Cellular Signalling 27 (2015) 14991508

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Cellular Signalling
journal homepage: www.elsevier.com/locate/cellsig

Intersectin adaptor proteins are associated with actin-regulating protein

WIP in invadopodia
Tetyana Gryaznova , Sergii Kropyvko, Mariia Burdyniuk, Olga Gubar, Valentyna Kryklyva,
Liudmyla Tsyba, Alla Rynditch
Institute of Molecular Biology and Genetics, 150 Zabolotnogo Street, Kyiv 03680, Ukraine

a r t i c l e

i n f o

Article history:
Received 5 March 2015
Accepted 15 March 2015
Available online 19 March 2015
Keywords:
Intersectin
WIP
Invadopodia
Actin cytoskeleton
N-WASP

a b s t r a c t
Invasive cancer cells form actin-rich membrane protrusions called invadopodia that degrade extracellular matrix
and facilitate cell invasion and metastasis. WIP (WASP-interacting protein) together with N-WASP (neural
Wiskott-Aldrich syndrome protein) are localized in invadopodia and play a crucial role in their formation.
Here we show that WIP interacts with endocytic adaptor proteins of the intersectin (ITSN) family, ITSN1 and
ITSN2. The interaction is mediated by the SH3 domains of ITSNs and the middle part of the WIP proline-rich
motifs. We have also demonstrated that ITSN1, WIP and N-WASP can form a complex in cells. Endogenous
ITSN1 and ITSN2 are located in invasive protrusions of MDA-MB-231 breast cancer cell line. Moreover, data
from immunouorescent analysis revealed co-localization of ITSN1 and WIP at sites of invadopodia formation
and in clathrin-coated pits. Together, these ndings provide insights into the molecular mechanisms of
invadopodia formation and identify ITSNs as scaffold proteins involved in this process.
2015 Elsevier Inc. All rights reserved.

1. Introduction
Actin cytoskeleton remodeling is important for numerous cellular
processes such as cell migration, invasion, adhesion, endo-/exocytosis,
cell division and fusion [1,2]. Reorganization of actin laments is
regulated by highly integrated signaling cascades that transduce
extracellular stimuli to the actin laments. The implication of various
scaffold proteins that function as platforms for the assembly of
multiprotein complexes is essential for this process [3]. Adaptor/scaffold
proteins of the intersectin (ITSN) family are considered to be a link between external stimuli and processes of endo- and exocytosis, signal
transduction and cytoskeleton rearrangements. Two ITSN family members, ITSN1 and ITSN2, are widely expressed in mammal tissues and
have similar domain structures [4,5]. Each has two main isoforms,
short and long, produced by alternative splicing [6]. The short isoform
(ITSN-S) comprises two EH (Eps15 homology) domains, a coiledcoil
region (CCR) and ve SH3 (Src 3 homology) domains. The long isoform
(ITSN-L) includes additionally the DH (Dbl homology) domain, the PH
(pleckstrin homology) domain and C2 domain [4,5]. Each of the
intersectin domains is implicated in the interaction with various

Abbreviations: ITSN, intersectin; WIP, WASP-interacting protein; N-WASP, neural

Wiskott-Aldrich syndrome protein; SH2, Src homology 2 domain; SH3, Src homology 3
domain; PRM, proline-rich motif.
Corresponding author at: Institute of Molecular Biology and Genetics, 150 Zabolotnogo
Street, Kyiv 03680, Ukraine. Tel.: +380 44 200 0327.
E-mail address: t.griaznova@gmail.com (T. Gryaznova).

http://dx.doi.org/10.1016/j.cellsig.2015.03.006
0898-6568/ 2015 Elsevier Inc. All rights reserved.

endocytic and signaling proteins overall numbering more than 30 partners [7]. Several additional splice variants affecting domain structure
and ligand-binding specicity of ITSN1 have also been reported [8,9].
ITSNs are associated with several neurodegenerative pathologies
and cancers [1013]. In particular, ITSN1-S is involved in migration
and invasion of human glioma cells [14]. The overexpression of ITSN1-L
in rodent broblasts has been shown to promote transforming activity [15]. However, Specht et al. showed that increased levels of ITSN2
transcripts are associated with prolonged disease-free survival of patients with breast cancer [13].
Both ITSN family members are involved in the regulation of actin
remodeling. They assemble pivotal components of the actin nucleation
complex such as the Wiskott-Aldrich syndrome protein (WASP) and
its ubiquitously expressed variant N-WASP, that activate the Arp2/3
(actin-related proteins 2 and 3) complex and stimulate actin lament
nucleation [16,17]. Moreover, ITSN-L is a specic guaninenucleotide
exchange factor for the small GTPase Cdc42, and the latter in turn activates N-WASP [18,19]. In cells, both WASP, which is primarily expressed
in hematopoietic cells, and N-WASP are associated with WIP (WASPinteracting protein) [2022]. WIP is a widely expressed actin-binding
protein that regulates stability, location and function of WASP and
N-WASP and participates in cellular signaling, endocytosis and actin
cytoskeleton remodeling [2325]. Current results indicate a potential
role of WIP in cancer cell invasion and metastasis. Expression analysis
of the WIPF1 gene in glioma, colorectal cancer and breast cancer
shows a signicant correlation between low WIP expression and
improved prognosis for patients [26]. Recent studies denote that WIP

