Scaffold proteins ITSN1 and ITSN2 interact with nuclear RNA-binding proteins
Aim. To identify novel ITSN1 and ITSN2 partners among RNA-binding proteins (RBPs) involved in regulation of mRNA processing. Methods. GST pull-down, immunoprecipitation assays and bioinformatics analysis was used to identify other RBPs that could interact with ITSN1 and ITSN2 proteins. Results. ITSN...
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Інститут молекулярної біології і генетики НАН України
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| Цитувати: | Scaffold proteins ITSN1 and ITSN2 interact with nuclear RNA-binding proteins / S.V. Pankivskyi, N.V. Senchenko, P.B. Busko, A.V. Rynditch // Вiopolymers and Cell. — 2019. — Т. 35, № 2. — С. 81-90. — Бібліогр.: 32 назв. — англ. |
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irk-123456789-1543972019-07-07T13:03:43Z Scaffold proteins ITSN1 and ITSN2 interact with nuclear RNA-binding proteins Pankivskyi, S.V. Senchenko, N.V. Busko, P.B. Rynditch, A.V. Structure and Function of Biopolymers Aim. To identify novel ITSN1 and ITSN2 partners among RNA-binding proteins (RBPs) involved in regulation of mRNA processing. Methods. GST pull-down, immunoprecipitation assays and bioinformatics analysis was used to identify other RBPs that could interact with ITSN1 and ITSN2 proteins. Results. ITSN1 and ITSN2 SH3 domains interacted in vitro with nuclear RBPs SAM68, WBP11, and LARP6. ITSN1 and ITSN2 also co-precipitated with SAM68 and LARP6 from 293 cell lysates. Finally, the bioinformatics analysis identified more than 500 nuclear RBPs that contained several SH3 domain-interacting proline motifs and could bind ITSN1/2. Conclusions. ITSN1 and ITSN2 SH3 domains bind nuclear RBPs SAM68, LARP6, and WBP11 in vitro, form complexes with SAM68 and LARP6 in 293 cells, and potentially could interact with other nuclear RBPs containing SH3 domain-interacting motifs. Мета. Виявити нових партнерів ITSN1 і ITSN2 з-поміж РНК-зв'язуючих білків (RBP), що беруть участь в регуляції процесингу мРНК. Методи. Взаємодії були проаналізовано з використанням GST pull-down assay та імунопреципітації, тоді як біоінформатичний аналіз було використано для ідентифікації інших RBP, які могли б взаємодіяти із білками ITSN1 та ITSN2. Результати. Було показано, що SH3 домени білків ITSN1 та ITSN2 взаємодіють з ядерними RBP SAM68, WBP11 і LARP6. Крім того, було виявлено, що ITSN1 та ITSN2 копреципітувались із SAM68 і LARP6 із лізатів клітин лінії 293. Біоінформатичний аналіз показав, що існує більше 500 ядерних RBP, які містять кілька пролінових мотивів, що можуть взаємодіяти із SH3 доменами білків ITSN1/2. Висновки. SH3 домени білків ITSN1 і ITSN2 взаємодіють із ядерними RBP SAM68, LARP6 і WBP11 in vitro, утворюють комплекси із SAM68 і LARP6 в клітинах лінії 293 і потенційно можуть взаємодіяти з іншими ядерними RBP, що містять мотиви, які зв’язуються із SH3 доменами. Цель. Найти новых партнеров ITSN1 и ITSN2 среди РНК-связывающих белков (RBP), участвующих в регуляции процессинга мРНК. Методы. Взаимодействия были проанализированы с использованием GST pull-down assay и иммунопреципитации, тогда как биоинформатический анализ был проведен для идентификации других RBP, которые могли бы взаимодействовать с белками ITSN1 и ITSN2. Результаты. Было показано, что SH3 домены белков ITSN1 и ITSN2 взаимодействуют с ядерными RBP SAM68, WBP11 и LARP6. Кроме того, было обнаружено, что ITSN1 и ITSN2 копреципитировались с SAM68 и LARP6 из лизатов клеток линии 293. Биоинформатический анализ показал, что существует более 500 ядерных RBP, содержащие несколько пролиновых мотивов, которые могут взаимодействовать с SH3 доменами белков ITSN1/2 в ядре клетки. Выводы. SH3 домены белков ITSN1 и ITSN2 взаимодействуют с ядерными RBP SAM68, LARP6 и WBP11 in vitro, образуют комплексы с SAM68 и LARP6 в клетках линии 293 и потенциально могут взаимодействовать с другими ядерными RBP, содержащими мотивы, которые связываются с SH3 доменами. 2019 Article Scaffold proteins ITSN1 and ITSN2 interact with nuclear RNA-binding proteins / S.V. Pankivskyi, N.V. Senchenko, P.B. Busko, A.V. Rynditch // Вiopolymers and Cell. — 2019. — Т. 35, № 2. — С. 81-90. — Бібліогр.: 32 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.000999 http://dspace.nbuv.gov.ua/handle/123456789/154397 577.22 en Вiopolymers and Cell Інститут молекулярної біології і генетики НАН України |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| collection |
