Focal adhesion kinase (FAK1) regulates SHB phosphorylation and its binding with a range of signaling proteins
Aim. To investigate an effect of the Focal adhesion kinase 1 (FAK1) expression on the level of tyrosine phosphorylation of an adaptor protein SHB and to find functional consequences of this posttranslational modification. Methods. Recombinant DNA construction, protein expression and purification, hu...
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| Cite this: | Focal adhesion kinase (FAK1) regulates SHB phosphorylation and its binding with a range of signaling proteins / O.V. Dergai, A.M. Yaruchik, A.V. Rynditch // Вiopolymers and Cell. — 2016. — Т. 32, № 1. — С. 34-40. — Бібліогр.: 22 назв. — англ. |
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Dergai, O.V. Yaruchik, A.M. Rynditch, A.V. 2019-06-12T18:05:27Z 2019-06-12T18:05:27Z 2016 Focal adhesion kinase (FAK1) regulates SHB phosphorylation and its binding with a range of signaling proteins / O.V. Dergai, A.M. Yaruchik, A.V. Rynditch // Вiopolymers and Cell. — 2016. — Т. 32, № 1. — С. 34-40. — Бібліогр.: 22 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.00090A https://nasplib.isofts.kiev.ua/handle/123456789/152765 577.22 Aim. To investigate an effect of the Focal adhesion kinase 1 (FAK1) expression on the level of tyrosine phosphorylation of an adaptor protein SHB and to find functional consequences of this posttranslational modification. Methods. Recombinant DNA construction, protein expression and purification, human cell transfection, western blot. Results. The expression of FAK1 induces the massive tyrosine phosphorylation of SHB adaptor and enhances its interaction in vitro with SH2 domains of a range of the signaling proteins such as PI3K, ABL, CRK and PLCG1. Additionally we have found that Epstein-Barr virus protein LMP2A can partially mimic the FAK1-mediated effect strongly elevating the efficiency and SHB interaction with the mentioned above proteins. While the expression of individual proteins elevated SHB phosphorylation level, the co-expression of LMP2A and FAK1 did not display a synergetic effect. Conclusions. FAK1 as well as LMP2A induce the SHB tyrosine phosphorylation and enhance its interaction with a set of the signaling proteins. Мета. Дослідити ефект надексперсії кінази фокальної адгезії (FAK1) на рівнь фосфорилювання залишків тирозину адапторного білка SHB та знайти функціональне значення цієї посттрансляційної модифікації. Методи. Конструювання рекомбінантних ДНК, експресія та очистка рекомбінантних білків, трасфекція клітинних ліній, вестерн блот. Результати. Надексперсія FAK1 в клітинах лінії 293 викликає масивне фосфорилювання залишків тирозину адапторного білка SHB та значно підсилює його взаємодію in vitro з SH2-доменами низки сигнальних білків, таких як PI3K, ABL, CRK та PLCg1. Крім того, білок LMP2A вірусу Епштейна-Барр може посилювати in vitro зв’язування SHB з вищезазначеними білками, так само, як і FAK1. Тоді як надекспресія окремих білків FAK1 та LMP2A підвищувала рівні фосфорилювання SHB, їх ко-експерсія не мала синергічного ефекту. Висновки. Експресія FAK1 та LMP2A індукує фосфорилювання залишків тирозину адапторного білка SHB та підсилює його взаємодію з SH2-доменами низки сигнальних білків. Цель. Исследовать эффект суперэкспрессии киназы фокальной адгезии (FAK1) на уровень фосфорилирования остатков тирозина адаптерного белка SHB и найти функциональное значение этой посттрансляционной модификации. Методы. Конструирование рекомбинантных ДНК, экспрессия и очистка рекомбинантных белков, трансфекция клеточных линий, вестерн блот. Результаты. Суперэксперссия FAK1 в клетках линии 293 приводит к многократному повышению уровней фосфорилирования остатков тирозина адаптерного белка SHB, что в свою очередь приводит к усилению взаимодейсвия с SH2-доменами ряда сигнальных белков, таких как PI3K, ABL, CRK та PLCg1. Кроме