CD150-mediated Akt signalling pathway in normal and malignant B cells
Aim of this article is to study upstream and downstream events in CD150-mediated Akt signaling pathway in normal human B cells, EBV-transformed lymphoblastoid (LCL) and malignant Hodgkin’s lymphoma (HL) B cell lines.
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
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| Цитувати: | CD150-mediated Akt signalling pathway in normal and malignant B cells / M. Yurchenko, L.M. Shlapatska, O.L. Romanets, D. Ganshevskiy, E. Kashuba, A. Zamoshnikova, Yu.V. Ushenin, B.A. Snopok, S.P. Sidorenko // Experimental Oncology. — 2011. — Т. 33, № 1. — С. 9-18. — Біліогр.: 53 назв. — англ. |
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irk-123456789-323122012-04-17T12:21:06Z CD150-mediated Akt signalling pathway in normal and malignant B cells Yurchenko, M. Shlapatska, L.M. Romanets, O.L. Ganshevskiy, D. Kashuba, E. Zamoshnikova, A. Ushenin, Yu.V. Snopok, B.A. Sidorenko, S.P. Original contributions Aim of this article is to study upstream and downstream events in CD150-mediated Akt signaling pathway in normal human B cells, EBV-transformed lymphoblastoid (LCL) and malignant Hodgkin’s lymphoma (HL) B cell lines. 2011 Article CD150-mediated Akt signalling pathway in normal and malignant B cells / M. Yurchenko, L.M. Shlapatska, O.L. Romanets, D. Ganshevskiy, E. Kashuba, A. Zamoshnikova, Yu.V. Ushenin, B.A. Snopok, S.P. Sidorenko // Experimental Oncology. — 2011. — Т. 33, № 1. — С. 9-18. — Біліогр.: 53 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/32312 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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DSpace DC |
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English |
| topic |
Original contributions Original contributions |
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Original contributions Original contributions Yurchenko, M. Shlapatska, L.M. Romanets, O.L. Ganshevskiy, D. Kashuba, E. Zamoshnikova, A. Ushenin, Yu.V. Snopok, B.A. Sidorenko, S.P. CD150-mediated Akt signalling pathway in normal and malignant B cells Experimental Oncology |
| description |
Aim of this article is to study upstream and downstream events in CD150-mediated Akt signaling pathway in normal human B cells, EBV-transformed lymphoblastoid (LCL) and malignant Hodgkin’s lymphoma (HL) B cell lines. |
| format |
Article |
| author |
Yurchenko, M. Shlapatska, L.M. Romanets, O.L. Ganshevskiy, D. Kashuba, E. Zamoshnikova, A. Ushenin, Yu.V. Snopok, B.A. Sidorenko, S.P. |
| author_facet |
Yurchenko, M. Shlapatska, L.M. Romanets, O.L. Ganshevskiy, D. Kashuba, E. Zamoshnikova, A. Ushenin, Yu.V. Snopok, B.A. Sidorenko, S.P. |
| author_sort |
Yurchenko, M. |
| title |
CD150-mediated Akt signalling pathway in normal and malignant B cells |
| title_short |
CD150-mediated Akt signalling pathway in normal and malignant B cells |
| title_full |
CD150-mediated Akt signalling pathway in normal and malignant B cells |
| title_fullStr |
CD150-mediated Akt signalling pathway in normal and malignant B cells |
| title_full_unstemmed |
CD150-mediated Akt signalling pathway in normal and malignant B cells |
| title_sort |
cd150-mediated akt signalling pathway in normal and malignant b cells |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2011 |
| topic_facet |
Original contributions |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/32312 |
| citation_txt |
CD150-mediated Akt signalling pathway in normal and malignant B cells / M. Yurchenko, L.M. Shlapatska, O.L. Romanets, D. Ganshevskiy, E. Kashuba, A. Zamoshnikova, Yu.V. Ushenin, B.A. Snopok, S.P. Sidorenko // Experimental Oncology. — 2011. — Т. 33, № 1. — С. 9-18. — Біліогр.: 53 назв. — англ. |
| series |
Experimental Oncology |
| work_keys_str_mv |
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| first_indexed |
2025-07-03T12:49:38Z |
| last_indexed |
2025-07-03T12:49:38Z |
| _version_ |
1836630124581093376 |
| fulltext |
Experimental Oncology ��� ����� ���� ��arc����� ����� ���� ��arc�� ��arc�� �
CD150-MEDIATED AKT SIGNALLING PATHWAY IN NORMAL
AND MALIGNANT B CELLS
M. Yurchenko1,*, L.M. Shlapatska1, O.L. Romanets 1, D. Ganshevskiy 1, E. Kashuba1,2,
A. Zamoshnikova1, Yu.V. Ushenin3, B.A. Snopok3, S.P. Sidorenko1
1R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology NAS of Ukraine,
Kyiv 03022, Ukraine
2Microbiology and Tumorbiology Center, Karolinska Institutet, Stockholm 171 77, Sweden
3V.E. Lashkarev Institute of Semiconductors Physics NAS of Ukraine, Kyiv 03028, Ukraine
Aim: To study upstream and downstream events in CD150-mediated Akt signaling pathway in normal human B cells, EBV-
transformed lymphoblastoid (LCL) and malignant Hodgkin’s lymphoma (HL) B cell lines. Methods: To access protein-protein
interaction we applied immunoprecipitation, Western blot analysis and surface plasmon resonance (SPR) technique. A novel
modification of SPR technique using reduced glutathione bound to golden surface was proposed. Immunostaining and isolation
of cytoplasmic fractions and nuclear extracts were performed to detect proteins’ localization in cells. Western blot analysis was
performed to follow up the phosphorylation of proteins on specific sites and proteins’ expression level. Results: It was shown that
CD150 ligation induced Akt activation in normal tonsillar B cells (TBC), SH2D1A positive LCL and HL B cell lines. The p85α
subunit of PI3K co-precipitated with CD150 cytoplasmic tail. This direct association depends on tyrosine phosphorylation and
is mediated by N terminal SH2 domain of p85α. CD150 initiated phosphorylation of FoxO1 transcription factor in normal B cells
as well as in LCL MP-1 and HL cell line L1236. At the same time, CD150 ligation triggered GSK-3β kinase phosphorylation
only in immortalized LCL MP-1 and HL cell line L1236. Conclusions: We have demonstrated that CD150 receptor could trigger
PI3K-mediated Akt signaling pathway in normal, EBV-transformed and malignant B cells. CD150-mediated phosphorylation of Akt
downstream targets GSK-3β and FoxO1 in EBV-transformed and HL cells could be one of the mechanisms to avoid apoptosis and
support survival program in these immortalized B cells.