DSpace DC |
| language |
English |
| topic |
Structure and Function of Biopolymers Structure and Function of Biopolymers |
| spellingShingle |
Structure and Function of Biopolymers Structure and Function of Biopolymers Pankivskyi, S.V. Senchenko, N.V. Busko, P.B. Rynditch, A.V. Scaffold proteins ITSN1 and ITSN2 interact with nuclear RNA-binding proteins Вiopolymers and Cell |
| description |
Aim. To identify novel ITSN1 and ITSN2 partners among RNA-binding proteins (RBPs) involved in regulation of mRNA processing. Methods. GST pull-down, immunoprecipitation assays and bioinformatics analysis was used to identify other RBPs that could interact with ITSN1 and ITSN2 proteins. Results. ITSN1 and ITSN2 SH3 domains interacted in vitro with nuclear RBPs SAM68, WBP11, and LARP6. ITSN1 and ITSN2 also co-precipitated with SAM68 and LARP6 from 293 cell lysates. Finally, the bioinformatics analysis identified more than 500 nuclear RBPs that contained several SH3 domain-interacting proline motifs and could bind ITSN1/2. Conclusions. ITSN1 and ITSN2 SH3 domains bind nuclear RBPs SAM68, LARP6, and WBP11 in vitro, form complexes with SAM68 and LARP6 in 293 cells, and potentially could interact with other nuclear RBPs containing SH3 domain-interacting motifs. |
| format |
Article |
| author |
Pankivskyi, S.V. Senchenko, N.V. Busko, P.B. Rynditch, A.V. |
| author_facet |
Pankivskyi, S.V. Senchenko, N.V. Busko, P.B. Rynditch, A.V. |
| author_sort |
Pankivskyi, S.V. |
| title |
Scaffold proteins ITSN1 and ITSN2 interact with nuclear RNA-binding proteins |
| title_short |
Scaffold proteins ITSN1 and ITSN2 interact with nuclear RNA-binding proteins |
| title_full |
Scaffold proteins ITSN1 and ITSN2 interact with nuclear RNA-binding proteins |
| title_fullStr |
Scaffold proteins ITSN1 and ITSN2 interact with nuclear RNA-binding proteins |
| title_full_unstemmed |
Scaffold proteins ITSN1 and ITSN2 interact with nuclear RNA-binding proteins |
| title_sort |
scaffold proteins itsn1 and itsn2 interact with nuclear rna-binding proteins |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| publishDate |
2019 |
| topic_facet |
Structure and Function of Biopolymers |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/154397 |
| citation_txt |
Scaffold proteins ITSN1 and ITSN2 interact with nuclear RNA-binding proteins / S.V. Pankivskyi, N.V. Senchenko, P.B. Busko, A.V. Rynditch // Вiopolymers and Cell. — 2019. — Т. 35, № 2. — С. 81-90. — Бібліогр.: 32 назв. — англ. |
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Вiopolymers and Cell |
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81
S. V. Pankivskyi, N. V. Senchenko, P. B. Busko
© 2019 S. V. Pankivskyi et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Bio-
polymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium,
provided the original work is properly cited
UDC 577.22
Scaffold proteins ITSN1 and ITSN2 interact with nuclear
RNA-binding proteins
S. V. Pankivskyi, N. V. Senchenko, P. B. Busko, A. V. Rynditch
Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03143
s.v.pankivskyi@imbg.org.ua
Aim. To identify novel ITSN1 and ITSN2 partners among RNA-binding proteins (RBPs)
involved in the regulation of mRNA processing. Methods. The interactions were revealed
using GST pull-down and immunoprecipitation assays whereas bioinformatics analysis was
used to identify other RBPs that could interact with proteins ITSN1 and ITSN2. Results. It
was shown that ITSN1 and ITSN2 SH3 domains interacted with nuclear RBPs SAM68,
WBP11, and LARP6 in vitro. Next, it was found that ITSN1 and ITSN2 co-precipitated with
SAM68 and LARP6 from 293 cells lysates. Finally, the bioinformatics analysis identified more
than 500 nuclear RBPs that contain several SH3 domain-interacting proline motifs and could
bind ITSN1/2. Conclusions. ITSN1 and ITSN2 SH3 domains bind nuclear RBPs SAM68,
LARP6, and WBP11 in vitro, form complexes with SAM68 and LARP6 in 293 cells, and
potentially could interact with other nuclear RBPs containing SH3 domain-interacting motifs.
K e y w o r d s: ITSN1, ITSN2, RNA-binding proteins.