того, белок LMP2A вируса Эпштейна-Барр может усиливать in vitro взаимодействие SHB с указанными белками, аналогично FAK1. Тогда как суперэкспрессия отдельных белков FAK1 та LMP2A приводила к интенсификации фосфорилирования SHB, их ко-экспрессия не обладала синергическим эффектом. Выводы. Экспрессия FAK1 и LMP2A индуцирует фософрилирование остатков тирозина адаптерного белка SHB и усиливает его взаимодействие с SH2-доменами ряда сигнальных белков. This work was supported by joint project between NAS of Ukraine and CNRS “From Molecular to Cellular Events in Human Pathologies” (N0113U002831). en Інститут молекулярної біології і генетики НАН України Вiopolymers and Cell Genomics, Transcriptomics and Proteomics Focal adhesion kinase (FAK1) regulates SHB phosphorylation and its binding with a range of signaling proteins Кіназа фокальної адгезії (FAK1) регулює фосфорилювання адаптерного білка SHB та його взаємодію з низкою сигнальних білків Киназа фокальной адгезии (FAK1) регулирует фосфорилирование адаптерного белка SHB и его взаимодействие с рядом сигнальных белков Article published earlier |
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Focal adhesion kinase (FAK1) regulates SHB phosphorylation and its binding with a range of signaling proteins |
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Focal adhesion kinase (FAK1) regulates SHB phosphorylation and its binding with a range of signaling proteins Dergai, O.V. Yaruchik, A.M. Rynditch, A.V. Genomics, Transcriptomics and Proteomics |
| title_short |
Focal adhesion kinase (FAK1) regulates SHB phosphorylation and its binding with a range of signaling proteins |
| title_full |
Focal adhesion kinase (FAK1) regulates SHB phosphorylation and its binding with a range of signaling proteins |
| title_fullStr |
Focal adhesion kinase (FAK1) regulates SHB phosphorylation and its binding with a range of signaling proteins |
| title_full_unstemmed |
Focal adhesion kinase (FAK1) regulates SHB phosphorylation and its binding with a range of signaling proteins |
| title_sort |
focal adhesion kinase (fak1) regulates shb phosphorylation and its binding with a range of signaling proteins |
| author |
Dergai, O.V. Yaruchik, A.M. Rynditch, A.V. |
| author_facet |
Dergai, O.V. Yaruchik, A.M. Rynditch, A.V. |
| topic |
Genomics, Transcriptomics and Proteomics |
| topic_facet |
Genomics, Transcriptomics and Proteomics |
| publishDate |
2016 |
| language |
English |
| container_title |
Вiopolymers and Cell |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| format |
Article |
| title_alt |
Кіназа фокальної адгезії (FAK1) регулює фосфорилювання адаптерного білка SHB та його взаємодію з низкою сигнальних білків Киназа фокальной адгезии (FAK1) регулирует фосфорилирование адаптерного белка SHB и его взаимодействие с рядом сигнальных белков |
| description |
Aim. To investigate an effect of the Focal adhesion kinase 1 (FAK1) expression on the level of tyrosine phosphorylation of an adaptor protein SHB and to find functional consequences of this posttranslational modification. Methods. Recombinant DNA construction, protein expression and purification, human cell transfection, western blot. Results. The expression of FAK1 induces the massive tyrosine phosphorylation of SHB adaptor and enhances its interaction in vitro with SH2 domains of a range of the signaling proteins such as PI3K, ABL, CRK and PLCG1. Additionally we have found that Epstein-Barr virus protein LMP2A can partially mimic the FAK1-mediated effect strongly elevating the efficiency and SHB interaction with the mentioned above proteins. While the expression of individual proteins elevated SHB phosphorylation level, the co-expression of LMP2A and FAK1 did not display a synergetic effect. Conclusions. FAK1 as well as LMP2A induce the SHB tyrosine phosphorylation and enhance its interaction with a set of the signaling proteins.
