localization
(arrow head) in YZ and XZ planes. Scale bar=10µm. (f) Subcellular localization of
AFAP1L1, cortactin and F-actin in U2OS osteosarcoma cells stably expressing eGFP-
tagged AFAP1L1. Co-localization of eGFP-AFAP1L1 (green, (ii)), cortactin (amber, (iv))
and F-actin (red, (iii)) rich sub-nuclear invadopodia is indicated (arrow heads), nuclei were
stained with Hoechst 33342 (blue, (i)). Maximum image projection in XY plane and regions
of co-localization indicated (arrow head). Merged image (v) shows location of line scan (vi)
for quantification of co-localization. Scale bar=15µm. (g) Subcellular localization of eGFP-
AFAP1L1 in COS7 kidney epithelia cells. Co-localization of eGFP-AFAP1L1 (green) and
F-actin (red) rich structures, nuclei stained with Hoechst 33342 (blue). Maximum image
projection in XY plane shown. Scale bar=10µm. (h) Analysis of direct interaction of the C-
terminal region of AFAP1L1 (G-A-CT; aa 630-777) with F-actin by co-sedimentation
Page 25 of 29
analysis. Coomassie stained SDS-PAGE gel of high-speed (100,000 x g) precipitate (ppt)
and supernatants (SN) from F-actin incubated with GST or GST-AFAP1L1-CT (G-A-CT).
(i) Yeast two-hybrid analysis of AFAP1L1 self-association. Yeast reporter activation of full-
length, C-terminal deleted (aa 1-627) and N-terminal deleted (aa 630-777) LexA fusions of
AFAP1L1 co-expressing full-length AFAP1L1 as a VP16 fusion. (j) Analysis of domains
directing the subcellular localization of AFAP1L1 in U2OS sarcoma cells. The C-terminal
leucine-zipper (L) and α-helical (H) domains are both required for localization of AFAP1L1
to actin-rich structures in sarcoma cells. Localization of eGFP-AFAP1L1 full-length (i) and
deletion constructs (ii-vi) (green), F-actin (red) rich structures, and nuclei (Hoechst 33342;
blue) in transiently transfected U2OS cells. Maximum image projection in XY plane shown.
Scale bar=10µm.
Figure 2. RNAi knockdown of AFAP1L1 in sarcoma cells inhibits cell spreading,
migration, invasion, and invadopodia formation. (a) Immunoblot analysis of AFAP1L1
protein levels after 72h of RNAi-mediated knockdown with oligonucleotides S43843 (S3),
S43844 (S4) or non-targeting control RNAi (NT) in U2OS and MG-63 cells. (b) Analysis of
cell spreading in serum free and serum containing cultures at 4h post plating of U2OS
cells with (S3, S4) or without (NT) RNAi-mediated knockdown of AFAP1L1. Results are
mean ± S.D., n=3, **p<0.01. (c) Real-time assay for cell attachment and spreading using
cell impedance measurements with E-plates on an xCELLigence (ACEA Biosciences).
U2OS cells plated in serum free and serum containing media with (S3, S4) or without (NT)
RNAi-mediated knockdown of AFAP1L1. Results are mean ± S.D., n=3. (d) Modified
Boyden chamber migration assay. U2OS cells with (S3, S4) or without (NT) RNAi-
mediated knockdown of AFAP1L1 were seeded in serum free media (top chamber) and
assayed for migration to serum containing media (bottom chamber), enumerated after 16h
by counting Hoechst 33342 stained nuclei on the bottom chamber side of the membrane
(8µm pore size). Results are mean ± S.D., n=3, **p<0.01. (e) RNAi mediated knockdown
of AFAP1L1 in U2OS sarcoma cells reduces the number of cells showing clusters of
invadopodia. U2OS cells after 72h knockdown of AFAP1L1 (S3, S4) or non-targeting
control (NT) were assayed for invadopodia clusters by F-actin staining. Results are mean
± S.D., n=3, *p<0.05, **p<0.01. (f) RNAi mediated knockdown of AFAP1L1 in MG-63
sarcoma cells alters the clustering of invadopodia and their ability to reform with
stimulation (serum+vanadate) after serum starvation. MG-63 cells after 72h knockdown of
AFAP1L1 (S3, S4) or non-targeting control (NT) were assayed for invadopodia clusters by
F-actin staining in growing cells (top panels, quantitation in right graph) and serum
Page 26 of 29
starvation (middle panels) and re-stimulation with serum/vanadate (bottom panels,
quantitation in right graph). Results are mean ± S.D., n=3, *p<0.05. (g) RNAi mediated
knockdown of AFAP1L1 in U2OS and MG-63 sarcoma cells reduces their invasion in
modified Boyden chamber assays with a Matrigel matrix coating. U2OS and MG-63 cells
with (S3, S4) or without (NT) RNAi-mediated knockdown of AFAP1L1 were seeded in
serum free media (top chamber) and assayed for invasion through Matrigel matrix coated
filters (8µm pore size) in to serum containing media (bottom chamber, 10% FBS),
enumerated after 48h by counting Hoechst 33342 stained nuclei on the bottom chamber
side of the membrane. Results are mean ± S.D., n=3, *p<0.05.