Introduction
Scaffold proteins represent a group of biomol-
ecules that are involved in the modulation of
various cellular processes. These proteins are
composed of multiple protein-interacting do-
mains and serve as platforms for the formation
of functional protein complexes [1]. Intersectins
(ITSN1 and ITSN2) are evolutionarily con-
served scaffold proteins encoded by two paral-
ogous genes. Short isoforms of ITSN1
(ITSN1s) and ITSN2 (ITSN2s) possess identi-
cal domain structure and consist of two Eps15
homology domains (EH) that bind Asp-Pro-
Phe (NPF) motifs, a coiled-coil region provid-
ing homo- and heterodimerization and five
SH3 domains that interact with proline-rich
motifs [2]. Long isoforms of ITSN1 (ITSN1l)
and ITSN2 (ITSN2l) contain additional
C-terminal DH-PH-C2 domains involved in
the specific activation of GTPase Cdc42 [3].
ITSN1s, ITSN2s and ITSN2l are expressed
ubiquitously whereas ITSN1l is expressed only
in neurons [4]. ITSN1/2 are known to interact
Structure and Function
of Biopolymers
ISSN 1993-6842 (on-line); ISSN 0233-7657 (print)
Biopolymers and Cell. 2019. Vol. 35. N 2. P 81–90
doi: http://dx.doi.org/10.7124/bc.000999
82
S. V. Pankivskyi, N. V. Senchenko, P. B. Busko et al.
with proteins involved in clathrin-mediated
endocytosis and actin cytoskeleton remodeling,
as well as with components of some cell sig-
naling cascades including MAPK-, PI3KC2β-,
and JNK-mediated pathways [4].
ITSN1 and ITSN2 were found to be in-
volved in oncogenesis. First, ITSN1 overex-
pression induces the oncogenic transformation
of rodent fibroblasts [5] and stimulates the
invadopodia formation [6]. Second, overex-
pression of ITSN1 is associated with the de-
velopment of neuroblastoma [7, 8] and glio-
blastoma [9, 10, 11]. However, the ectopic
expression of ITSN1 suppresses the prolifera-
tion, growth and migration of lung cancer
cells [12]. Similarly, high levels of ITSN2 in
breast cancer patients are associated with pro-
longed disease-free survival [13].
Previously, the analysis of HeLa nuclei
phosphoproteome identified ITSN1 peptides
in the nuclei of HeLa cells [14]. More re-
cently, ITSN1 has been found to undergo nu-
cleus-cytoplasmic shuttling via CRM1- and
importin α-dependent pathways and has been
shown to co-localize with lamin A/C [15]. The
data supports our observations revealing nu-
clear localization of ITSN1 (unpublished data).
However, the role of nuclear localization of
ITSN1 is unknown.
Several high-throughput studies based on
the results of two-hybrid screenings [16, 17]
and phage display [18] identified ITSN1/2
proteins as putative partners of nuclear RNA-
and DNA-binding proteins whereas the role of
the protein complexes in a cell is unknown.
We suggested that ITSN1/2 SH3 domains play
an essential role in the interaction between
ITSN1/2 and RNA-binding-proteins (RBPs)
as multiple RBPs possess proline-rich motifs
that directly interact with SH3 domains where-
as ITSN1/2-RBPs complexes might be in-
volved in novel nucleus-cytoplasm crosstalk
pathways.
Therefore, the current work aimed to confirm
the interactions between ITSN1/2 proteins and
RBPs identified in different high-throughput
studies using GST pull-down and immunopre-
cipitation assays. Moreover, using a bioinfor-
matics approach, we analyzed RBPs present in
human proteome to find additional proline-rich
motifs as potential ITSN1/2-binding sites.
Materials and Methods
Expression constructs. The plasmids encoding
human GST-fused SH3 domains-containing
fragment of ITSN1 and ITSN2 were described
previously [19, 20]. SAM68-GFP was a kind
gift of Dr. D. J. Elliott [21], WBP11-GFP was
received from Dr. M.Bollen [22], and LARP6-
GFP was obtained from Dr. L. M. Schwartz
[23].
Antibodies. Rabbit polyclonal antibodies
against ITSN1 and ITSN2 were described pre-
viously [19, 20]. Polyclonal anti-GFP antibod-
ies were a kind gift of Dr. V. V. Filonenko.
Secondary HRP-labeled anti-rabbit antibodies
were purchased from Promega.
Cell culture and transfection. 293 cells were
maintained in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10 %
fetal bovine serum (Sigma), 50 mg/ml penicil-
lin and 100 mg/ml streptomycin. The cells
were transiently transfected using JetPEI trans-
fection reagent (Polyplus Transfection) accord-
ing to recommended protocol and were pro-
cessed 24 h following transfection.
Pull-down assay. The recombinant GST-
fused proteins were produced in Escherichia
83
Scaffold proteins ITSN1 and ITSN2 interact with nuclear RNA-binding proteins
coli Top10 cells and purified using glutathione-
Sepharose 4B (GE Healthcare) according to
the manufacturer‘s protocol. Lysates of tran-
siently transfected 293 cells were prepared in
extraction buffer containing 20 mM Tris-HCl
pH 7.4, 150 mM NaCl, 0.5 % Triton X-100, 1
mM EDTA and 1 mM phenylmethylsulfonyl-
fluoride (PMSF) and centrifuged for 10 min at
12.000 rpm at 4°C. For pull-down experi-
ments, 5–10 μg of GST or GST-fused proteins
were bound to 30 μl of 50 % glutathione-
Sepharose 4B beads and incubated with the
293 cell lysates for 1 h at 4°C. After extensive
washing, the beads were boiled in Laemmli
sample buffer.