Key Words: CD150 receptor, PI3 kinase, Akt, GSK-3β, FoxO1, immortalized B cells, Hodgkin’s lymphoma.
CD�5� �IPO-�/SLA�� is a member of CD� family
of t�e immonoglobulin �Ig� superfamily of surface
receptors [��5]. It is expressed on activated T and
B lymp�ocytes� dendritic cells and monocytes [��
6]. Low level of CD�5� expression was also found
on natural killer T cells� platelets� basop�iles and ma-
ture dendritic cells [7� �]. T�is receptor is upregulated
via antigen receptors� CD4�� Toll-like receptors and
by mitogenes or cytokines �reviewed in [�]�. T�ere
are experimental evidences t�at CD�5� and SH�D�A
adaptor protein are co-expressed during a narrow
window of B cell maturation� and SH�D�A may be in-
volved in regulation of B cell differentiation via switc�-
ing of CD�5�-mediated signaling pat�ways [��� ��].
CD�5� is co-expressed wit� SH�D�A adaptor protein
in some B-lymp�oblastoid cell lines and lymp�oma
cells wit� activated B cell p�enotype� suc� as Hodg-
kin’s lymp�oma �HL� and ABC-type diffuse large B cell
lymp�oma [��� ��].
Tumor cells of classical HL are known as Hodgkin
and Reed-Sternberg cells �HRS�� and originate from
preapoptotic germinal center B cells t�at do not ex-
press functional B cell receptor �BCR� [��� �4]. HRS
cells are able to escape t�e regulation mec�anisms
aimed to eliminate B cells lacking functional BCR. Sev-
eral aberrantly activated signaling pat�ways �ave
been identified t�at contribute to t�e rescue of HRS
cells from apoptosis: JAK-STAT [�5� �6]� �APK/ERK
initiated by CD��-� CD4�-� RANK [�7] and CD�5� [��]
signaling� PI�K/AKT linked to CD4� [��]� L�P� and
L�P�a in EBV-positive cases [�����]; NF-kappaB due
to aberrant expression� mutations� CD4�� CD�� stimu-
lation �reviewed in [��� �4� ��]�.
Activation of t�e p�osp�atidylinositol �-kinase
�PI�K� pat�way �as been linked wit� tumor cell growt��
survival and resistance to t�erapy in several cancer
types [�4]. T�e main downstream PI�K effector� w�ic�
controls t�e survival of normal and malignant cells�
is Akt/PKB [�5]. Ligation of tumor necrosis factor �TNF�
family receptors could induce Akt p�osp�orylation/ac-
tivation in normal [�6] and HRS cells [��]. Previously�
we �ave demonstrated t�at Akt could be activated via
CD�5� in DT4� model system� lymp�oblastoid cell line
�P-� [��� �7] and HL cells L���6 [��]. T�e �ypot�esis
was proposed t�at CD�5�-SH�D�A association could
play decisive role in CD�5�-mediated Akt signaling.
In current study we furt�er explored t�e upstream and
downstream events in CD�5�-mediated Akt pat�way
in normal and malignant B cells.
Received: November 15, 2010.
*Correspondence: Fax: +380442581656;
E-mail: myurchenko@hotmail.com
Abbreviations used: BCR – B cell receptor; CD150ct – CD150 cy-
toplasmic tail; Cyt – cytoplasmic �raction; �� – �od�kin�s lym-Cyt – cytoplasmic �raction; �� – �od�kin�s lym-�� – �od�kin�s lym-
phoma; �RS – �od�kin�s and Reed-Sternber�; ��S� – immu-��S� – immu-
notyrosine based switch moti�; �C� - lymphoblastoid cell line;
NE – nuclear extract; NS�2 – N-terminal S�2 domain; P�3K
- phosphatidylinositol 3-kinase; S�2D1� – S�2 domain contain-S�2D1� – S�2 domain contain-
in� protein 1�; SPR – sur�ace plasmon resonance; �BC – tonsillar
B cells; �C� – total cell lysate; �NF – tumor necrosis �actor.
Exp Oncol ����
��� �� ����
ORIGINAL CONTRIBUTIONS
�� Experimental Oncology ��� ����� ���� ��arc��
MATERIALS AND METHODS
Cell lines. T�e B lymp�oblastoid cell lines �B-LCL�
�P-�� CESS� T5-�� fres�ly infected LCLs from pe-
rip�eral blood B cells and HL cell line of B cell origin
L���6 were maintained in RP�I �64� medium con-
taining ��% FBS� � m� L-glutamine� and antibiotics.
Human tissue specimens. After receiving
informed consent in accordance wit� t�e Declara-
tion of Helsinki� fres� tonsils were obtained from
�� patients undergoing tonsillectomy. Tonsillar B cells
were isolated by density fractionation on discontinu-
ous Lymp�oprep �Axis-S�ield PoC AS� Norway� and
Percoll �Sigma� USA� gradients as described [��].
�6.5���.6% of cells in CD�- populations were of B cell
lineage since expressed CD�� and/or CD��� antigens.
GST proteins preparation. GST-fusion con-
structs of CD�5� cytoplasmic tail �CD�5�ct� were
prepared earlier and described in [��� �7]. cDNA
of p�5α regulatory subunit was a kind gift of Dr. I. Gout
�University College� London� UK�. N-terminal SH� do-
main of p�5α regulatory subunit was cloned into pGEX-
�T vector using specific primers� and sequenced
�we t�ank for t�e �elp Dr. Vladimir Kas�uba� �TC�
Karolinska institutet� Sweden�. T�e plasmid encoding
GST-SH�D�A was kindly provided by Dr. Kim Nic�ols
�T�e C�ildren’s Hospital of P�iladelp�ia� P�iladelp�ia�
PA� USA�. Plasmids containing GST-CD�5�ct� GST-
SH�D�A and GST-NSH�-p�5α were transformed into
t�e Escherichia coli strains BL��DE� �Invitrogen� USA�
to produce non-p�osp�orylated fusion proteins� and
GST-CD�5�ct wit� and wit�out point mutations were
transformed to TKX� strain �Stratagene� USA� for pro-
duction of tyrosine-p�osp�orylated fusion proteins.
Surface plasmon resonance (SPR) instrumen-
tation and SPR analysis. T�e substrates and cell
design of scanning SPR spectrometer “BioSuplar”
�“PLAS�ON-��5”� �V. Las�karev Institute of Semicon-
ductor P�ysics� NAS of Ukraine� Kyiv� Ukraine� as well
as its ot�er c�aracteristics are described in [��]. T�is
spectrometer� w�ic� �as open measurement arc�itec-
ture� was used to study protein�protein interactions.