Figure 3. AFAP1L1 binds and is phosphorylated by Lyn, and tyrosine kinase
activation promotes total AFAP1L1 localization to invadopodia. Interaction of Lyn with
AFAP1L1 and the ability of the kinase to phosphorylate AFAP1L1. (a) Using the yeast two-
hybrid technology the SH3 domain of Lyn binds to AFAL1L1. (b) Co-immunoprecipitation
of Lyn and AFAP1L1 from transiently transfected COS7 cells. Maximum co-
immunoprecipitation occurs with hyperactive Lyn (LynY508F mutant) that also induces a
maximal phosphorylation of AFAP1L1. (c) Co-localization of Lyn and AFAP1L1 in actin
rich structures (white arrowheads) in transiently transfected COS7 cells. (scale bar =
10µm). (d) Lyn can directly phosphorylate AFAP1L1. Inactive (Y397F) and hyperactive
(Y508F) Lyn were immunoprecipitated from transiently transfected COS7 cells and using
in in vitro kinase assays with purified recombinant AFAP1L1 (as a GST fusion). (e)
Subcellular localization of eGFP-AFAP1L1 (green) in live cell cultures of U2OS
osteosarcoma cells during vanadate stimulation. Sub-nuclear invadopodia are indicated
(arrow heads), nuclei stained with Hoechst 33342 (blue). Maximum image projection in XY
plane. Scale bar=10µm.
Figure 4. AFAP1L1 has oncogenic potential and significantly alters the cytoskeleton,
cell attachment/spreading and migration/invasion. (a) Soft agar anchorage
independent growth assay of NIH3T3 cells expressing AFAP1L1. Phase-contrast images
of vector only control (Con) and cells expressing AFAP1L1 after 7 days growth in soft agar
(i), with enumeration of colonies formed (ii), results are mean ±S.D., n=3, **p<0.01. (b)
Effect of expression of AFAP1L1 alone or in combination with wild-type Lyn (LynWT),
inactive Lyn (LynY397F) or hyperactive Lyn (LynY508F) on NIH3T3 cell morphology.
Phase contrast images are shown, scale bar = 20µm. (c) Wound healing/scratch assay of
NIH3T3 cells expressing AFAP1L1 and control cells. The number of cells in quadrants that
Page 27 of 29
had migrated to wards the scratch was enumerated at 16h post initiation of scratch assay.
Results are mean ± S.D., n=8, **p<0.01. (d) Real-time assay for cell attachment and
spreading using cell impedance measurements with E-plates on an xCELLigence (ACEA
Biosciences). HEK293 cells expressing AFAP1L1 or control (Con) cells plates in the
presence or absence of serum as indicated. Results are mean, n=3. (e) Analysis of
spontaneous cell movement in low confluence cultures of HEK293 cells expressing
AFAP1L1 and control cells (Con). Individual cell movement (n=30) for each cell type was
analysed at 1min intervals over 16min using Gradientech Tracking Tool (v1.70,
Gradientech). (f) Morphological analysis of HEK293 cells expressing eGFP-tagged
AFAP1L1 or vector only control (Con) gown on solid extracellular matrix (egg white) for 7
days. Fluorescent images are shown, scale bar = 50µm. (g) Modified Boyden chamber
migration assay. HEK293 cells expressing AFAP1L1 or control (Con) cells in serum free
media (top chamber) migrating to serum containing media (bottom chamber) were
enumerated after 16h. Results are mean ± S.D., n=3, **p<0.01. Results are mean, n=3. (h)
Real-time assay for cell migration using cell impedance measurements with CIM-plates
(8µm pore size) on an xCELLigence (ACEA Biosciences). HEK293 cells expressing
AFAP1L1 or control (Con) cells in serum free media migrating to serum containing media.