Immunoprecipitation assay. The immuno-
precipitation was performed as described pre-
viously [20]. In brief, 293 cells were lysed in
IP buffer (150 mM NaCl, 20 mM Tris, pH 7.5,
10 % glycerol, 0.5 % NP40, protease inhibitors
cocktail (Roche) and centrifuged for 15 min
at 12,000 rpm. The supernatant (2 mg of pro-
teins) was incubated with 2 µg of anti-ITSN1
or anti-ITSN2 antibodies and 20 µl of Protein
G-conjugated agarose beads (Santa Cruz
Biotechnology) for 4 h at 4°C. Then, beads
were washed three times with IP buffer and
boiled in Laemmli sample buffer.
Western blot. Protein samples in Laemmli
buffer were resolved by SDS-PAGE and trans-
ferred to nitrocellulose membranes (Bio-Rad).
The membranes were blocked with 5 % non-fat
milk in TBS-T (1xTBS, 0.1 % Tween 20) for
1 h, incubated with anti-GFP, anti-ITSN1 or
anti-ITSN2 antibodies for 1 h and washed.
Next, membranes were incubated with HRP-
conjugated secondary anti-rabbit antibodies
for 1 h. Immunoreactive bands were detected
using ECL reagents. Chemiluminescence was
captured with Molecular Imager ChemiDoc™
XRS+ (Bio-Rad).
Bioinformatics analysis. A list of human
RNA-binding proteins was composed using
RBPs Databases ATtRACT [24] (last accessed
December 21, 2018) and RBPDB [25] (last
accessed December 21, 2018) and QuickGO
annotation service [26] (last accessed January
28, 2019) where proteins annotated with GO
terms “RNA binding” (GO:0003723) and “ex-
perimental evidence used in manual assertion”
(ECO:0000269) were obtained. A list of pro-
teins that localize in a cell nucleus was also
obtained from QuickGO service using GO
term “Nucleus” (GO:0005634). Amino acid
sequences of the selected RBPs were acquired
from the UniProt database (https://www.uni-
prot.org/, last accessed December 21, 2019).
Proline-rich motifs (PxxP, RxxPxxP, PxxPxR)
that potentially interact with SH3 domains
were searched in the amino acid sequences of
RBPs automatically using Python scripts.
Results and Discussion
The putative interaction between ITSN1/2 and
several RNA-binding proteins was studied in
vitro. For the purpose, a GST pull-down assay
was performed to analyze the binding of
ITSN1/2 SH3 domains to the putative part-
ners. The selected RNA-binding proteins in-
cluded SAM68 (Src-Associated substrate in
Mitosis of 68 kDa), WBP11 (WW Domain
Binding Protein 11), and LARP6 (La
Ribonucleoprotein Domain Family Member
6). All these proteins contain several PxxP
motifs that can potentially interact with
ITSN1/2 SH3 domains. As a result, recombi-
nant proteins representing SH3-containing
fragments of ITSN1 (ITSN1SH3) and ITSN2
84
S. V. Pankivskyi, N. V. Senchenko, P. B. Busko et al.
Fig. 1. The SH3 domains of ITSN1 (A, B, C) and ITSN2 (D, E, F) interact with RNA-binding proteins SAM68 (A,
D), WBP11 (B, E) and LARP6 (C, F). Following GST pull-down assay and SDS-PAGE, GST-fused SH3 domains of
ITSN1 and ITSN2 were visualized using Ponceau staining whereas GFP-fused RBPs were detected using α-GFP an-
tibodies. The relative amounts of RBP bound to GST or GST-ITSN1/2SH3 were calculated using ImageJ software.
Histograms represent mean ± SD values obtained from three separate assays. WB – Western blotting, TCL – total cell
lysate, a.u. – arbitrary units.
A
D
B
E
C
F
85
Scaffold proteins ITSN1 and ITSN2 interact with nuclear RNA-binding proteins
(ITSN2SH3) fused to GST were immobilized
on the glutathione sepharose and incubated
with the lysates of 293 cells overexpressing
one of the target proteins. Recombinant GST
alone was used as a negative control.
Following gel electrophoresis, GST-fused
ITSN1SH3 and ITSN2SH3 were detected with
Ponceau staining whereas precipitated pro-
teins were detected by Western blot. The anal-
ysis demonstrated that ITSN1 SH3 domains
precipitated all studied RNA-binding proteins
(Fig. 1 A, B, C) whereas SAM68 showed the
most significant binding. Similar data were
obtained for ITSN2 SH3 domains that showed
binding to all analyzed RNA-binding and
strong interaction with SAM68 (Fig. 1 D, E,
F). These data suggest that ITSN1 and ITSN2
interacted with RBPs SAM68, WBP11 and
LARP6 in vitro.