T�e peristaltic pump provided a constant solution flow
�> 5� μl/min� t�roug� t�e experimental cell. SPR kinet-
ics was analyzed wit� a model t�at takes into account
t�e �eterogeneous processes at t�e interface using
a stretc�ed exponential function [��].
Surface modification and protein immobiliza-
tion. T�e surface cleaning and activation procedure �for
planar metal electrodes of SPR c�ips� eac� wit� a metal
layer 5�� Št�ick consisting of a 45�-Šlayer of gold
on a 5�-Å c�romium ad�esive layer� was carried out
at room temperature as follows. To remove organic con-
tamination� t�e c�ips were was�ed in a fres�ly prepared
mixture of aqueous �NH4��S�O� ��.�� �� and H�O� ���%�
��5:6 vol:vol� for �� min. Traces of �eavy metal ions were
removed wit� a fres�ly prepared mixture H�O� ���%�
and HCl ��7%� in water ��:�:�� vol:vol:vol� for �� min.
T�e surface was modified by t�iols СOOH-�CH����-SH
��.� μМ� and CH�-�CH���-SH �� μМ�� �Sigma� USA�
in ratio �:��� ��� �� �7 °С� [��]. C�ips were was�ed
by et�anol. Cadmium acetate ��� m� solution� room
temperature� was used for activation of carboxyl groups
at least for � � prior experiment. Cadmium acetate was
used as an immobilization bridge� w�ic� �as selectiv-
ity to t�iogroup of glutat�ione �as ligand� and interacts
wit� carboxyl groups on c�arged t�iols. Glutat�ione
immobilization: � m� solution of reduced glutat�i-
one �Sigma� USA� in TBE buffer �рН �.�� was applied
on c�ips’ surface for � � at room temperature. Proteins
were immobilized in PBS buffer.
Biochemical methods and antibodies. Cell ly-
sis� SDS-PAGE� Western blotting� stimulation of cells
by anti-CD4� �G��-5� kind gift of Prof. Edward Clark�
University of Was�ington� Seattle� WA� USA�� anti-Ig�
�AffiniPure F�ab’�� fragment goat anti-�uman Ig�
Fc5μ fragment specific� Jackson ImmunoResearc�
laboratories� West Grove� PA� USA�� anti-CD�5� mAb
�IPO-�� produced in IEPOR NASU� Kiev� Ukraine��
CD�5� immunoprecipitations were performed as de-
scribed earlier [��� �7]. Western blot results were
visualized using a ��x LumiGLO® Reagent and ��x Per-
oxide �Cell Signaling Tec�nology� Beverly� �A� USA�.
Goat antisera against actin� was purc�ased from Santa
Cruz Biotec�nology �Santa Cruz� CA� USA�. Rabbit
anti-SH�D�A serum was a kind gift of Dr. Kim Nic�ols
�T�e C�ildren’s Hospital of P�iladelp�ia� P�iladelp�ia�
PA� USA�. Rabbit anti-pAkt �Ser47�� and �T�r�����
anti-Akt� anti-pFoxO��T�r�4�/Fox��a�T�r���� anti-
FoxO�� anti-FoxO�a� anti-pGSK-�β �Ser��� anti-PI�K
p�5α antibody� anti-PARP� anti-Bcl-� were purc�ased
from Cell Signaling Tec�nology �Beverly� �A� USA�.
Immunostaining. T�e double staining for
CD�5� receptor and SH�D�A adaptor protein was
done as follows: cells were spun on glass slides
��� x ��4 per slide�; fixed in cold met�anol-acetone
��:�� at ��� oC for � �; re�ydrated wit� PBS for � �;
stained wit� anti-CD�5� �IPO-�� mAb �5 μg/ml� for
� �� followed by � was�es in PBS; incubated wit�
�orse anti-mouse Texas Red-conjugated antibodies
�VectorLab� CA� USA�� and was�ed � times. Cells were
stained wit� anti-SH�D�A antibodies �dilution �:����
for � �� t�ree was�es in PBS� incubated wit� mouse
anti-rabbit FITC-conjugated antibodies �DAKO� Den-
mark�� and mounted wit� ��% glycerol solution in PBS
t�at contained �.5% ��4-diazabicyclo-��.�.��octane
�Sigma�. Bisbenzimide �Hoec�st ���5�� was added
at a concentration of �.4 μg/ml to t�e last secondary
antibody for DNA staining. T�e images were recorded
on a DAS microscope Leitz D� RB �Leica Inc.� Deer-
field� IL� USA� wit� a Hamamatsu dual mode cooled
CCD camera C4��� �Hamamatsu City� Japan�.
Isolation of cytoplasmic fraction and nuclear
extracts. Total cell lysates �TCL� were prepared from
5 x ��6 of cells in RIPA lysis buffer ��5� m� NaCl�
�� m� Tris-HCl �pH �.��� � m� EGTA� � m� EDTA
�pH �.��� �% Tryton X-���� protease in�ibitors’ cocktail
�Sigma� USA�� � m� P�SF�� and sonicated on ice for
�5 s �Sonicator �SE Soniprep �5� Plus� �SE �UK�
Limited� UK�� spun for �5 min� �7��� g� +4 oC� su-
pernatants transferred to fres� tubes. For subcellular
Experimental Oncology ��� ����� ���� ��arc����� ����� ���� ��arc�� ��arc�� ��
fractionation� control and stimulated cells ��� x ��6�
were lysed in buffer A �5� m� NaCl� �� m� HEPES
�pH �.��� 5�� m� sucrose� � m� EDTA �pH �.���
�.�% Tryton X-���� protease in�ibitors’ cocktail
�Sigma� USA�� � m� P�SF�� vortexed and spun for
� min ��75 g� +4 oC�. Cytoplasmic fractions �Cyt�
of supernatant were collected to fres� tubes. Pellets
�nuclei� were was�ed twice by �.5 ml of cold buffer B
�5� m� NaCl� �� m� HEPES �pH �.��� �5% glycerol�
�.� m� EDTA �pH �.��� and spun for � min ��75 g�
+4 oC�. Pellets were resuspended in ��� μl of buffer C
��5� m� NaCl� �� m� HEPES �pH �.��� �5 % glycerol�
�.� m� EDTA �pH �.��� protease in�ibitors’ cocktail
�Sigma� USA�� � m� P�SF�� and sonicated on ice for
�5 s� spun for �5 min� �7��� g� +4 oC� and superna-
tants were transferred to fres� tubes. TCL� NE and Cyt
fractions were stored at -�� oC. Protein concentration
was measured in all samples �TCL� NE� Cyt�� and total
amount of �� μg of protein was loaded to eac� well for
Western blot analysis.