Мета. Дослідити ефект надексперсії кінази фокальної адгезії (FAK1) на рівнь фосфорилювання залишків тирозину адапторного білка SHB та знайти функціональне значення цієї посттрансляційної модифікації. Методи. Конструювання рекомбінантних ДНК, експресія та очистка рекомбінантних білків, трасфекція клітинних ліній, вестерн блот. Результати. Надексперсія FAK1 в клітинах лінії 293 викликає масивне фосфорилювання залишків тирозину адапторного білка SHB та значно підсилює його взаємодію in vitro з SH2-доменами низки сигнальних білків, таких як PI3K, ABL, CRK та PLCg1. Крім того, білок LMP2A вірусу Епштейна-Барр може посилювати in vitro зв’язування SHB з вищезазначеними білками, так само, як і FAK1. Тоді як надекспресія окремих білків FAK1 та LMP2A підвищувала рівні фосфорилювання SHB, їх ко-експерсія не мала синергічного ефекту. Висновки. Експресія FAK1 та LMP2A індукує фосфорилювання залишків тирозину адапторного білка SHB та підсилює його взаємодію з SH2-доменами низки сигнальних білків.
Цель. Исследовать эффект суперэкспрессии киназы фокальной адгезии (FAK1) на уровень фосфорилирования остатков тирозина адаптерного белка SHB и найти функциональное значение этой посттрансляционной модификации. Методы. Конструирование рекомбинантных ДНК, экспрессия и очистка рекомбинантных белков, трансфекция клеточных линий, вестерн блот. Результаты. Суперэксперссия FAK1 в клетках линии 293 приводит к многократному повышению уровней фосфорилирования остатков тирозина адаптерного белка SHB, что в свою очередь приводит к усилению взаимодейсвия с SH2-доменами ряда сигнальных белков, таких как PI3K, ABL, CRK та PLCg1. Кроме того, белок LMP2A вируса Эпштейна-Барр может усиливать in vitro взаимодействие SHB с указанными белками, аналогично FAK1. Тогда как суперэкспрессия отдельных белков FAK1 та LMP2A приводила к интенсификации фосфорилирования SHB, их ко-экспрессия не обладала синергическим эффектом. Выводы. Экспрессия FAK1 и LMP2A индуцирует фософрилирование остатков тирозина адаптерного белка SHB и усиливает его взаимодействие с SH2-доменами ряда сигнальных белков.
|
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0233-7657 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/152765 |
| citation_txt |
Focal adhesion kinase (FAK1) regulates SHB phosphorylation and its binding with a range of signaling proteins / O.V. Dergai, A.M. Yaruchik, A.V. Rynditch // Вiopolymers and Cell. — 2016. — Т. 32, № 1. — С. 34-40. — Бібліогр.: 22 назв. — англ. |
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34
O. V. Dergai , A. M. Yaruchik, A. V. Rynditch
© 2016 O. V. Dergai et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers 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
Focal adhesion kinase (FAK1) regulates SHB phosphorylation
and its binding with a range of signaling proteins.
O. V. Dergai , A. M. Yaruchik, A. V. Rynditch
Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Zabolotnogo Str., Kyiv, Ukraine, 03680
o.dergai@gmail.com
Aim. To investigate an effect of the Focal adhesion kinase 1 (FAK1) expression on the level of tyrosine phos-
phorylation of an adaptor protein SHB and to find functional consequences of this posttranslational modifica-
tion. Methods. Recombinant DNA construction, protein expression and purification, human cell transfection,
western blot. Results. Expression of FAK1 induces massive tyrosine phosphorylation of SHB adaptor and
enhances its interaction in vitro with SH2 domains of a range of the signaling proteins such as PI3K, ABL,
CRK and PLCG1. Additionally we have found that Epstein-Barr virus protein LMP2A can partially mimic the
FAK1-mediated effect strongly elevating the efficiency and SHB interaction with the above-mentioned pro-
teins. While the expression of individual proteins elevated SHB phosphorylation level, the co-expression of
LMP2A and FAK1 did not display a synergetic effect. Conclusions. FAK1 as well as LMP2A induce SHB
tyrosine phosphorylation and enhance its interaction with a set of the signaling proteins.