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is one of the key components of podosomes and invadopodia (actin-rich

membrane protrusions with matrix degradation activity present in
normal and cancer invasive cells respectively) [2729]. Prominent
components and specic markers of invadopodia such as cortactin and

Tks5 (Tyr kinase substrate with ve SH3 domains) [30] are binding
partners of WIP. Cortactin directly binds WIP while interaction with
Tks5 was predicted by mass-spectrometry [31,32]. It was previously
shown that actin polymerization driven by the interaction between

Fig. 1. ITSN1 interacts with WIP in vivo and in vitro. (A) ITSN1 and WIP form a complex in mouse brain. The WIP/ITSN1 complex was immunoprecipitated from mouse brain lysate with
anti-WIP antibodies. The precipitated proteins were detected by Western blotting with anti-ITSN1-EH2. (B) ITSN1 and WIP co-precipitated in MDA-MB-231 cells. The cells were
transfected with plasmid encoding HA-tagged WIP. Cell extracts were subjected to immunoprecipitation with anti-ITSN1-EH2 antibodies. Protein complexes were detected by Western
blotting, using anti-HA and anti-ITSN1/Ese1 antibodies. (C) ITSN1 directly interacts with WIP. Recombinant GST-fusion proteins containing all ve SH3 domains of ITSN1 (GST-ITSN1-SH3(A-E))
or GST alone were immobilized on glutathione Sepharose and incubated with puried recombinant WIP-6xHis. The pulled down fraction was assessed by Western blotting using anti-WIP
antibodies. GST-fusion proteins were visualized with Ponceau S staining. (D) ITSN1-S co-localizes with WIP in CCPs. NIH 3T3 cells were plated on coverslips and co-transfected with
mCherry-ITSN1-S, HA-WIP and GFP-clathrin. HA-WIP was stained with anti-HA antibodies and visualized with Alexa Fluor 633-conjugated secondary antibodies. White arrows indicate
structures that are positive for all three proteins. Scale bar: 20 m. (E) Partial co-localization of endogenous ITSN1, WIP and cortactin in human U87-MG glioblastoma cells. Cells were seeded
on bronectin-coated glass coverslips and subjected to immunouorescence analysis. Endogenous WIP, ITSN1 and cortactin were detected with anti-WIP, anti-ITSN1 and anti-cortactin
antibodies followed by visualization with anti-goat Alexa Fluor 633-conjugated, anti-rabbit Dylight 488-conjugated and anti-mouse Texas Red-conjugated secondary antibodies, respectively.
Scale bar: 10 m.

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N-WASP, Arp2/3 complex and their upstream regulators, Cdc42, Nck1

and WIP is required for invadopodia formation [27]. Thus, it seemed
probable that ITSNs (as partners of most of these components) may be
involved in this process. We also supposed a possible interaction
between ITSNs and WIP, because they participate in the same processes
such as endocytosis and actin cytoskeleton rearrangement, and possess
putative interaction motifs.
Recently WIP was identied as an ITSN2 binding protein in the yeast
two-hybrid screen [33]. Here we report that WIP is a binding partner for
ITSN1 and ITSN2 in vitro and in vivo. We found that ITSN1, WIP and
N-WASP can form complexes. Moreover, we have shown that endogenous ITSNs are localized in invasive protrusions of the breast cancer
cell line MDA-MB-231. We propose that investigation of the ITSN/WIP
interaction can help identify the molecular mechanisms of invadopodia
formation in metastatic cells.
2. Materials and methods
2.1. Expression constructs
The cDNA fragments encoding human wild type WIP (aa 13500,
Genbank accession number NP_001070737.1) were cloned into the
pCMV-HA vector (Clontech) with an additional HA-tag at the 3 end
and into the pET28c(+) vector (Novagen). The construct encoding
WIP(aa 13450) was generated by PCR from a construct carrying
wild type WIP with subsequent cloning into the pCMV-HA vector with
an additional HA-tag. The constructs encoding WIP(aa 13215),
WIP(aa 318500) and WIP(aa 216317) were generated from the
abovementioned plasmid by deletion of the DNA fragments encoding
amino acid residues 216500, 13317 and 216317, respectively. The
full coding sequence of N-WASP (Genbank accession number NP_
003941.3) was cloned into the pcDNA4/HisMaxB vector (Invitrogen)
with an Omni-tag sequence.
Expression constructs containing all ve SH3 domains of ITSN1 as
well as individual GST-fused SH3 domains of ITSN1 were described
previously [34,35]. Plasmids encoding the GST-fused SH3 domains of
ITSN2, Omni-ITSN2-S, Omni-ITSN1-S, GFP-ITSN1-L and mCherryITSN1-S were described previously [3639]. GFP-clathrin was a kind
gift of Dr. S. Havrylov (Warsaw, Poland).
2.2. Antibodies
Rabbit polyclonal antibodies against the EH2 domain of human
ITSN1 (anti-ITSN1-EH2) were described previously [37]. Polyclonal
antibodies against the ITSN2 (anti-ITSN2) were produced in mouse
immunized with the recombinant His-tagged protein comprising
amino acid residues 349499 of human ITSN2. Monoclonal antiITSN1/Ese1 antibody (611574) was from BD Biosciences.
Polyclonal WIP (G-20): sc-16882 and N-WASP (H-100): sc-20770
antibodies and monoclonal anti-Omni (D-8): sc-7270 antibody
were purchased from Santa Cruz Biotechnology. Monoclonal anti-HA,