RESULTS
p85α subunit of PI3K co-precipitates with
CD150 cytoplasmic tail. Previously� we �ave s�own
t�at CD�5�-mediated signals lead to Akt S47� p�os-
p�orylation in �uman B-lymp�oblastoid cell line
�P-� [�7] and c�icken B cell line DT4� [��]. �oreover�
using DT4� model system it was s�own t�at CD�5�-
mediated Akt pat�way was dependent on SH�D�A
binding to CD�5�� was negatively regulated by Lyn and
Btk� and positively - by Syk [��]. Also� anot�er member
of CD� family� CD�44 ��B4�� upon tyrosine p�osp�ory-
lation was s�own to recruit not only SH�D�A� but also
associate wit� t�e p�5α regulatory subunit of PI�K
[��]. To answer t�e question w�et�er CD�5� could
form a complex wit� PI�K in B cells� we performed
immunoprecipitations of CD�5� by specific mAb
�IPO-�� from tonsillar B cell �TBC� and �P-� lysates.
Western blot analysis of t�ese immunoprecipitates
demonstrated t�at p�5α regulatory subunit of PI�K
co-precipitated wit� CD�5� from bot� examined
lysates �Fig. ��.
MP-1
TBC
p85α PI3K
p85α PI3K
ly
sa
te
an
ti-
M
O
PC
an
ti-
CD
15
0
Fig. 1. PI�K p�5α regulatory subunit precipitated wit� CD�5� re-
ceptor bot� from �P-� and tonsillar B cells �TBC�. Western blot
analysis wit� anti-p�5α of CD�5� immunoprecipitates. Ig isotype
matc�ed �OPC�� antibody served as negative control
CD150ct directly interacts with p85α subunit
N terminal SH2 domain of PI3K. To test w�et�er
p�5α regulatory subunit of PI�K directly binds
CD�5� cytoplasmic tail we applied surface plasmon
resonance �SPR� approac�. For SPR-based analy-
sis of protein-protein interactions we used tyrosine
p�osp�orylated and non-p�osp�orylated GST-fused
cytoplasmic tail of CD�5� �GST-CD�5�ct� wit� point
mutations �Y/F substitution� in ITS� motifs� described
earlier [��]. Also� fusion proteins of GST-SH�D�A�
GST-fused N-terminal SH� domain of p�5α regula-
tory subunit of PI�K �GST-NSH�-p�5alp�a� were used
in t�is study. GST-NSH�-p�5alp�a was c�osen since
it was demonstrated for p�5-associated receptors EGF
and PDGF t�at t�e affinity of t�e N-SH� domain of p�5α
for t�e receptors correlated wit� t�e steady-state level
of p�5-receptor complex [�4]. T�e quantitative ratio
of protein partners was calculated based on molecular
weig�t of interacting proteins.
Interaction between GST-fusion proteins
of CD�5�ct �GST-CD�5�ct� and SH�D�A �GST-
SH�D�A� was s�own to be �:� for non-p�osp�oryl-�D�A� was s�own to be �:� for non-p�osp�oryl-D�A� was s�own to be �:� for non-p�osp�oryl-�A� was s�own to be �:� for non-p�osp�oryl-A� was s�own to be �:� for non-p�osp�oryl-�:� for non-p�osp�oryl-
ated GST-CD�5�ct �Fig. �� a�� and �:� for tyrosine
p�osp�orylated GST-CD�5�ctPY and GST-SH�D�A
�Fig. �� b�. T�is ratio corresponds to t�e data obtained
by bioc�emical studies� s�owing t�at immunotyrosine
based switc� motif �ITS�� wit� non-p�osp�orylated
tyrosine Y��� could bind SH�D�A� and t�at bot�
ITS�s wit� Y��� and Y��7 could bind adaptor protein
SH�D�A w�en p�osp�orylated [��� �5]. T�us� our
results obtained by SPR approac� wit� glutat�ione
immobilized on golden c�ips� completely matc� wit�
previously obtained results for CD�5�-SH�D�A inter-
action� and� t�erefore� t�is approac� could be used for
studies of GST-CD�5�ct direct interaction wit� ot�er
SH�-domain containing proteins.
Using t�is approac� we examined GST-NSH�-
p�5alp�a interaction wit� non-p�osp�orylated
�Fig. �� c� and tyrosine p�osp�porylated GST-CD�5�ct
�Fig. �� d�. It was found t�at NSH� domain of p�5α sub-SH� domain of p�5α sub-domain of p�5α sub-p�5α sub- sub-
unit interacts only wit� tyrosine p�osp�orylated recep-
tor �see Fig. �� d�. To determine� w�ic� tyrosines �Y����
Y��7� in CD�5�ct are essential for CD�5� - NSH� p�5α
interaction we used fusion proteins of CD�5�ct wit�
point mutations Y/F. It was s�own t�at mutation Y��7F
did not alter CD�5�ct-p�5α SH� interaction. However�
double mutant Y���F/Y��7 did not bind p�5α SH� do-α SH� do- SH� do-
main �data not s�own�. T�us� t�e first ITS� motif con-�data not s�own�. T�us� t�e first ITS� motif con-data not s�own�. T�us� t�e first ITS� motif con-�. T�us� t�e first ITS� motif con-T�us� t�e first ITS� motif con-
taining Y��� s�ould be available and p�osp�orylated
for NSH�-p�5α interaction wit� CD�5�ct.
T�us� we demonstrated by SPR t�at p�5α subunit
of PI�K via its N terminal SH� domain could directly
bind CD�5�ct� and t�at t�is interaction takes place
in normal TBC and lymp�oblastoid cell line �P-�. T�e
direct interaction of CD�5� wit� p�5α subunit of PI�K
could be a starting point for regulation of PI�K activa-
tion and subsequent Akt activation via CD�5�.
CD150-mediated Akt activation in lympho-
blastoid cell lines. �P-� and CESS lymp�oblastoid
cell lines bot� express CD�5� and SH�D�A proteins.
Here we demonstrate t�at CD�5� ligation led to p�os-
p�orylation of bot� Akt activation sites �T��� and S47��
in �P-� cells �Fig. ��. In CESS cells CD�5� ligation
induce only slig�t Akt p�osp�orylation on T��� and
S47�� and CD�5� signals �ad no effect on Akt p�os-
p�orylation levels in T5-� cells and fres�ly obtained
�� Experimental Oncology ��� ����� ���� ��arc��
from p�erip�eral blood B cells LCLs �see Fig. ��. T�ese
cell lines differed by adaptor protein SH�D�A expres-
sion levels: �P-� cells expressed SH�D�A protein
at �ig� level� CESS cells and LCLs � at muc� lower
level� and T5-� cells did not express SH�D�A at all
�[��] and data not s�own�.