K e y w o r d s: FAK1, SHB, LMP2A, phosphorylation.
Introduction
Focal adhesion kinase 1 (FAK1) is an ubiquitously
expressed non-receptor tyrosine kinase which is lo-
calized to focal adhesion where a cell is attached to
the extracellular matrix. FAK1 operates downstream
of the cell surface receptors, such as integrins and
receptor tyrosine kinases i.e. EGFR, PDGFR,
G-protein coupled receptors (GPCR), EPHA2, ne-
trin receptors and LDL receptors [1,2]. Upon incom-
ing activation signal from the above mentioned re-
ceptors FAK1 associates with kinase Src, gets phos-
phorylated by latter and autophosphorylated to gain
the maximum of intrinsic kinase activity. Activated
FAK1 phosphorylates ACTN1, ARHGEF7, GRB7,
RET, WASL and promotes phosphorylation of
BCAR1, GIT2 and SHC1, many of these proteins
are crucial regulators of the cytoskeleton reorganiza-
tion, cell spreading, migration, cell cycle progres-
sion, and prevention of apoptosis [3-5]. FAK1 is
comprised of a centrally located kinase domain
flanked by the large N- and C-terminal non-catalytic
domains (Fig. 1).
The N-terminal region of FAK1 contains FERM
homology domain, whereas the C-terminal region
of FAK1 contains two proline-rich motifs and a fo-
cal adhesion targeting (FAT) domain that mediates
its discrete localization to focal adhesions upon
FAK1 activation [6]. In addition to the function as
a kinase, FAK1 also serves as a scaffolding protein.
FAK1 contains th e binding sites for many signal-
ing proteins (Fig.1). FAK1 was shown to interact
with EGFR, PDGFR, Src, Shc, phosphotidylinosi-
tol 3-kinase (PI3K), and phospholipase Cg1 (PLC
g1), signaling adaptors GRB2 and GRB7, paxillin,
tallin [1, 4, 7].
FAK1 interacts with and gets activated by signal-
ing scaffold SHB[8]. FAK1 and SHB in a concert
Genomics, Transcriptomics
and Proteomics
ISSN 1993-6842 (on-line); ISSN 0233-7657 (print)
Biopolymers and Cell. 2016. Vol. 32. N 1. P 34–40
doi: http://dx.doi.org/10.7124/bc.00090A
35
Focal adhesion kinase (FAK1) regulates SHB phosphorylation and its binding with a range of signaling proteins.
regulate the cell spreading and migration [8]. SHB
is an ubiquitously expressed protein of about 56 kDa
comprising N-terminal proline-rich domain (PRD)
which interacts with SH3 domain of PI3K, phos-
photyrosin-binding domain (PTB) and C-terminally
located SH2 domain that binds the platelet derived
growth factor receptor (PDGFR) and IL-2 receptor
and T-cell receptor [8]. Recently SHB was shown to
modulate the FAK1 signaling activity in ABL-BCR
lymphoproliferative disorder [9]. SHB operates
downstream of plasma membrane receptor kinases
and participates in AKT/PI3K signaling pathway
[10, 11].
Here we show that FAK1 can regulate interaction
of SHB scaffold with a range of SH2 domain con-
taining signaling molecules such as CRK, PI3K and
с-ABL through the tyrosine phosphorylation of
SHB. It has been found that expression of viral pro-
tein LMP2A can partially mimic the effect of FAK1
overexpression enhancing binding of SHB to the
SH2-domains of CRK, PI3K, GRB2, PLC g1 and
с-ABL. An intriguing observation appeared that
FAK1 can partially block LMP2A-induced phos-
phorylation of SHB.
Material and Methods
Antibodies and plasmids
Monoclonal antibody to the FLAG epitope (clone
M2) was from Sigma; monoclonal α-omni (D-8) and
α-pTyr (pY99) antibodies were from Santa Cruz
Biotechnology; a monoclonal α-HA (MMS-101P)
antibody was from Covance.