Results are mean, n=3.
Figure-5. AFAP1L1 phosphorylated by Lyn generates specific pY motifs for binding
Vav2 (Y136) and Nck2 (Y566). (a) Interaction of Vav2 with AFAP1L1 through both its SH3
and SH2 domains. (i) Yeast two-hybrid assay of Vav2 SH3 domain interacting with a
proline rich sequence in AFAP1L1. (ii) Phospho-tyrosine yeast two-hybrid assay of Vav2
SH2 domain binding the phosphorylated Y136 motif of AFAP1L1. (iii) Co-
immunoprecipitation of Vav2 and AFAP1L1 from transiently transfected COS7 cells.
Maximum co-immunoprecipitation occurs with vanadate stimulation to induce
phosphorylation of AFAP1L1, the Y136F mutation of AFAP1L1 significantly reduces co-
immunoprecipitation of Vav2 and AFAP1L1. (iv) Co-localization of Vav2 and AFAP1L1 in
actin rich structures (white arrowheads) in transiently transfected COS7 cells stimulated
with vanadate. (scale bar = 10µm). (v) Direct binding of labeled Vav2-SH2 domain via
protein blotting of lysates from transiently transfected COS7 cells expressing hyperactive
Lyn (Y508F) and either wild-type AFAP1L1 of the Y136F mutant. (b) Interaction of Nck2
with AFAP1L1 through both its SH2 domain. (i) Phospho-tyrosine yeast two-hybrid assay
of Nck2 SH2 domain binding the phosphorylated Y566 motif of AFAP1L1. (ii) Co-
immunoprecipitation of Nck2 and AFAP1L1 from transiently transfected COS7 cells. Co-
Page 28 of 29
immunoprecipitation occurs with vanadate stimulation to induce phosphorylation of
AFAP1L1, the Y566F mutation of AFAP1L1 eliminates detectable co-immunoprecipitation
of Nck2 and AFAP1L1. (iii) Co-localization of Nck2 and AFAP1L1 in actin rich structures
(white arrowheads) in transiently transfected COS7 cells stimulated with vanadate. (scale
bar = 10µm). (iv) Direct binding of labeled Nck2-SH2 domain via protein blotting of lysates
from transiently transfected COS7 cells expressing hyperactive Lyn (Y508F) and either
wild-type AFAP1L1 of the Y566F mutant. (c) In transiently transfected COS7 cells
phosphorylation of AFAP1L1 by LynY508F induces significant cytoskeletal changes that
are dependent on both the Vav2 and Nck binding Y136 and Y566 motifs. Phosphorylation
of the AFAP family member AFAP1 by LynY508F induces podosomes in COS7 cells. (d)
Immunoblot analysis of AFAP1L1 immunoprecipitated from U2OS cells stimulated with
vanadate (VO4) probed for tyrosine phosphorylation (pY) and compartmentalization of
AFAP1L1 to cytosolic/membrane (Cyt) and F-actin (Fil) subcellular fraction. (e)
Immunoblot analysis of AFAP1L1 immunoprecipitated for association of Vav2 from U2OS
cells stimulated with epidermal growth factor and fibronectin (EGF/FN) for the times (min)
indicated. (f) Immunoblot analysis of Nck2 and AFAP1L1 immunoprecipitated from U2OS
and MG-63 cells stimulated with vanadate (VO4). (g) Immunoblot analysis of Vav2 and
AFAP1L1 immunoprecipitated from U2OS and MG-63 cells stimulated with vanadate
(VO4), compared to total levels in lysates (L). (h) Subcellular localization of AFAP1L1
(green) and ((i), left panel) Vav2 (red) or ((ii) right panel) Nck2 in U2OS osteosarcoma
cells after vanadate stimulation (1h). Sub-nuclear invadopodia are indicated (arrow heads),
nuclei stained with Hoechst 33342 (blue). Maximum image projection in XY plane. Scale
bar=10µm.