CD150 and SH2D1A localization in lympho-
blastoid cell lines. Studies performed on regulation
of CD�5�-mediated Akt activation in DT4� model sys-
tem [��] and Hodgkin’s lymp�oma cell lines [��� ��]
�ave s�own t�at CD�5�-mediated Akt activation could
be regulated by SH�D�A expression and its cellular
localization. Using immunofluorescent staining �ere
we found t�at in �P-� and CESS cells SH�D�A was
localized in cytoplasm close to t�e cellular membrane
�Fig. 4�. Practically all �P-� cells in contrary to ��%
of CESS cells were SH�D�A positive �see Fig. 4�.
As CD�5�-SH�D�A interaction was s�own to be crucial
for Fyn recruitment and CD�5� tyrosine p�osp�oryla-
tion at least in T cells [�6� �7] we examined t�e p�os-
p�orylation of receptor in bot� cell lines. It was s�own
t�at CD�5� �ad lower level of tyrosine p�osp�orylation
in CESS cells t�an in �P-� cells �data not s�own�.
T�us� t�e level of SH�D�A expression directly corre-
lated wit� tyrosine p�osp�orylation. Since p�5α PI�K
binds only to tyrosine p�osp�orylated CD�5�� obtained
data could explain t�e lack of CD�5�-mediated Akt
p�osp�orylation in cells� w�ic� do not express SH�D�A
or �ave low expression level of t�is adaptor protein.
Akt activation in primary B cells. CD�5�-
mediated Akt p�osp�orylation was studied in dense
�naive and memory B cells� and buoyant fractions
�activated cells� of TBC. It was s�own t�at Akt kinase
could be p�osp�orylated via CD�5� bot� in naïve and
activated primary TBC �Fig. 5a, b�. As a control� cells
were stimulated via B cell receptor. It was found t�at
stimulation of dense TBC via CD�5� and Ig� �ad ad- �ad ad-
ditive effect on Akt p�osp�orylation �Fig. 5a, c, grap��.
Akt distribution between cytoplasmic and
nuclear fractions in LCL and HL cell lines. Subcel-
lular localization of Akt kinase is very important for its
function. In resting unstimulated cells Akt molecules
resides bot� in t�e cytoplasm and nucleus [��]. Activa-
tion of Akt occurs in proximity of plasma membrane and
�as been s�own to be followed by pAkt translocation
to cytosol and nucleus [��]. Suc� translocation allows
pAkt to p�osp�orylate its downstream targets� among
w�ic� t�ere are cytoplasmic as well as nuclear proteins.
In current study we addressed t�e question �ow
Akt kinase was distributed between nucleus and cy-
64.9
64.8
64.7
64.6
64.5
64.4
64.3
64.2
64.9
64.8
64.7
64.6
64.5
64.4
64.7
64.6
64.5
64.4
64.9
64.8
64.7
64.6
64.5
64.4
64.3
64.2
64.1
An
gl
e,
d
eg
re
e
An
gl
e,
d
eg
re
e
An
gl
e,
d
eg
re
e
An
gl
e,
d
eg
re
e
0 20 40 60 80 100 120 140 160 180-20 0 20 40 60 80 100 120
0 0 10 20 30 40 50 60-1020 40 60 80 100 120
140 160-20
Time, minTime, min
Time, minTime, min
a b
c
d
Fig. 2. Representative SPR responses of protein-protein interactions �a-d�. Non-tyrosine p�osp�orylated GST-CD�5�ct interacted wit�
GST-SH�D�A protein in ratio �:� �a�. Tyrosine p�osp�orylated GST-CD�5�ct �GST-CD�5�ctPY� interacted wit� GST-SH�D�A protein
in ratio �:� �b�. Non-tyrosine p�osp�orylated GST-CD�5�ct did not bind to GST-NSH�-p�5alp�a �c�. GST-CD�5�ctPY interacted wit�
GST-NSH�-p�5alp�a in ratio �:� �d�
Experimental Oncology ��� ����� ���� ��arc����� ����� ���� ��arc�� ��arc�� ��
toplasm in EBV-transformed cells �P-� and HL cells
�L���6� before and after stimulation via surface
receptors CD4� and CD�5�. Before ligation of recep-
tors� Akt kinase was mainly localized in cytoplasmic
fraction in L���6 HL cells� and partially in t�e nuclear
extract �Fig. 6� a�. Upon stimulation of L���6 cells via
CD�5� and CD4� receptors� Akt was completely ex-
ported from t�e nucleus. In �P-� cells� Akt kinase was
localized exceptionally in cytoplasm �Fig. 6� b�. Signals
via CD4� or CD�5� receptor �ad no influence on Akt
distribution between nucleus and cytoplasm �Fig. 6� b�.
CD150-mediated phosphorylation of Akt down-
stream targets in normal and malignant B cells. Akt
kinase regulates t�e activity of multiple target proteins
in cells� w�ic� results in promotion of cell survival
and in�ibition of apoptosis. Among t�em are stress-
activated kinases� BAD� Fork�ead family of transcrip-
tion factors� IκB kinase� GSK-�� mTOR� c-Raf� B-Raf�
Nur77� etc �reviewed in [�4]�.
GSK-� serine/t�reonine protein kinase is one
of t�e crucial Akt target proteins. Two �omologous
mammalian GSK-� isoforms� encoded by different
genes� are known � GSK-�α and GSK-�β. Akt could
p�osp�orylate GSK-�α on S�� and GSK-�β on S�� and
inactivate t�ese proteins [��]. We examined if CD�5�-
mediated Akt activation could lead to p�osp�orylation
of GSK-�β in primary TBC� LCL �P-� and HL cell line
L���6. It was found t�at in primary TBC� bot� in dense
and buoyant fractions� t�e basal level of GSK-�β
p�osp�orylation on S� was very �ig�� and it was not
altered by stimulation via CD�5� receptor �Fig. 7� a�.
At t�e same time� CD�5�-mediated Akt activation
bot� in �P-� and L���6 cells resulted in upregulation
of GSK-�β S� p�osp�orylation wit� kinetics similar
to Akt S47� p�osp�orylation �Fig. 7� b, c�.