Flag-LMP2A and omni-SHB-HA encoding plas-
mids were described previously [12]. The plasmids
encoding GST-fused SH2 domains of PLCG1, PI3K,
ABL, GRB2 and CRK were described before [13].
The plasmid encoding HA-FAK1 was kindly
provided by Dr. V. M. Golubovskaya [14].
Cell culture and transfection
293 cells were maintained in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10%
fetal bovine serum, 50 U/ml penicillin and 100 μg/ml
streptomycin. The cells were transiently transfected
using JetPEI (Polyplus Transfection) according to the
manufacturer’s instructions and processed 24h after
transfection.
Immunoprecipitation
Cell lysis and immunoprecipitation were performed
as described previously [15].
Proteins expression, purification and GST-pull
down assay
GST-fused SH2-domains of PLCG1, PI3K, ABL,
GRB2 and CRK were expressed in E.coli BL21 DE3
and affinity purified using glutathione-sepharose 4B
(GE Healthcare) according to the manufacturer’s in-
structions and further used for the
GST-pull down assays as described before [13].
Results and Discussion
Taking into consideration the fact that SHB regu-
lates the FAK1 [8] activity through its tyrosine phos-
phorylation we asked whether there is a reciprocal
loop of SHB regulation by FAK1. At first we per-
formed in silico prediction of the FAK1 capability to
phosphorylate SHB undertaken by means of GPS3.0
Fig. 1. A schematic representation of FAK1 and SHB proteins.
The most important phosphotyrosine sites of FAK1 are shown
above with indication of proteins which recognize them. FAK1-C
term lacking N-terminal portion with kinase domain is demon-
strated below the full-length one. SHB comprises the proline-rich
domain (PRD), phosphotyrosine-binding domain (PTB) and SH2
domain, the two latter domains of SHB are hosphotyrosine-recog-
nition modules. The tyrosine residues predicted by GPS3.0 to be
phosphorylated by FAK1 are show above SHB scheme.
36
O. V. Dergai , A. M. Yaruchik, A. V. Rynditch
software (Group-based Prediction System) [16].
According to this analysis 8 tyrosine residues were
predicted to be phosphorylated by FAK1 at high
stringency, namely Y96, Y162, Y178 Y246, Y272,
Y297, Y336, Y444 (Fig.2A). So, we took advantage
of the experimental data from PhosphoSitePlus data-
base (http://www.phosphosite.org/) and found that 5
tyrosine residues (namely Y96, Y246, Y272, Y297,
Y336) out of 8 predicted ones were found to be
phosphorylated in human and mouse samples.
Moreover, the indicated five tyrosine residues are
conserved in Vertebrates in contrast to tyrosines at
the positions Y162, Y178, Y444 (Fig. 2A) To test
experimentally the hypothesis that FAK1 may phos-
phorylate SHB we co-expressed the full-length HA-
tagged FAK1 or its truncated omni-tagged FAK1-
Cterm lacking kinase domain (see Fig. 1) together
with omni-tagged SHB in 293 cells. The basal level
of SHB phosphorylation in 293 cells was below of
the detection limit. Despite the equal amount of pre-
cipitated SHB (Fig.2B, second panel), a level of its
phosphorylation is greatly increased in the cells
transfected with full-length FAK1 but not in the cells
with expression of FAK1-Cterm lacking a kinase do-
main (Fig.2B, first panel).
Next, we asked whether the FAK1-induced phos-
phorylation of SHB has a functional consequence.
It’s widely accepted that phosphorylation in general
serves to switch proteins conformations or to create
interaction interfaces which can be recognized by
the specialized phospho-binding protein domains.