Fig. 2. The SH3 domains of ITSN1 interact with the middle part of WIP PRMs. (A) WIP binds
to the SH3A, SH3C and SH3E domains of ITSN1 in vitro. Immobilized on glutathione beads,
GST-fused SH3 domains of ITSN1 were incubated with lysates of MDA-MB-231 cells
overexpressing HA-WIP. Precipitated HA-WIP was detected using anti-HA antibodies.
GST-fusion proteins were visualized by Coomassie staining. (B) A schematic representation
of wild-type human WIP and WIP deletion constructs. VH, N-terminus verprolin homology
domain; PRMs, proline-rich motifs; WH2, actin-binding WASP-homology 2 domain; WBD,
WASP-binding domain; ABM-2, prolin-binding actin-based motility 2 region. All deletion
constructs and WIPwt contain a C-terminal HA-tag. (C) GST-fused ITSN1-SH3(A-E)
domains were incubated with the lysates of MDA-MB-231 cells overexpressing
WIP(aa 13215), WIP(216-317), WIP(aa 318500) and WIPwt. The precipitated proteins
were detected with anti-HA antibodies. GST-fusion proteins were detected by Ponceau S
staining. (D) ITSN1 interacts with the WIP(aa 13450) deletion mutant that does not interact
with N-WASP. Lysates of MDA-MB-231 cells expressing the WIP(aa 13450) mutant were
incubated with GST-ITSN1-SH3(A-E) domains or GST alone. WIP(aa 13-450) was detected
with anti-HA antibodies. GST-fusion proteins were visualized by Ponceau S staining.

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anti-cortactin (p80/85) clone 4F11 and anti--actin antibodies were

purchased from Covance, Millipore (Upstate) and Sigma, respectively.
Secondary horseradish peroxidase-labeled anti-rabbit, anti-mouse
and anti-goat antibodies were purchased from Promega. Donkey
anti-goat Alexa Fluor 633, goat anti-mouse Alexa Fluor 633, donkey
anti-rabbit Alexa Fluor 555, donkey anti-goat Alexa Fluor 555, donkey
anti-mouse Alexa Fluor 488 and Alexa 647-phalloidin were purchased
from Invitrogen. Horse anti-mouse Texas Red and sheep anti-rabbit
Dylight 488 were from Vector Laboratories and Seratec, respectively.

2.3. Protein expression, pull-down assay and Western blot analysis

The recombinant His- or GST-fused proteins were expressed in
Escherichia coli BL21 cells and afnity puried using NiNTA Agarose
(Qiagen) and glutathione-Sepharose 4B (GE Healthcare) according to
the manufacturer's instructions. Lysates of transiently transfected
MDA-MB-231 cells were prepared in extraction buffer containing
20 mM TrisHCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA,
1 mM phenylmethylsulfonyluoride (PMSF) and protease inhibitor
cocktail from Roche and centrifuged for 20 min at 12,000 g at 4 C. For
pull-down experiments, 5 g of GST or GST-fused proteins were
bound to 30 l of 50% glutathione-Sepharose 4B beads and incubated
with cell lysates for 1 h at 4 C. After extensive washing, the beads