Immunoprecipitation assay was used to ob-
tain the formation of complexes between
ITSN1/2 proteins and selected RBPs in 293
cells. For this purpose, antibodies specific to
ITSN1 or ITSN2 were immobilized on protein
G-containing agarose beads and were incubated
with the lysates of 293 cells overexpressing one
of the target proteins. ITSN1/ITSN2-containing
protein complexes were precipitated and ana-
lyzed using Western blot analysis. The data
demonstrated that both ITSN1 and ITSN2 pre-
cipitated SAM68 and LARP6 suggesting that
ITSN1 and ITSN2 form complexes with SAM68
and LARP6 in cells (Fig. 2 A, C, D, F). However,
ITSN1 and ITSN2 failed to precipitate WBP11
Fig. 2. Endogenous ITSN1 (A, C) and ITSN2 (D, F) precipitate RBPs SAM68 (A, D) and LARP6 (C, F) but did not
precipitate WBP11 (B, E) overexpressed in 293 cells. Following immunoprecipitation assay and SDS-PAGE, precipi-
tated proteins ITSN1s (short isoform) or ITSN2s and ITSN2l (short and long isoforms, respectively) were visualized
using respective antibodies and ECL detection. Next, the same nitrocellulose membrane was re-incubated with α-GFP
antibodies to identify co-precipitated RBPs. IP – immunoprecipitation, NRS – normal rabbit serum, WB – Western
blot, TCL – total cell lysate.
D E
C
F
A B
86
S. V. Pankivskyi, N. V. Senchenko, P. B. Busko et al.
(Fig. 2 B, E) that might be caused by the absence
of specific external (i.e., mitogenic stimulation)
or internal (i.e. posttranslational modification)
factors facilitating the interaction.
As multiple RNA-binding proteins contain
proline-rich regions, we suggested that ITSN1
and ITSN2 could interact with other RBPs. As
a result, we used bioinformatics analysis to
screen amino acid sequences of human RBPs
for the presence of SH3 domain-interacting
motifs. Using information from RBPs data-
bases and QuickGO service, we identified
1383 RBPs whereas 823 RBPs localized in the
nucleus. Next, the presence of proline motifs
(PxxP and RxxPxxP/PxxPxR) that are spe-
cifically recognized by ITSN1/2 SH3 domains
[27] was studied. The analysis demonstrated
that most of RBPs contained at least one PxxP
motif and almost 28 % of RBPs contained at
least one RxxPxxP or PxxPxR motif. The re-
sults of the analysis are visualized in the form
of Venn diagram (Figure 3A) demonstrating
the number of unique and overlapping RBPs
according to the studied properties.
However, as one motif might be insufficient
for the interaction, or it may be localized inside
a protein fold, the number of PxxP and
RxxPxxP/PxxPxR motifs in RBPs was ana-
lyzed. As we were interested in the identifica-
tion of putative ITSN1 partners localized in
the nucleus, the number of proline motifs in
nuclear RBPs is shown (Figure 3B). According
to the frequency distribution, nuclear RBPs
were characterized by various numbers of pro-
line motifs. Putative ITSN1/2 partners that
were identified in the current study (SAM68,
WBP11, and LARP6) significantly differed in
the number of proline motifs. Similar fre-
Fig. 3. Multiple RNA binding proteins contain SH3 domain-interacting proline motifs. A – Venn diagram demonstrat-
ing a number of all RBPs, nuclear RBPs, and RBPs containing at least one indicated proline motif found in human
proteome. B – A histogram showing the frequency of occurrence of certain number of proline motifs identified in
nuclear RBPs.
A B
87
Scaffold proteins ITSN1 and ITSN2 interact with nuclear RNA-binding proteins
quency distribution was obtained for cytoplas-
mic RBPs (data not shown). As a result, it
might be suggested that other nuclear and
cytoplasmic RBPs containing functional pro-
line motifs could form complexes with ITSN1
and ITSN2 similar to Sam68 and LARP6.
However, the experimental evidences are re-
quired to confirm the putative interactions.
The present data revealed the interaction
between ITSN1/2 proteins and RNA-binding
proteins SAM68 and LARP6. Sam68 is in-
volved in the regulation of mRNA processing
including its transcription and splicing [28]. In
particular, SAM68 modulates the transcrip-
tional activity of acetyl-transferase CBP [29],
regulates the expression of cyclins D1 and E
[30], controls the activation of c-myc targeted
genes [31], and is involved in the regulation of
androgen receptor-mediated transcription [32].