One of the well-known class of phosphotyrosine rec-
ognition modules is SH2-domain (Src homology2),
which specifically binds the phosphotyrosine resi-
dues [17]. Some of the established SHB binding sub-
strates such as GRB2, ABL, PLCg1 contain SH2-
domain [18]. We studied whether the FAK1-induced
phosphorylation of SHB can modulate its interaction
with SH2 domains of the binding partners. To do so,
we used a set of GST-fused SH2-domains as a bait in
pull-down assay to precipitate SHB from the cell ex-
tracts transfected with HA-FAK1. As Fig. 3A shows
the overexpression of FAK1 enhanced the interac-
tion of SHB with SH2 domains of CRK, PI3K and
ABL while no interaction with SH2 domain of GRB2
was detected. This fact may evidence that the FAK1-
dependent SHB phosphorylation specifically trig-
gers the association of different SH2 domains con-
taining signaling proteins with SHB. Additionally,
Fig.2. Overexpression of FAK1 kinase induces tyrosine phos-
phorylation of SHB protein. A. Table representing a list of phos-
photyrosine sites of SHB predicted with highest stringency by
GPS3.0 to be phosphorylated by FAK1 kinase. Category “mam-
mals” contains Homo sapiens, Mus musculus, Rattus norvegicus,
Canis lupus. B. 293 cells were co-transfected with omni-SHB-
HA, HA-FAK1 or omni-FAK1-Cterm. 24h after transfection cells
were harvested, lysed and omni-SHB-HA immunoprecipitated
with monoclonal a-omni antibodies from the respective cell ex-
tracts. Precipitated material was analyzed by western blot: tyro-
sine phosphorylated species were detected by monoclonal a-phos-
photyrosine antibody, total omni-SHB-HA was visualized by a-
omni antibody. Two lower panel show immunodetection of omni-
SHB-HA, full-length and omni-FAK1-Cterm in cell extracts.
http://www.phosphosite.org/
37
Focal adhesion kinase (FAK1) regulates SHB phosphorylation and its binding with a range of signaling proteins.
the interaction of SHB with SH2 domain of PLCg1
was significantly increased upon the FAK1 overex-
pression but the binding of PLCg1 SH3 domain to
SHB was decreased suggesting that the tyrosine
phosphorylation of SHB can regulate binding prop-
erties of its proline-rich sequences (Fig.3A,
lanes 9, 10).
The interaction of SHB with PI3K that is mediated
by SH2 domains of latter is of great interest as SHB
is required for the activation of the PI3K/AKT path-
way [11] and the regulation of cell survival [18]. ABL
is a proto-oncogene that encodes a protein tyrosine
kinase involved in a variety of cellular processes, in-
cluding cell division, adhesion, differentiation, and
response to stress. In the signaling pathways ABL is
located upstream of PI3K/AKT [19]. A functional
significance of phosphorylation-dependent interac-
tion with ABL kinase is enigmatic but recently SHB
has been shown to enhance FAK1 activity in the
BCR-ABL aberrant cells that caused accelerated dis-
ease progression emphasizing the importance of
ABL/SHB interplay [9]. It was demonstrated that the
interactions between SHB and ABL regulates the
ABL kinase activity. The data suggest that ABL binds
to tyrosine phosphorylated SHB via a concerted ef-
fort involving both ABL SH3- and SH2-domains and
these interactions modulate cell death in response to
the genotoxic agents [20]. In general the place of
SHB in signaling network is depicted at the Fig. 3D.
The activated receptor tyrosine kinases transduce a
Fig.3. FAK1 and LMP2A regulate association of SHB with SH2-domains. A. Bacterially expressed GST-fused SH2 (A, B and C) or
SH3-PLCg1 (A, lanes 9 and 10) of indicated proteins were immobilized on glutathione-beads and incubated with 293 lysates co-
transfected with omni-SHB-HA and HA-FAK1 (A and B) or Flag-LMP2A (C) or vector. D. Schematic representation of SHB role in
RTKs- and integrin-driven signaling. SHB may directly interact with depicted proteins, with red dashed arrow is shown newly dis-
covered phosphorylation event, FAK1-induced interactions are shown with bold back arrows. Indirect effects are shown with dashed
arrows. WCE stands for the whole cell extract.