were boiled in Laemmli sample buffer. The proteins were resolved in

SDSPAGE, transferred to nitrocellulose membranes (Bio-Rad) and
blocked in 5% nonfat milk. The membranes were incubated with primary
antibodies for 1 h followed by incubation with peroxidase-conjugated
secondary antibodies for 40 min. Immunoreactive bands were detected
using ECL reagents. Chemiluminescence was captured with Molecular
Imager ChemiDoc XRS+ (Bio-Rad).
2.4. Immunoprecipitation
For immunoprecipitation (IP), the cells and mouse brain were lysed
in IP buffer (20 mM TrisHCl pH 7.4, 150 mM NaCl, 1% Nonidet P40, 10%
glycerol, 2 mM EDTA and protease inhibitor cocktail). The lysates were
incubated with antibodies and protein A/G PLUS-Agarose (Santa Cruz
Biotechnology) prewashed in IP buffer. After incubation for 3 h at 4 C,
the beads were washed three times with IP buffer. Bound proteins
were eluted by boiling in Laemmli sample buffer and then analyzed by
SDSPAGE and Western blotting.
2.5. Cell culture and transfection
MDA-MB-231, U87-MG and NIH 3T3 cell lines were maintained in
Dulbecco's modied Eagle's medium (DMEM) supplemented with 10%

Fig. 3. ITSN1 interacts with the WIP/N-WASP complex in the breast cancer cell line MDA-MB-231 in vivo. (A) Endogenous proteins ITSN1-S, WIP and N-WASP co-precipitate in
MDA-MB-231 cells. Cell extracts were subjected to immunoprecipitation with anti-WIP antibodies. Protein complexes were analyzed by Western blotting using anti-ITSN1-EH2,
anti-N-WASP and anti-WIP antibodies. (B) ITSN1-L forms complexes with WIP and N-WASP. MDA-MB-231 cells were co-transfected with plasmids encoding GFP-ITSN1-L, HA-WIP and
Omni-N-WASP. Cell extracts were subjected to immunoprecipitation with anti-ITSN1-EH2 rabbit antibodies. Protein complexes were analyzed by Western blotting using anti-GFP, anti-HA
and anti-Omni antibodies. (C) ITSN1, WIP and N-WASP co-localize at the plasma membrane in MDA-MB-231 cells. The cells were co-transfected with mCherry-ITSN1-S, HA-WIP and
Omni-N-WASP. WIP and N-WASP were detected with anti-HA and anti-Omni antibodies followed by visualization with Alexa Fluor 633-conjugated and Alexa Fluor 488-conjugated
secondary antibodies, respectively. White arrows indicate structures that are positive for all the three proteins. Scale bar: 10 m.

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fetal bovine serum, 50 U/ml penicillin and 100 g/ml streptomycin. The
cells were transiently transfected with JetPEI (polyethyleneimine,
Polyplus Transfection) according to manufacturer's instructions and
processed 24 h after transfection.

2.6. Immunouorescence and confocal microscopy

The cells were plated on coverslips and transfected with the appropriate plasmid DNA using the JetPEI reagent. 24 h post-transfection, the
cells were washed with ice-cold PBS (phosphate buffered saline), xed
in 4% formaldehyde for 15 min and washed three times in PBS containing 0.2% Triton X-100. The cells were blocked with 2% bovine serum
albumin (BSA), 0.2% Triton X-100 in PBS for 30 min at room temperature and incubated with the appropriate antibodies. The slides were
mounted using PVA-DABCO (Fluka) and confocal images were taken
using a Zeiss LSM510 microscope.

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2.7. Invadopodia gelatin degradation assay

Coverslips were acid-washed and coated with poly-L lysine (Sigma)
for 20 min, washed with PBS and crosslinked with 0.5% glutaraldehyde
(Sigma) for 15 min. The coverslips were then inverted on Oregon Green
488-conjugated uorescent gelatin (Molecular Probes) for 15 min in
the dark. After washing with PBS, the coverslips were then incubated
with 5 mg/ml sodium borohydride for 3 min at room temperature
followed by another washing with PBS and sterilization with 70%
ethanol for 15 min. Finally, uorescent gelatin-coated coverslips were
incubated with complete medium for 1 h at 37 C before plating cells.
MDA-MB-231 cells were cultured on uorescent gelatin-coated coverslips for 6 h and then stained with the appropriate antibodies and
phalloidin to visualize invadopodia and gelatin degradation sites.
The cells were xed with 4% formaldehyde, blocked with 2% BSA in
PBS containing 0.1% Triton X-100 and incubated with the appropriate
primary and uorescent secondary antibodies or phalloidin. Phalloidin