As a regulator of alternative splicing, SAM68
induces the production of prooncogenic iso-
forms of CD44, CCND1, ASF/SF2, and BIRC5
[28]. Additionally, the interaction between
SAM68 and some SH3 domain-containing pro-
teins (i.e., kinases and methyltransferases) af-
fects its RNA- and protein-binding properties
[28]. Moreover, the SAM68 overexpression is
observed in different cancer types (breast, glio-
blastoma, prostate) [28]. LARP6 was found to
stimulate the expression of metalloprotease
MMP-9. The overexpression of LARP6 is also
observed in breast cancer tumors and cell lines
[23, 29]. Additionally, LARP6 stimulates an-
giogenesis and tumor growth [23]. On the oth-
er hand, accumulation of ITSN1 in the nucleus
leads to the alteration of the expression of im-
mediate response genes (MYC, EGR1, and FOS)
whereas the ITSN1 knockdown causes the in-
creased production of proapoptotic isoform of
ASF/SF2 (unpublished data). As ITSN1 lacks
RNA- and DNA-binding motifs, it could be
suggested that the effect of ITSN1 on the ob-
served nucleus-specific processes is mediated
by RNA-binding proteins including SAM68 or
LARP6 and could be associated with pro- or
anti-oncogenic signalling. The identification of
multiple proline motifs-containing RBPs sup-
ports the suggestion that ITSN1/2 proteins
might interact with other proteins involved in
RNA processing although the functional con-
sequence of possible interactions is still un-
known. As a result, it can be suggested that the
interaction between ITSN proteins and nuclear
RBPs could serve as a link between signaling
processes in the cytoplasm and RNA processing
in the nucleus. However, further analysis is
needed to confirm the predictions and find out
their functional role.
Conclusion
The SH3 domains of ITSN1 and ITSN2 inter-
acted with the nuclear RNA-binding proteins
SAM68, WBP11 and LARP6 in vitro whereas,
in 293 cells, ITSN1/2 scaffolds formed the
protein complexes with SAM68 and LARP6.
Moreover, human proteome contains a large
number of other nuclear RBPs that have puta-
tive SH3 domain-interacting proline-rich re-
gions which could potentially interact with
ITSN1 and ITSN2. Overall, it could be sug-
gested that ITSN-RBPs complexes could be
involved in the regulation of mRNA processing
although the molecular basis of the potential
relationship requires further analysis.
Acknowledgments
The authors thank Dr. D. J. Elliott for GFP-
tagged SAM68, Dr. M. Bollen for GFP-tagged
88
S. V. Pankivskyi, N. V. Senchenko, P. B. Busko et al.
WBP11, Dr. L. M. Schwartz for GFP-tagged
LARP6, and Dr. V. V. Filonenko for the kind
gift of polyclonal anti-GFP antibodies.
Funding
This work was supported by the grant
№0115U002947 “Mechanisms of invadopodia
functioning and expression of their compo-
nents in the breast cancer disease” as a part of
the interdisciplinary project of NAS of Ukraine
“The molecular and cellular biotechnology in
medicine, industry and agriculture”.
REFERENCES
1. Good MC, Zalatan JG, Lim WA. Scaffold proteins:
hubs for controlling the flow of cellular information.
Science. 2011;332(6030):680–6.
2. Tsyba L, Nikolaienko O, Dergai O, Dergai M, No-
vokhatska O, Skrypkina I, Rynditch A. Intersectin
multidomain adaptor proteins: regulation of func-
tional diversity. Gene. 2011;473(2):67–75.
3. Hussain NK, Jenna S, Glogauer M, Quinn CC,
Wasiak S, Guipponi M, Antonarakis SE, Kay BK,
Stossel TP, Lamarche-Vane N, McPherson PS. En-
docytic protein intersectin-l regulates actin assembly
via Cdc42 and N-WASP. Nat Cell Biol. 2001;
3(10):927–32.
4. Herrero-Garcia E, O’Bryan JP. Intersectin scaffold
proteins and their role in cell signaling and endocy-
tosis. Biochim Biophys Acta Mol Cell Res.
2017;1864(1):23–30.
5. Wang JB, Wu WJ, Cerione RA. Cdc42 and Ras co-
operate to mediate cellular transformation by inter-
sectin-L. J Biol Chem. 2005;280(24):22883–91.
6. Gryaznova T, Kropyvko S, Burdyniuk M, Gubar O,
Kryklyva V, Tsyba L, Rynditch A. Intersectin adaptor
proteins are associated with actin-regulating protein
WIP in invadopodia. Cell Signal. 2015;27(7):1499–
508.
7. Russo A, O’Bryan JP. Intersectin 1 is required for
neuroblastoma tumorigenesis. Oncogene. 2012;
31(46):4828–34.
8. Harris J, Herrero-Garcia E, Russo A, Kajdacsy-
Balla A, O’Bryan JP, Chiu B. Silencing Intersectin
1 Slows Orthotopic Neuroblastoma Growth in Mice.
J Pediatr Hematol Oncol. 2017;39(8):e413-e418.
9. Gu F, Zhang H, Qin F, Liu X, Li W, Fu L, Ying G,
Li B, Zhang M, Ma Y. Intersectin1-S, a multidomain
adapter protein, is essential for malignant glioma
proliferation. Glia. 2015;63(9):1595–605.
10. Ma Y, Wang B, Li W, Ying G, Fu L, Niu R, Gu F. Re-
duction of intersectin1-s induced apoptosis of human
glioblastoma cells. Brain Res. 2010; 1351: 222–8.