FoxOs transcription factors constitute important
downstream targets of t�e PI�K/Akt signaling pat�-
way. FoxOs are regarded as tumor suppressor genes
�reviewed in [4�]�. FoxOs regulate transcription of pro-
apoptotic genes� w�ile Akt kinase protects cells from
apoptosis by p�osp�orylating FoxOs� w�ic� target
t�em to �4-�-�-mediated nuclear export and/or pro-
teosome-mediated degradation [4��4�]. T�e sites for
Akt p�osp�orylation in FoxO� are T�4� S�56 and S����
and for FoxO�a are T��� S�5� and S��5 �reviewed in
[4�]�. Here we explored CD�5�-mediated p�osp�ory-
lation of FoxO�/FoxO�a on T�4/T�� in comparison
pAkt (S473)
pAkt (T308)
actin
pAkt (S473)
pAkt (T308)
pAkt (S473)
pAkt
pAkt (S473)
actin
actin
0
MP-1
CESS
T5-1
LCL
63 —
63 —
37 —
63 —
63 —
63 —
63 —
37 —
63 —
37 —
15 30 60 90Stimulation, min
Fig. 3. CD�5�-mediated Akt p�osp�orylation on serine
47� �S47�� and t�reonine ��� �T���� in lymp�oblastoid cell
lines �a�. Akt kinase was markedly p�osp�orylated on bot�
sites S47� and T��� in �P-� cells� only slig�tly p�osp�orylated
in CESS cells �a, upper panels�. T�e level of Akt p�osp�orylation
was not altered in T5-� and LCL �b� lower panels�. Equal loading
was monitored by anti-actin or anti-Akt Western blot
MP-1 CESS
Fig. 4. Double immunostaining of CD�5� and SH�D�A demonstrated t�at t�ese two proteins are co-expressed and co-localized close to t�e
cellular membrane in all �P-� cells. Only ��% of CESS cells were SH�D�A positive� and SH�D�A was also co-localized wit� CD�5�� x4��
�4 Experimental Oncology ��� ����� ���� ��arc��
to stimulation via CD4� and Ig� receptors. We found
t�at only FoxO�� but not FoxO�a is expressed in TBC�
�P-� and HL cells L���6 on protein level �data not
s�own�. It was s�own t�at FoxO� p�osp�orylation
level on T�4 was upregulated via CD�5� and Ig�� but
not CD4� in dense fraction �naïve and memory cells�
of TBC �Fig. �� a� upper panel�. In buoyant TBC t�e level
of pFoxO� was not altered by eit�er CD�5� or CD4� li-
gation� but was only en�anced by ligation of Ig� �see
Fig. �� a� lower panel�.
T�e basal p�osp�orylation level of FoxO� on T�4 was
quite �ig� in LCL �P-�. Nevert�eless signals via CD�5� and
CD4� upregulated t�e level of pFoxO� in �� and 6� min
upon stimulation �Fig. �� b� grap��. T�e effect of CD�5� li-
gation was more pronounced t�an of CD4� �see Fig. ��
b� grap��.
Similarly� in HL cells L���6 bot� signals via
CD�5� or CD4� receptors significantly en�anced t�e
p�osp�orylation of FoxO� T�4 in �5 and 6� min upon
receptors’ ligation� �owever� effect of CD4� crosslink-
ing was more prominent �Fig. �� c�.
DISCUSSION
T�e serine/t�reonine kinase Akt/PKB is a crucial
regulator of divergent cellular processes� including
proliferation� differentiation and apoptosis [�4]. Akt
signaling is often deregulated in cancer� leading
to constitutively active Akt kinase [�4].
T�e p�osp�orylation of Akt �pAkt S47�� was found
to be upregulated in HL derived cell lines and in HRS
cells in 64% [��] and ���% [44] of primary lymp� node
sections of HL. In all tested cases� Akt was detected
bot� in HRS cells and t�e surrounding reactive cells�
w�ile active p�osp�orylated form of Akt was expressed
only by t�e HRS cells [��]. T�is �ig� level of basal Akt
p�osp�orylation t�oug�t to be maintained by signals
via tumor necrosis factor �TNF� family receptors CD4��
RANK� and CD�� [��]. We �ave s�own previously
t�at CD�5� receptor mediated signaling could also
contribute to Akt activation in HL cells [��� ��]. Here
we demonstrated t�at t�is signaling pat�way could
be activated via CD�5� in primary normal B cells�
as well as in LCL and HL cell lines.
Previously� it was s�own t�at CD�5�ct interac-
tion wit� adaptor protein SH�D�A was necessary for
CD�5� interaction wit� protein kinases and ITS�s’
p�osp�orylation [�6� 45]. Our data suggested t�at
CD�5�-SH�D�A association and SH�D�A localization
play decisive role in activation of Akt signaling pat�way
upon CD�5� ligation �Fig. �� 4�.
Based on our data it could be suggested t�at
SH�D�A adaptor protein association wit� CD�5�ct
allows tyrosine p�osp�orylation of CD�5�ct by cellular
pAkt (S473)
pAkt (S473)
actin
pAkt (S473)
actin
pAkt (S473)
Akt
1
Stimulation,
15 min
Stimulation,
15 min
Stimulation,
15 min
Dense TBC
Buoyant TBC
Co
nt
ro
l
Ig
M
CD
15
0
Ig
M
+
CD
15
0
IgM CD150 IgM + CD150
Co
nt
ro
l
Ig
M
CD
15
0
Ig
M
+
CD
15
0
63 —
37 —
63 —
63 —
63 —
37 —
3 5 7 92 4 6 8 11 1210 13
0 15 60 15 605 30 5 30 15 305 60
a
b
c
0
2
4
6
8
10
12
14
16
18
1 2 3 4 5 6 7 8 9 10 11 12 13
Fo
ld
Fig. 5. CD�5� receptor ligation induced Akt kinase p�osp�oryla-
tion �S47�� in TBC. Crosslinking of Ig� and CD�5� on dense �a�c�
and buoyant �b� TBC resulted in Akt p�osp�orylation on S47�.
Co-ligation of Ig� wit� CD�5� on t�ese cells �ad additive effect
�b and c� grap��. Equal loading was monitored by anti-actin
or anti-Akt Western blot. T�e level of pAkt was normalized against
t�e level of Akt using TotalLab program �c� grap��.