Fig. 4. ITSN2 and WIP are associated in cells. (A) MDA-MB-231 cells were transfected with Omni-ITSN2-S and HA-WIP. Cell extracts were subjected to immunoprecipitation with anti-HA
antibodies. Protein complexes were analyzed by Western blotting with anti-WIP and anti-Omni antibodies. (B) WIP interacts with the SH3 domains of ITSN2 in vitro. GST-fused SH3
domains of ITSN2 immobilized on glutathione beads were incubated with extracts of MDA-MB-231 cells overexpressing HA-WIP. GST-fused proteins were visualized by Ponceau S staining.
HA-WIP was detected with anti-HA antibodies. (C) The SH3A, SH3C and SH3E domains mediate ITSN2 interaction with N-WASP. The GST-SH3 domains of ITSN2 or GST alone immobilized
on glutathione beads were incubated with lysates of MDA-MB-231 expressing Omni-N-WASP. Bound proteins were detected with antibodies against Omni. GST-fused proteins were
visualized by Coomassie staining. (D) WIP(aa 13450) interacts with the SH3A, SH3C and SH3E domains of ITSN2. HA-WIP(aa 13450) was overexpressed in MDA-MB-231 cells and
the lysates were incubated with the GST-fused SH3 domains of ITSN2 or GST alone. WIP(aa 13450) was detected with anti-HA antibodies. GST-fused proteins were stained with
Coomassie. (E) Partial co-localization of endogenous ITSN2 and WIP in human U87-MG cells. Cells were seeded on glass coverslips and subjected to immunouorescence analyses.
WIP and ITSN2 were detected with anti-WIP, and anti-ITSN2 antibodies followed by visualization with Alexa Fluor 555-conjugated and Alexa Fluor 488-conjugated secondary antibodies,
respectively. Scale bar: 15 m.

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Fig. 5. ITSN proteins are localized at sites of invadopodia in MDA-MB-231 cells. (A) Immunouorescent staining of endogenous ITSN1 in invadopodia. ITSN1 was stained with anti-ITSN1-EH2
antibodies and visualized with Alexa 555-conjugated secondary antibodies. F-actin was detected with Alexa 647-phalloidin. Arrows indicate co-localization of ITSN1 and actin in
invadopodia. (B) Endogenous ITSN2 localizes to invadopodia. ITSN2 was stained with anti-ITSN2 antibodies followed by visualization with Alexa 555-conjugated secondary antibodies.
F-actin was detected with Alexa 647-phalloidin. Arrows indicate sites of ITSN2 and actin co-localization in invadopodia. (C) ITSN1 co-localizes with WIP at sites of invadopodia. ITSN1
and WIP were stained with anti-ITSN1-EH2 and anti-WIP antibodies and visualized with Alexa 555-conjugated and Alexa 633-conjugated secondary antibodies, respectively. Arrows
denote co-localization of ITSN1 and WIP at sites of invadopodia. Each lower row in (A), (B) and (C) represents the magnications of the respective boxed areas containing invadopodia.
Scale bar: 10 m.

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was used to stain F-actin. Finally, coverslips were mounted using

PVA-DABCO (Fluka).

3. Results
3.1. ITSN1 interacts with WIP in vivo and in vitro
The interaction of WIP and ITSN1 was initially predicted by the online service Scansite (http://scansite.mit.edu). To determine whether
ITSN1 and WIP form a complex in vivo, we performed immunoprecipitation assays using mouse brain protein lysate and found that two major
ITSN1 isoforms (ITSN1-S and ITSN1-L) precipitated with anti-WIP
antibodies (Fig. 1A). Moreover, hemagglutinin HA-tagged WIP
(HA-WIP) transiently expressed in the breast cancer cell line MDAMB-231 also co-precipitated with endogenous ITSN1-S and ITSN1-L
using antibodies against the EH2 domain of ITSN1 (Fig. 1B).
In cells, WIP is associated with WASP and N-WASP [20,22]. Coprecipitation experiments revealed that more than 80% of WIP and
WASP exist as a complex [25]. Furthermore, N-WASP has been shown
to bind directly to the SH3 domains of ITSN1-L and to up-regulate its
guanine nucleotide exchange factor (GEF) activity toward the small
GTPase Cdc42 [16]. Since N-WASP interacts with both ITSN1 and WIP
we checked whether ITSN1 and WIP bind directly or whether their interaction is mediated by N-WASP. ITSN1 contains ve SH3 domains
that bind numerous proline-rich motifs (PRMs) of target proteins. To
conrm that ITSN1 directly interacts with WIP pull-down experiments
were performed with puried recombinant GST-fused ITSN1 SH3(A-E)
domains and His-tagged WIP (Fig. 1C). Recombinant WIP readily coprecipitated with immobilized GST-ITSN1-SH3(A-E) domains. Our
data conrm that the interaction between WIP and ITSN1 is direct and
not mediated by N-WASP.
Immunouorescent analysis was then performed to investigate
the subcellular distribution of the proteins studied. The embryonic
mouse broblast cell line, NIH 3T3, was co-transfected with mCherry-