38
O. V. Dergai , A. M. Yaruchik, A. V. Rynditch
signal to ABL, SRC, phospholipase C and PI3K that
results in the cell proliferation or survival depending
on the signaling context. SHB is required for the sig-
naling downstream of RTKs and its role depends on
the interaction with the protein-partners such as ABL,
SRC, GRB2, PI3K and PLCg1. FAK1 is activated by
integrins (Fig. 3D) and indirectly by RTKs signaling
that leads to the cytoskeleton changes and cell migra-
tion [18]. We suggest that the FAK1-induced SHB
phosphorylation may be a part of signaling cross-talk
between RTKs- and integrin-controlled pathways.
Additionally, the augmentation of FAK activity in
human tumors, in which it is often overexpressed, oc-
curs through its elevated expression [1], so described
here the FAK1-induced effects may be a part of path-
ological phenotype, e.g. the resistance to apoptosis
and invasiveness.
Previously we have shown that the expression of
viral protein LMP2A induces the tyrosine phosphor-
ylation of SHB [12]. To address whether the LMP2A-
induced phosphorylation of SHB may have an im-
pact on the SHB binding to the SH2-domain con-
taining proteins we applied a before mentioned ap-
proach. As Fig. 3B shows the expression LMP2A
strongly enhanced the binding of SHB to a range of
the SH2 domains of such proteins as CRK, GRB2,
PI3K, PLCg1 and ABL. One can suggest that FAK1
and LMP2A caused the phosphorylation of non-
overlapping sets of tyrosine residues as far as the
LMP2A expression enhanced the SHB association
with GRB2 but for FAK1 (Fig3 A and B). The de-
scribed effects of LMP2A and FAK1 expression on
the SHB in vitro interaction with before mentioned
SH2 and SH3 domains could be explained in two
ways: 1) both FAK1 and LMP2A cause the SHB
phosphorylation at certain sites that are recognized
by respective SH2 domains or 2) FAK1 and LMP2A
serve as a scaffold to bring together SHB and before
mentioned domains. The later statement is supported
by our unpublished observation that LMP2A and
FAK1 can interact with SH2-domains of PI3K, ABL
and GRB2. From the other hand, the fact that FAK1
negatively regulated SHB binding to the SH3-
domain of PLCg1 challenges the idea that FAK1
serves simply as an adaptor. Unfortunately, so far it
is difficult to uncouple the phosphorylation-induced
and potential scaffolding effects of FAK1 on the
SHB interaction with its partners.
As far as the expression of both LMP2A and
FAK1 strongly upregulated the SHB tyrosine phos-
phorylation we expected to detect a synergetic effect
of co-expression of both proteins with SHB. In addi-
tion while the expression of individual proteins ele-
vated SHB phosphorylation level, the co-expression
of LMP2A and FAK1 had remarkable negative co-
operation effect (Fig.4). The reason for this kind of
conclusion is the fact that the most robust effect on
the SHB phosphorylation was caused by LMP2A
while the co-expression of FAK1 decreased this ef-
fect to the FAK1-induced level. One could suggest
that FAK1 partially inhibits the activity of Syk and
Lyn tyrosine kinases which carry out the LMP2A-
induced SHB phosphorylation [12].
SHB is required for the tonic and serum-indepen-
dent AKT activation caused by Epstein-Barr virus
Fig.4. Expression of FAK1 and LMP2A displays negative coop-
eration effect on the tyrosine phosphorylation levels of SHB.
293 cells were co-transfected with omni-SHB-HA, HA-
FAK1+control vector or HA-FAK1 and Flag-LMP2A. Levels of
SHB tyrosine phosphorylation were estimated as described at
figure 2B.