Akt
actin
PARP
Bcl-2
L1236
63 —
37 —
140 —
25 —
30
Cyt NE
CD150 CD40 CD150 CD40
Co
nt
ro
l
TC
L
Cy
t
NE
60 30 60 30Co
nt
ro
l
60 30 60
Akt
actin
PARP
Bcl-2
MP-1
63 —
37 —
140 —
25 —
30
Cyt NE
CD150 CD40 CD150 CD40
Co
nt
ro
l
TC
L
Cy
t
NE
60 30 60 30Co
nt
ro
l
60 30 60
a
b
Fig. 6. Western blot analysis of Akt kinase localization in cyto-
plasmic fraction �Cyt� and nuclear extracts �NE� of L���6 �a�
and �P-� �b� cells. Akt kinase was localized bot� in Cyt and
NE of L���6 cells� and was completely exported from t�e nucleus
upon L���6 stimulation via CD�5� and CD4� receptors �a� up-
per panel�. In �P-� cells� Akt was localized only in cytoplasmic
fraction �Cyt�� �b� upper panel�. Poly �ADP-ribose� polymerase
�PARP� and Bcl-� expression levels were c�ecked to control t�e
purity of fractions �TCL � total cell lysate�. Anti-actin Western blot
was performed to control t�e protein loading to gels
Experimental Oncology ��� ����� ���� ��arc����� ����� ���� ��arc�� ��arc�� �5
tyrosine kinases �Lyn in B cells [��]�� w�ic� permit direct
interaction of CD�5�ct and p�5α regulatory subunit
of PI�K. Immunoprecipitation of CD�5� from �P-� cells
and TBC demonstrated t�at CD�5� co-precipitated
wit� p�5α �Fig. ��. By SPR approac�� we demonstrated
t�at t�is interaction was direct� dependent on CD�5�ct
p�osp�orylation �Fig. �� c,d�� and ITS� motif containing
Y��� was necessary for NSH�-p�5α interaction wit�
CD�5�ct. T�us� CD�5�-initiated Akt activation in B cells
was mediated by direct interaction of CD�5�ct wit� p�5α
regulatory subunit of PI�K.
For SPR analysis we applied modification of met�-
od first proposed by Boltovets et al. [��]� w�en glu-
tat�ione is fixed on t�e golden surface of sensor c�ips.
T�is low-cost and effective modification of met�od
for GST-protein immobilization on sensor c�ips could
pGSK-3β (S9)
pAkt (S473)
actin
pGSK-3β (S9)
pAkt (S473)
actin
pGSK-3β (S9)
pAkt (S473)
actin
Stimulation,
15 min
CD150
stimulation, min
Dense
TBC
Buoyant
TBC
MP-1
Control CD150
63 —
37 —
49 —
a
b
c
63 —
37 —
49 —
63 —
37 —
49 —
0 15 6030 90
pGSK-3β (S9)
pAkt (S473)
actin
CD150
stimulation, min
L1236 63 —
37 —
49 —
0 15 6030 90
Fig. 7. CD�5�-mediated p�osp�orylation of Akt target protein
GSK-�β kinase in TBC� �P-� and L���6 cell lines. Bot� dense
and buoyant TBC �ad �ig� basal level of GSK-�β p�osp�oryla-
tion� w�ic� was not altered by CD�5�-initiated signaling �a�. T�e
level of pGSK-�β was upregulated in �P-� �b� and L���6 cells
�c� upon stimulation via CD�5�. Anti-pAkt Western blot was per-
formed to monitor Akt p�osp�orylation upon ligation of CD�5�.
Equal loading was monitored by anti-actin Western blot
Control IgMCD40CD150
pFoxO1 (T24)
pFoxO1
pFoxO1 (T24)
pFoxO1 (T24)
pFoxO1 (T24)
pAkt (S473)
actin
pFoxO1 (T24)
pAkt (S473)
actin
pFoxO1 (T24)
pAkt (S473)
actin
pFoxO1 (T24)
pAkt (S473)
actin
Stimulation,
15 min
Stimulation,
min
Dense
TBC
Buoyant
TBC
63 —
37 —
80 —
63 —
37 —
80 —
63 —
37 —
80 —
L1236
MP-1
30
CD150 CD40
Control
Control
60 30 60
Stimulation,
min
63 —
37 —
80 —
30
CD150 CD40
60 30 60
a
b
c
0
1
2
3
4
0
1
2
0
1
2
0
3
6
9
12
15
Fo
ld
Fo
ld
Fo
ld
Fo
ld
Fig. 8. CD�5�-mediated p�osp�orylation of Akt target protein
FoxO� transcription factor on T�4 in TBC� �P-� and L���6 cell
lines. T�e basal level of pFoxO� was �ig� bot� in dense and
buoyant TBC fractions �a� control�. Ligation of receptors medi-
ated t�e upregulation of pFoxO� level in dense cells� and �ad
almost no influence on pFoxO� level in buoyant cells �a�. LCL
�P-� was c�aracterized by �ig� basal level of FoxO� p�osp�ory-
lation� w�ic� was upregulated by CD�5�- and CD4�-mediated
signaling �b�. CD�5� and CD4� induced strong upregulation
of pFoxO� level in HL cell line L���6 �c�. T�e level of pFoxO� was
normalized against actin or FoxO� levels using TotalLab program
�grap�s�. Anti-pAkt Western blot was performed to monitor Akt
p�osp�orylation upon ligation of CD�5� receptor. Equal loading
was monitored by anti-actin or anti-FoxO� Western blot
�6 Experimental Oncology ��� ����� ���� ��arc��
be efficiently applied to study ot�er protein-protein
interactions using GST fusion proteins as interacting
partners wit�out using specific antibodies.
Several downstream effectors of Akt signaling� in-
cluding GSK-�α/β� and mTOR substrates 4E-BP� and
p7� S6 kinase� were s�own to be p�osp�orylated
in primary HL cells [44]. GSK-� is one of t�e crucial Akt
target protein. GSK-� p�osp�orylates a broad range
of substrates and is implicated in multiple biological
processes apart from its well studied function as regu-
lator of glycogen synt�esis. GSK-�β �as been s�own
to regulate cyclin D� proteolysis and subcellular local-
ization during t�e cell division cycle� t�ereby triggering
rapid cyclin D� turnover [46]. Also GSK-� controls
transcription factor c-myc proteolysis and subnuclear
localization as p�osp�orylation of c-�yc T5� facilitates
its rapid proteolysis by t�e ubiquitin pat�way [47].
We �ave demonstrated t�at GSK-�β was p�os-
p�orylated upon CD�5� ligation in �P-� and HL cell
line L���6� but not in normal TBC �Fig. 7�. It seems
like regulation of GSK-�β p�osp�orylation level/
inactivation via CD�5� receptor is more critical for
immortalized tumor cells �HL� and EBV-transformed
cells �LCL�� w�ic� use all cellular signaling mac�inery
to avoid apoptosis and support t�eir proliferation.