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ITSN1, HA-WIP and GFP-clathrin constructs. Consistent with previous

reports, ITSN1 was found mainly in the perinuclear region and at the
plasma membrane in clathrin-coated pits (CCPs) [37,40]. Signicant
co-localization of overexpressed ITSN1 and WIP was observed in CCPs
at the plasma membrane (Fig. 1D).
It was previously shown that ITSN1-S is involved in migration and
invasion of human glioma cells [14]. We examined the subcellular
localization of endogenous WIP and ITSN1 in highly invasive human
U87-MG glioblastoma cells which form invadopodia. Overlapping
signals of these proteins were detected in the perinuclear zone and
throughout the cytoplasm (Fig. 1E). WIP is a cortactin binding protein.
Interaction between these proteins serves to regulate cortical actin
dynamics in lamellipodia, actin-rich protrusions that play crucial role
in cell motility and migration [31]. Cortactin localizes at the cell periphery in the regions of active actin remodeling [41]. We have demonstrated partial co-localization of endogenous ITSN1, WIP and
cortactin in U87-MG cells (Fig. 1E). Together, these ndings indicated
that ITSN1/WIP complexes are localized in clathrin-coated pits and
regions of actin cytoskeleton rearrangements.

3.2. Mapping the regions of ITSN1 and WIP interaction

To explore the ITSN1/WIP binding sites, we performed in vitro
binding assays with the individual GST-fused SH3 domains of ITSN1.
Among the ve ITSN1 SH3 domains, only the SH3A, SH3C and SH3E
domains mediated interaction with WIP in vitro (Fig. 2A). Both splice
variants of the SH3A domain (neuron-specic and ubiquitously
expressed) readily precipitated with WIP.
Human WIP consists of the N-terminal highly conserved verprolin
homology (VH) domain, followed by the multiple proline-rich motifs
(PRMs), three actin-based motility 2 domains (ABM-2) for prolin
binding and the WASP-binding domain (WBD) at the C-terminus [25].
The PRMs of WIP are involved in the interaction with the SH3 domains
of various adaptor proteins such as Nck and CrkL, kinases such as Syk

Fig. 6. A model of ITSN/WIP-dependent regulation of the Arp2/3 complex activation via N-WASP and Cdc42. ITSN directly associates with the N-WASP/WIP complex via its SH3 domains. At
the same time, ITSN-L functions as a specic GEF for the GTPase Cdc42. The DH domain of ITSN-L interacts with the GDP-bound Cdc42 and promotes its conversion to the GTP-bound state
[16]. ITSN also interacts with CdGAP and inhibits its GAP activity [51]. After activation, Cdc42 acquires the capacity to bind N-WASP and therefore to activate it. Activated N-WASP binds to
the Arp2/3 complex that leads to the initiation of Arp2/3-dependent actin polymerization [16]. WIP maintains N-WASP in an inactive state. In this conformation N-WASP is unable to initiate Arp2/3-dependent actin polymerization even in the presence of an active Cdc42 [22]. Interaction between ITSN1 and WIP can lead to the inactivation of WIP that enables Cdc42 to
activate N-WASP and the Arp2/3 complex.

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and PKC, as well as with the actin-binding proteins cortactin and