Figure 6. Signalling through AFAP1L1 regulates Rac, PAK and MLC2. (A) Rac1 and
RhoA activation assays from COS7 cells transiently transfected with HA-tagged AFAP1L1
and inactive Lyn (LynY397F) or hyperactive Lyn (LynY508F), or stimulated with vanadate
(VO4). PAK1-PBD beads were used to pull-down active Rac1, while Rhotekin-PBD beads
were used to pull-down active RhoA and immunoblots probed for Rac1 and RhoA,
respectively. Relative Rac1 and RhoA activity is measured as a ratio of Rac1/RhoA in the
pull-down relative to total Rac1/RhoA in the lysate. Assays performed in duplicate (mean
±S.E.M.). (B) Alterations to phosphorylation status of p21 activated kinases PAK1/2/3 and
myosin light chain-2 (MLC2) in cells expressing AFAP1L1 and stimulated with vanadate.
Immunoblot analysis of COS7 cells transiently transfected with or without HA-tagged
AFAP1L1 and stimulated with vanadate (VO4) for the times indicated. The level of
Page 29 of 29
phosphorylated PAK to total PAK was quantitated by densitometry (graph). (C) RNAi
mediated knockdown of AFAP1L1 in U2OS sarcoma cells regulates PAK activity.
Immunoblot analysis of U2OS cell lysates after 72h of RNAi-mediated knockdown with
oligonucleotides S43843 (S3), S43844 (S4) or non-targeting control RNAi (NT), stimulated
with or without vanadate (VO4) for 1 h. (D) RNAi mediated knockdown of AFAP1L1 in
U2OS sarcoma cells regulates PAK activity and MLC2 phosphorylation and subcellular
compartmentalization. Immunoblot analysis of cytosolic (Cyt) and F-actin containing (Fil)
subcellular fractions of U2OS cell lysates after 72h of RNAi-mediated knockdown with
oligonucleotides S43843 (S3), S43844 (S4) or non-targeting control RNAi (NT), stimulated
with or without vanadate (VO4) for the times indicated.
Figure 7. Schematic of AFAP1L1 signalling network in sarcoma cells. Schematic
model of pathways intersected by AFAP1L1 in sarcoma cells to regulate invadopodia and
invasion/migration. Activation through ligation of growth factors and extracellular matrix
(GF/ECM) activate Lyn/Src family tyrosine kinases (Lyn/SFK), the PI3kinase pathway and
protein kinase C (PKC), leading to phosphorylation of AFAP1L1 on serines (S) in the inter-
PH domain region, tyrosines (Y) at Y136 and Y566 and promotion of location to
phospholipid enriched membranes. AFAP1L1 also has strong constitutive interaction with
F-actin via carboxyl-terminal leucine-rich (L) and a-helical (H) regions that also mediate
multimerization of AFAP1L1, which may facilitate cross-linking (X-linking) of F-actin fibers.
The coiled-coil (C) region of AFAP1L1 may provide additional signals for localization to
invadopodia localized F-actin. A proline-rich N-terminal region (P) of AFAP1L1 can bind
the SH3 domains of Src family kinases (SFK), Vav2, cortactin and tyrosine kinase
substrate-5 (Tks5). The SH2 domain of Vav2 binds to the phosphorylate Y136 motif and
can lead to activation of a Rac1 (or possibly cdc42) – Pak – MLCK pathway. Further the
SH2 domain of Nck2 binds to the phosphorylate Y566 motif and with Nck2 forming a
complex with cortactin, N-WASP, Arp2/3 and Tks5. Consequently Vav2 and Nck2
interaction with AFAP1L1 facilitates close connection of multiple cytoskeletal modulating
proteins to bring about regulation of migration and invasion in sarcoma cells.