In normal TBC GSK-�β is initially switc�ed off ��ig�ly
p�osp�orylated on in�ibitory site�� w�ic� is probably
needed for furt�er differentiation of t�ese cells.
In mammalian cells� t�e class O of fork �ead �fk��
transcription factors is �omologous to Caenorhab-
ditis elegans transcription factor DAF-�6 �abnormal
DAuer Formation-�6�� and consists of four members:
FoxO�� FoxO�� FoxO4 and FoxO6 [4�]. Several ki-
nases were s�own to p�osp�orylate FoxOs in different
sites: Akt� serum and glucocorticoid-inducible kinase
�SGK�� mammalian sterile ��-like kinase-� ��ST���
cyclin-dependent kinase-� �CDK��� p��-kDa ribo-
somal S6 kinase-� �Rsk-��� dual-specificity tyrosine-
p�osp�orylated and regulated kinase �A �DYRK�a�
�reviewed in [4�� 4�� 4�]�. As to t�e ot�er post-trans-
lational modifications� FoxOs activity/stability could
be regulated by acetylation and ubiquitination [5�].
In our study we s�owed t�at despite t�e �ig� basal
level of pFoxO�� CD�5� mediated FoxO p�osp�oryla-
tion in normal TBC and LCL �P-� �Fig. ��. �oreover�
CD�5� as well as CD4� was involved in inactivation
of pro-apoptotic FoxO� transcription factor in HL cell
line L���6� w�ic� could contribute to t�e survival
of HRS cells. In s�ould be emp�asized t�at all studied
B cells expressed FoxO�� but not FoxO�a on protein
level. It is an important finding since it was t�oug�t
t�at t�e dominant isoform expressed in lymp�ocytes�
at least on mRNA level� is FoxO�a [4�].
Recently� we �ave s�own t�at CD�5� mediates JNK
activation in normal and HL cells [��]. JNK kinases
could p�osp�orylate FoxO in transactivating domain
[5�]. Also� JNK �as been s�own to p�osp�orylate �4-
�-� proteins� w�ic� result in reduced �4-�-� binding
to partner proteins� including FoxOs [5�]. Regulation
of multiple posttranslational modifications of FoxOs via
CD�5�-mediated JNK and ot�er signaling pat�ways
s�ould be furt�er explored to get more clear under-
standing of t�e FoxOs activity regulation in normal and
malignant B cells.
Subcellular localization of Akt kinase is important
for downstream cytoplasmic and nuclear protein
targets p�osp�orylation. Using model cell lines it was
s�own t�at activation of Akt results in its nuclear
translocation wit�in �� to �� min after stimulation [5�].
Following BCR stimulation of mouse B cell line pAkt
resides bot� in t�e cytosol and nucleus [��]. In HL cell
lines K�-H� and L4�� pAkt was localized close to t�e
membrane or in cytosol [44]. Here we �ave s�own
t�at in unstimulated L���6 cells Akt kinase was pres-
ent bot� in cytoplasmic fraction and nuclear extract.
Upon stimulation of cells via CD�5� and CD4�� Akt
was completely exported from t�e nucleus �Fig. 6�.
It is consistent wit� t�e fact t�at in most studied cases
of primary HL in HRS cells pAkt was s�own to be pref-
erentially cytoplasmic [44]. T�is could be ac�ieved
by sustained signals via TNF receptors �including
CD4�� and CD�5�. However� t�e biological effect
of t�is nuclear-cytoplasmic translocation in HL cells
s�ould be studied furt�er. We �ave s�own previously
t�at sustained signaling via CD�5� and CD4� recep-
tors induced in�ibition of cellular proliferation and cell
deat� of L���6 cells [��]. We may �ypot�esize t�at
retaining of Akt in cytoplasm would prevent t�e p�os-
p�orylation of its nuclear targets in t�ese cells� w�ic�
could be t�e reason of inability of Akt survival pat�way
to in�ibit CD�5�-induced cell deat�.
Alt�oug� t�e PI�K/Akt pat�way in�ibits apoptosis
and promotes cell cycle progression and proliferation
[�4]� t�e consequences of its activation in HRS cells
remain currently unclear. T�oug� Dutton et al. [44]
s�owed t�at HL cell lines and primary HL tumor cases
were c�aracterized by �ig� levels of p�osp�orylated/
activated Akt� in�ibition of PI�K and mTOR �ad quite
modest effect on cell survival. At t�e same time�
ot�er aut�ors [��] demonstrated t�at PI�K in�ibitor
LY��4��� �ad anti-proliferative and pro-apoptotic ef-
fects on some studied HL cell lines. �oreover� CD�5�-
stimulation of L���6 HL cell line induced cell deat��
t�oug� t�is cell line was c�aracterized by simultaneous
fast and marked Akt activation via CD�5� receptor [��].
T�us� outcome of PI�K/Akt signaling cascade may
depend on interaction wit� ot�er signaling pat�ways
t�at are initiated by different stimuli from HL microen-
vironment.
Taken toget�er� in current study we �ave s�own t�at
CD�5� ligation induced Akt p�osp�orylation/activation
in normal TBC� LCLs and HL cell lines. It could be as-
sumed t�at CD�5�-mediated activation of Akt kinase
depends on CD�5�-SH�D�A interaction followed
by p�osp�orylation of CD�5�ct and attraction of p�5α
regulatory subunit of PI�K. Akt kinase� activated via
CD�5�� could p�osp�orylate its downstream target
FoxO� transcription factor in normal B cells� as well
as in immortalized HL and B-lymp�oblastoid cell lines.
At t�e same time Akt p�osp�orylation of its substrate
Experimental Oncology ��� ����� ���� ��arc����� ����� ���� ��arc�� ��arc�� �7
GSK-�β kinase was detected only in HL cell line
L���6 and LCL �P-�� but not in normal TBC. CD�5�-
mediated Akt signaling� followed by regulation of GSK-
�β and FoxO� activity in EBV-transformed and HL tu-
mor cells could interplay wit� ot�er CD�5�-initiated
signaling pat�ways �i.e. �APK� creating signaling
network favorable for maintaining t�e survival program
and escaping apoptosis of transformed B cells.
ACKNOWLEDGMENTS
We t�ank Dr. E.A. Clark for valuable discussion and
anti-CD4� antibodies� and Dr. K. Nic�ols for providing
anti-SH�D�A antibodies and GST-SH�D�A construct.
T�is work was supported by Ukrainian governmental
grants ����U��6647 and ���7U����44� and by Swed-
is� Institute in t�e frame of Visby program.
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