11. Ma Y, Wang B, Li W, Liu X, Wang J, Ding T, Zhang J,
Ying G, Fu L, Gu F. Intersectin1-s is involved in
migration and invasion of human glioma cells. J
Neurosci Res. 2011;89(7):1079–90.
12. Jeganathan N, Predescu D, Zhang J, Sha F, Bar-
dita C, Patel M, Wood S, Borgia JA, Balk RA, Pre-
descu S. Rac1-mediated cytoskeleton rearrange-
ments induced by intersectin-1s deficiency promotes
lung cancer cell proliferation, migration and metas-
tasis. Mol Cancer. 2016;15(1):59.
13. Specht K, Harbeck N, Smida J, Annecke K, Reich U,
Naehrig J, Langer R, Mages J, Busch R, Kruse E,
Klein-Hitpass L, Schmitt M, Kiechle M, Hoefler H.
Expression profiling identifies genes that predict
recurrence of breast cancer after adjuvant CMF-
based chemotherapy. Breast Cancer Res Treat.
2009;118(1):45–56.
14. Beausoleil SA, Jedrychowski M, Schwartz D, Elias
JE, Villén J, Li J, Cohn MA, Cantley LC, Gygi SP.
Large-scale characterization of HeLa cell nuclear
phosphoproteins. Proc Natl Acad Sci U S A. 2004;
101(33):12130–5.
15. Alvisi G, Paolini L, Contarini A, Zambarda C,
Di Antonio V, Colosini A, Mercandelli N, Timmo-
neri M, Palù G, Caimi L, Ricotta D, Radeghieri A.
Intersectin goes nuclear: secret life of an endocytic
protein. Biochem J. 2018;475(8):1455–1472.
16. Wong KA, Wilson J, Russo A, Wang L, Okur MN,
Wang X, Martin NP, Scappini E, Carnegie GK,
O’Bryan JP. Intersectin (ITSN) family of scaffolds
function as molecular hubs in protein interaction
networks. PLoS One. 2012;7(4):e36023.
17. Thalappilly S, Suliman M, Gayet O, Soubeyran P,
Hermant A, Lecine P, Iovanna JL, Dusetti NJ. Iden-
89
Scaffold proteins ITSN1 and ITSN2 interact with nuclear RNA-binding proteins
tification of multi-SH3 domain-containing protein
interactome in pancreatic cancer: a yeast two-hybrid
approach. Proteomics. 2008;8(15):3071–81.
18. Asbach B, Ludwig C, Saksela K, Wagner R. Com-
prehensive analysis of interactions between the
Src-associated protein in mitosis of 68 kDa and the
human Src-homology 3 proteome. PLoS One.
2012;7(6):e38540.
19. Nikolaienko O, Skrypkina I, Tsyba L, Fedyshyn Y,
Morderer D, Buchman V, de la Luna S, Drobot L,
Rynditch A. Intersectin 1 forms a complex with
adaptor protein Ruk/CIN85 in vivo independently
of epidermal growth factor stimulation. Cell Signal.
2009;21(5):753–9.
20. Novokhatska O, Dergai M, Tsyba L, Skrypkina I,
Filonenko V, Moreau J, Rynditch A. Adaptor proteins
intersectin 1 and 2 bind similar proline-rich ligands
but are differentially recognized by SH2 domain-
containing proteins. PLoS One. 2013;8(7):e70546.
21. Venables JP, Dalgliesh C, Paronetto MP, Skitt L,
Thornton JK, Saunders PT, Sette C, Jones KT, El-
liott DJ. SIAH1 targets the alternative splicing fac-
tor T-STAR for degradation by the proteasome. Hum
Mol Genet. 2004;13(14):1525–34.
22. Llorian M, Beullens M, Lesage B, Nicolaescu E,
Beke L, Landuyt W, Ortiz JM, Bollen M. Nucleocy-
toplasmic shuttling of the splicing factor SIPP1.
J Biol Chem. 2005;280(46):38862–9.
23. Shao R, Scully SJ Jr, Yan W, Bentley B, Mueller J,
Brown C, Bigelow C, Schwartz LM. The novel lupus
antigen related protein acheron enhances the devel-
opment of human breast cancer. Int J Cancer.
2012;130(3):544–54.
24. Giudice G, Sánchez-Cabo F, Torroja C, Lara-Pez-
zi E. ATtRACT-a database of RNA-binding proteins
and associated motifs. Database (Oxford).
2016;2016. pii: baw035.
25. Cook KB, Kazan H, Zuberi K, Morris Q, Hughes TR.
RBPDB: a database of RNA-binding specificities.
Nucleic Acids Res. 2011;39(Database issue):D301–8.
26. Huntley RP, Sawford T, Mutowo-Meullenet P, Shy-
pitsyna A, Bonilla C, Martin MJ, O’Donovan C. The
GOA database: gene Ontology annotation updates
for 2015. Nucleic Acids Res. 2015;43(Database
issue):D1057–63.