39
Focal adhesion kinase (FAK1) regulates SHB phosphorylation and its binding with a range of signaling proteins.
protein LMP2A [10] and in line with this observa-
tion it has been shown that LMP2A induces the SHB
tyrosine phosphorylation [12]. We suggest that de-
scribed here LMP2A-induced interaction of SHB
with SH2 domains of PI3K, ABL, PLCg1 may be
responsible for the downstream signaling towards
AKT and RAS activation. Moreover, it has been
shown that LMP2A induces the epithelial cell migra-
tion [21] in FAK1-dependent manner [22]. We as-
sume that signaling downstream of FAK1 and
LMP2A may converge on SHB. The fact that LMP2A
can induce the SHB phosphorylation that triggers
SHB binding to GRB2, PLCG1, PI3K and other pro-
teins in this way mimicking the FAK1 activity may
represent an important aspect of the LMP2A-induced
pathogenicity providing a link between the LMP2A-
driven SHB activation and cell migration.
Funding
This work was supported by joint project between NAS
of Ukraine and CNRS “From Molecular to Cellular
Events in Human Pathologies” (N0113U002831).
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Кіназа фокальної адгезії (FAK1) регулює
фосфорилювання адаптерного білка SHB та його
взаємодію з низкою сигнальних білків.
О. В. Дергай, А. М. Яручик, А. В. Риндич
Мета. Дослідити ефект надексперсії кінази фокальної адгезії
(FAK1) на рівнь фосфорилювання залишків тирозину адаптор-
ного білка SHB та знайти функціональне значення цієї пост-
трансляційної модифікації. Методи. Конструювання рекомбі-
нантних ДНК, експресія та очистка рекомбінантних білків,
трасфекція клітинних ліній, вестерн блот. Результати.
Надексперсія FAK1 в клітинах лінії 293 викликає масивне фос-
форилювання залишків тирозину адапторного білка SHB та
значно підсилює його взаємодію in vitro з SH2-доменами низки
сигнальних білків, таких як PI3K, ABL, CRK та PLCg1. Крім
того, білок LMP2A вірусу Епштейна-Барр може посилювати in
vitro зв’язування SHB з вищезазначеними білками, так само, як
і FAK1. Тоді як надекспресія окремих білків FAK1 та LMP2A
підвищувала рівні фосфорилювання SHB, їх ко-експерсія не
мала синергічного ефекту. Висновки. Експресія FAK1 та
LMP2A індукує фосфорилювання залишків тирозину адаптор-
ного білка SHB та підсилює його взаємодію з SH2-доменами
низки сигнальних білків.
К л юч ов і с л ов а: FAK1, SHB, LMP2A, фосфорилювання.
Киназа фокальной адгезии (FAK1) регулирует
фосфорилирование адаптерного белка SHB и его
взаимодействие с рядом сигнальных белков.
А. В. Дергай, А. М. Яручик, А. В. Рындич
Цель. Исследовать эффект суперэкспрессии киназы фокаль-
ной адгезии (FAK1) на уровень фосфорилирования остатков
тирозина адаптерного белка SHB и найти функциональное зна-
чение этой посттрансляционной модификации. Методы.
Конструирование рекомбинантных ДНК, экспрессия и очистка
рекомбинантных белков, трансфекция клеточных линий, ве-
стерн блот. Результаты. Суперэксперссия FAK1 в клетках ли-
нии 293 приводит к многократному повышению уровней фос-
форилирования остатков тирозина адаптерного белка SHB, что
в свою очередь приводит к усилению взаимодейсвия с SH2-
доменами ряда сигнальных белков, таких как PI3K, ABL, CRK
та PLCg1. Кроме того, белок LMP2A вируса Эпштейна-Барр
может усиливать in vitro взаимодействие SHB с указанными
белками, аналогично FAK1. Тогда как суперэкспрессия отдель-
ных белков FAK1 та LMP2A приводила к интенсификации
фосфорилирования SHB, их ко-экспрессия не обладала синер-
гическим эффектом. Выводы. Экспрессия FAK1 и LMP2A ин-
дуцирует фософрилирование остатков тирозина адаптерного
белка SHB и усиливает его взаимодействие с SH2-доменами
ряда сигнальных белков.
К л юч е в ы е с л ов а: FAK1, SHB, LMP2A, фосфорилирова-
ние.
Received 12.12.2015
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