Abp1 [31,4246]. To map the regions of WIP responsible for the interaction with ITSN1, a set of HA-tagged WIP deletion mutants was
constructed (Fig. 2B). WIP(aa 13215) contains the VH domain and
the N-terminal part of the PRMs that is responsible for binding to
cortactin. WIP(216317) lacks 101 amino acid residues in the central
part of the PRMs and WIP(aa 318500) contains the C-terminal part
of the PRMs. WIP(aa 13450) lacks 53 C-terminal residues of PRMs
responsible for WASP binding [47]. To map the region of WIP PRMs
that interacts with ITSN1 we performed GST pull-down experiments
(Fig. 2C, D). WIP(216317), WIP(aa 318500), WIP(aa 13450) and
WIPwt effectively co-precipitated with the SH3(AE) domains of
ITSN1 whereas no binding was observed for with WIP(aa 13215).
Thus, residues 318450 of WIP are important for the interaction with
ITSN1, whereas residues 216317 and 450503 are not critical.
3.3. ITSN1, N-WASP and WIP form a complex in cells
Since the WIP binding sites for ITSN1 and WASP do not overlap,
we suggested that a complex of all three proteins can exist in cells. Results of immunoprecipitation demonstrated that endogenous ITSN1-S
and N-WASP co-precipitate with WIP in the MDA-MB-231 cell line
(Fig. 3A). The same results were obtained with ITSN1-L when all three
proteins were overexpressed in MDA-MB-231 cell line. In this case,
both HA-WIP and Omni-N-WASP were detected in anti-ITSN1 precipitates (Fig. 3B).
Immunouorescent analysis was used to investigate the intracellular distribution of the proteins in MDA-MB-231 cells. mCherry-ITSN1-S,
HA-WIP and Omni-N-WASP displayed signicant levels of co-localization
(Fig. 3C). The results obtained suggest that ITSN1, WIP and N-WASP
form a complex in vivo.
3.4. ITSN2 and WIP are associated in cells
As ITSN2 shares high homology with ITSN1, we supposed that it also
could interact with WIP. To investigate this possibility, we performed
co-immunoprecipitation experiments with over-expressed proteins
and found that Omni-ITSN2-S co-precipitated with HA-WIP in the
MDA-MB-231 cell line (Fig. 4A).
To explore possible in vitro interaction of ITSN2 with WIP, GST
pull-down assays with individual GST-fused SH3 domains of ITSN2
were carried out. WIP interacts with the SH3A, SH3C and SH3E domains
of ITSN2 in vitro (Fig. 4B). It was previously reported that WASP interacts only with the SH3C and SH3E domains of ITSN2 [17]. Our experiments revealed that ubiquitously expressed N-WASP binds to the
SH3A, SH3C and SH3E domains of ITSN2 in vitro (Fig. 4C). As the
ITSN2 binding sites for WIP and N-WASP overlap, we performed
co-immunoprecipitation experiments with a WIP(aa 13450) deletion
mutant, incapable of binding N-WASP. WIP(aa 13450) readily precipitated with the same ITSN2 SH3 domains (Fig. 4D). Thus, as for ITSN1,
WIP/ITSN2 interaction is not mediated by N-WASP.
Immunouorescence analysis using anti-ITSN2 and anti-WIP antibodies demonstrated that endogenous proteins co-localized in U87-MG
cells (Fig. 4E).
3.5. ITSN proteins are localized at sites of invadopodia in metastatic breast
cancer cells
Previously, it was demonstrated that endogenous WIP localizes
at the distal tips of invasive protrusions of MDA-MB-231 cancer cells
[28]. Also it was identied that WIP in the complex with N-WASP is essential for invadopodia formation. Overexpression of the WBD of WIP in
metastatic MTLn3 cells resulted in a marked reduction in the number of
invadopodia [27]. Since ITSN1 directly interacts with WIP and N-WASP,
we investigated whether ITSN1 could be detected in the sites of
invadopodia. As metastatic breast cancer cell line MDA-MB-231 has an

invasive phenotype we used it for the invadopodia gelatin degradation

assay [48,49]. Degradation of the gelatin matrix caused by invadopodia
appears as dark spots under the cells in the gelatin. ITSN1 was enriched
in invadopodia (marked by the punctuate actin structures together with
gelatin degradation) (Fig. 5A). Moreover, signicant co-localization of
endogenous ITSN2 and actin was observed in invadopodia (Fig. 5B).
We then demonstrated that endogenous ITSN1 also co-localizes
with endogenous WIP at sites of invadopodia formation (Fig. 5C). Thus
ITSN proteins interact with WIP at invadopodia sites and this interaction
possibly could affect cell invasiveness.
4. Discussion
Scaffolding proteins of the ITSN family are key components of
the initiation stage of clathrin-mediated endocytosis [50]. They have
been shown to interact with numerous endocytic proteins including
AP2 (adaptor protein 2), Eps15, dynamin and the synaptojanin phosphatase [7]. ITSNs along with Eps15 are required for the clustering of
the membrane-sculpting protein FCHo that promotes the initiation of
clathrin-coated pit formation [50]. The long forms of ITSNs have guanine
nucleotide exchange activity toward the small GTPase Cdc42, which
plays an essential role in actin cytoskeleton rearrangements [18,19].
Moreover, ITSNs interact with the CdGAP (GTPase-activating protein)
that downregulates Cdc42 [51,36] as well as with WASP and N-WASP
that function as effectors for Cdc42 and as activators of the Arp2/3
complex [16,17].
Here we extend our understanding of the ITSN interaction network
in actin remodeling by identifying a novel protein partner WIP. Immunoprecipitation and in vitro binding experiments revealed that ITSNs
bind WIP and these interactions are mediated by the SH3A, SH3C and
SH3E domains of ITSNs. Our results show that the interaction between
ITSNs and WIP is direct and not mediated by N-WASP, which is constitutively associated with WIP in cells. In addition, we have demonstrated
that both ITSN1-S and ITSN1-L form a complex with WIP and N-WASP.
The immunouorescence analyses revealed partial co-localization of
ITSN1 and ITSN2 with WIP.
WIP is known to regulate actin cytoskeleton remodeling by interacting with actin [52] and nucleation promoting factors (NPFs) such as
N-WASP and cortactin [20,31]. NPFs initiate branched actin assembly
by activating the Arp2/3 complex. WIP has been shown to inhibit
Cdc42-mediated N-WASP-induced activation of the Arp2/3 complex
in vitro [20]. On the other hand, cortactin interaction with WIP
enhances cortactin-mediated activation of the Arp2/3 complex [31].
Thus, it is possible that WIP regulates activation of the Arp2/3 complex
via binding to N-WASP and cortactin. WIP interacts with cortactin via
residues 110170 of the N-terminal PRMs [31] and also with N-WASP
via residues 454485 of the -terminal end [47]. Here we report
that residues 318450 of WIP are important for the interaction with
ITSN1. Together these data suggest that ITSN1, cortactin and N-WASP
do not compete for WIP binding and may form a complex. Immunouorescence experiments revealed that ITSN1 as well as the actin binding
proteins WIP and cortactin are localized at the cell periphery in the
regions of the active actin remodeling. Thus, it is likely that the adaptor
protein ITSN1-L may serve as a platform for Cdc42, N-WASP, WIP,
CdGAP and cortactin proteins to regulate their activity in Arp2/3dependent actin polymerization (Fig. 6).
Previous studies revealed that ITSN1 is involved in oncological transformation [15]. Silencing of ITSN1-S inhibits migration and invasion of
human glioma cells and results in a decline of matrix metalloproteinase
(MMP) expression [14]. Data on the role of ITSN2 in carcinogenesis are
limited. Nevertheless, a correlation between high expression levels
of ITSN2 and improved prognosis for breast cancer patients has been
demonstrated [13].
In this study we found that endogenous ITSN1 is localized at sites of
invadopodia. Surprisingly, we identied that ITSN2 is also localized to
these membrane protrusions. At the present point we cannot explain