27. Teyra J, Huang H, Jain S, Guan X, Dong A, Liu Y,
Tempel W, Min J, Tong Y, Kim PM, Bader GD,
Sidhu SS. Comprehensive Analysis of the Human
SH3 Domain Family Reveals a Wide Variety of
Non-canonical Specificities. Structure. 2017;25(10):
1598-1610.e3.
28. Frisone P, Pradella D, Di Matteo A, Belloni E,
Ghigna C, Paronetto MP. SAM68: Signal transduc-
tion and RNA metabolism in human cancer. BioMed
Res Int. 2015; 2015: 528954.
29. Hong W, Resnick RJ, Rakowski C, Shalloway D,
Taylor SJ, Blobel GA. Physical and functional in-
teraction between the transcriptional cofactor CBP
and the KH domain protein Sam68. Mol Cancer
Res. 2002;1(1):48–55.
30. Taylor SJ, Resnick RJ, Shalloway D. Sam68 exerts
separable effects on cell cycle progression and apop-
tosis. BMC Cell Biol. 2004;5:5.
31. Yang JP, Reddy TR, Truong KT, Suhasini M, Wong-
Staal F. Functional interaction of Sam68 and het-
erogeneous nuclear ribonucleoprotein K. Oncogene.
2002;21(47):7187–94.
32. Rajan P, Gaughan L, Dalgliesh C, El-Sherif A,
Robson CN, Leung HY, Elliott DJ. The RNA-bind-
ing and adaptor protein Sam68 modulates signal-
dependent splicing and transcriptional activity of
the androgen receptor. J Pathol. 2008;215(1):67–77.
Скафолдні білки ITSN1 та ITSN2 взаємодіють
із ядерними РНК-зв’язуючими білками
С. В. Паньківський, Н. В. Сенченко, П. Б. Бусько,
А. В. Риндич
Мета. Виявити нових партнерів ITSN1 і ITSN2 з-поміж
РНК-зв’язуючих білків (RBP), що беруть участь в
регуляції процесингу мРНК. Методи. Взаємодії були
проаналізовано з використанням GST pull-down assay
та імунопреципітації, тоді як біоінформатичний аналіз
було використано для ідентифікації інших RBP, які
могли б взаємодіяти із білками ITSN1 та ITSN2.
Результати. Було показано, що SH3 домени білків
ITSN1 та ITSN2 взаємодіють з ядерними RBP SAM68,
WBP11 і LARP6. Крім того, було виявлено, що ITSN1
та ITSN2 копреципітувались із SAM68 і LARP6 із лі-
затів клітин лінії 293. Біоінформатичний аналіз пока-
90
S. V. Pankivskyi, N. V. Senchenko, P. B. Busko et al.
зав, що існує більше 500 ядерних RBP, які містять
кілька пролінових мотивів, що можуть взаємодіяти із
SH3 доменами білків ITSN1/2. Висновки. SH3 домени
білків ITSN1 і ITSN2 взаємодіють із ядерними RBP
SAM68, LARP6 і WBP11 in vitro, утворюють комплек-
си із SAM68 і LARP6 в клітинах лінії 293 і потенційно
можуть взаємодіяти з іншими ядерними RBP, що міс-
тять мотиви, які зв’язуються із SH3 доменами.
К л юч ов і с л ов а: ITSN1, ITSN2, РНК-зв’язуючі
білки.
Скаффолдные белки ITSN1 и ITSN2
взаимодействуют с ядерными РНК-
связывающими белками
С. В. Паньковский, Н. В. Сенченко, П. Б. Бусько,
А. В. Рындич
Цель. Найти новых партнеров ITSN1 и ITSN2 среди
РНК-связывающих белков (RBP), участвующих в ре-
гуляции процессинга мРНК. Методы. Взаимодействия
были проанализированы с использованием GST pull-
down assay и иммунопреципитации, тогда как биоин-
форматический анализ был проведен для идентифи-
кации других RBP, которые могли бы взаимодейство-
вать с белками ITSN1 и ITSN2. Результаты. Было
показано, что SH3 домены белков ITSN1 и ITSN2
взаимодействуют с ядерными RBP SAM68, WBP11 и
LARP6. Кроме того, было обнаружено, что ITSN1 и
ITSN2 копреципитировались с SAM68 и LARP6 из
лизатов клеток линии 293. Биоинформатический ана-
лиз показал, что существует более 500 ядерных RBP,
содержащие несколько пролиновых мотивов, которые
могут взаимодействовать с SH3 доменами белков
ITSN1/2 в ядре клетки. Выводы. SH3 домены белков
ITSN1 и ITSN2 взаимодействуют с ядерными RBP
SAM68, LARP6 и WBP11 in vitro, образуют комплексы
с SAM68 и LARP6 в клетках линии 293 и потенциаль-
но могут взаимодействовать с другими ядерными RBP,
содержащими мотивы, которые связываются с SH3
доменами.
К л юч е в ы е с л ов а: ITSN1, ITSN2, РНК-свя зы ваю-
мщие белки
Received 15.12.2018
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