T. Gryaznova et al. / Cellular Signalling 27 (2015) 14991508

this fact but we consider it should be intently investigated. Invadopodia

formation requires an actin core with scaffolding proteins, kinases
and MMPs. Recently it was shown that WIP is required for the matrix
invasion of breast cancer cells [28,29]. WIP-decient dendritic cells
possessed reduced secretion of proMMP2 and proMMP9. Moreover, it
was demonstrated that interaction between WIP and cortactin is essential for the recruitment of MMPs to the sites of focal extracellular
matrix degradation [53]. Here we demonstrate that ITSN1 and WIP
co-localize at sites of invadopodia. Therefore, interaction of ITSNs and
WIP could be important for the functioning and turnover of these invasive structures. It is known that Cdc42/N-WASP/WIP-dependent actin
polymerization via the Arp2/3 complex is required for invadopodia
formation [27]. Given the ability of ITSNs to activate Cdc42 and directly
interact with N-WASP and WIP, we suppose that ITSNs could be involved
in actin polymerization during invadopodia formation. A hypothetical
model of the role of ITSNs and WIP in invadopodia is presented in Fig. 6.
It was previously shown that ITSN1-L facilitates regulated exocytosis
in neuroendocrine cells by Cdc42 activation which in turn leads to
actin polymerization at the exocytic sites [54,55]. Initially high levels
of ITSN1-L expression were observed in neuronal cells [5658]. Using
immunoprecipitation experiments and RTPCR (data not shown)
we observed that ITSN1-L is also expressed in the cancer cell line
MDA-MB-231 which has an invasive phenotype. Thus, another role
that ITSN1-L and WIP may play in invadopodia is the regulation of
MMP secretion. Moreover, ITSN1 is a well-known component of the
endocytic complexes [40]. WIP was also shown to participate in endocytosis in yeast [23]. As we demonstrate a signicant co-distribution
of ITSN1/WIP complexes in CCPs, we suggest that they may function
together in compensatory endocytosis after MMP secretion. Thus,
ITSNs may function as important scaffold proteins of invadopodia
that inuence downstream signaling pathways related to invasion and
matrix degradation.
5. Conclusions
We have shown that scaffold proteins of the intersectin family,
ITSN1 and ITSN2 associates with WIP, a protein that is necessary for
invadopodium formation. We have identied domains responsible
for these interactions both in ITSN and WIP molecules. Data from immunouorescent analyse revealed co-localization of ITSN1 and WIP at sites
of invadopodia formation and in clathrin-coated pits. We have demonstrated that endogenous ITSN1 and ITSN2 are localized at sites of
invadopodia in MDA-MB-231 breast cancer cell line. The key endocytic
protein ITSN1 and WIP/N-WASP can form complex in cells.
Conict of interest statement
The authors declare no competing interests.
Acknowledgments
We thank Dr. V. Filonenko for the preparation of anti-ITSN2
antibodies. We also thank Dr. V. Gorchev and S. Karakhim for providing
expertise and help in obtaining the confocal images. We are grateful to
Dr. A.-L. Haenni for the critical reading of the manuscript.
Authorship
Contributions: T.G., S.K., M.B., O.G. and V.K. performed experiments,
analyzed data, designed research, and wrote the manuscript; L.T. and
A.R. analyzed data and wrote the manuscript.
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