PI3K/mTOR-dependent signaling pathway as a possible regulator of processing body assembly
Aim. To study the role of PI3K/mTOR signaling pathway in regulation of processing body (PB) assembly. Methods. During this study we employed cell imaging technique and Western blot analysis. Results. It was shown that treatment of cells with the specific inhibitors of PI3K/mTOR pathway leads to ch...
Saved in:
| Date: | 2011 |
|---|---|
| Main Authors: | , , , |
| Format: | Article |
| Language: | English |
| Published: |
Інститут молекулярної біології і генетики НАН України
2011
|
| Series: | Вiopolymers and Cell |
| Online Access: | https://nasplib.isofts.kiev.ua/handle/123456789/156374 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| Journal Title: | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| Cite this: | PI3K/mTOR-dependent signaling pathway as a possible regulator of processing body assembly / D.O. Gudkova, G.G. Panasyuk, I.O. Nemazanyy, V.V. Filonenko // Вiopolymers and Cell. — 2011. — Т. 27, № 5. — С. 369-372. — Бібліогр.: 14 назв. — англ. |
Institution
Digital Library of Periodicals of National Academy of Sciences of Ukraine| id |
nasplib_isofts_kiev_ua-123456789-156374 |
|---|---|
| record_format |
dspace |
| spelling |
nasplib_isofts_kiev_ua-123456789-1563742025-02-09T14:46:20Z PI3K/mTOR-dependent signaling pathway as a possible regulator of processing body assembly PI3K/mTOR-залежний сигнальний шлях як можливий регулятор утворення процесивних тілець PI3K/mTOR-зависимый сигнальный путь как возможный регулятор образования процессивных телец Gudkova, D.O. Panasyuk, G.G. Nemazanyy, I.O. Filonenko, V.V. Aim. To study the role of PI3K/mTOR signaling pathway in regulation of processing body (PB) assembly. Methods. During this study we employed cell imaging technique and Western blot analysis. Results. It was shown that treatment of cells with the specific inhibitors of PI3K/mTOR pathway leads to changes of PBs’ number and size within cells as well as proteasomal degradation of their scaffold protein RCD-8. Conclusions. We speculate that mTOR/PI3K pathway may regulate in part the dynamic of PB formation in the cell by affecting the stability of RCD-8 protein and therefore controle mRNA metabolism. Keywords: processing bodies, immunocytochemistry, mRNA degradation, mTOR, signaling pathway. Мета. Дослідити роль PI3K/mTOR-залежного сигнального шляху в регуляції утворення процесивних тілець. Методи. Використано методи імуноцитохімії та імуноблотингу. Результати. Показа - но, що обробка клітин специфічними інгібіторами PI3K/mTORсигнального шляху призводить до змін у кількості та розмірах процесивних тілець та протеасомної деградації одного з основ - них білків процесивних тілець RCD-8. Висновки. Ми припустили, що PI3K/mTOR-сигнальний шлях регулює динаміку утворення процесивних тілець у клітині, забезпечуючи стабільність скефолдного білка процесивних тілець RCD-8, і, як наслідок, нормалізує метаболізм РНК у цілому. Ключові слова: процесивні тільця, імуноцитохімія, деградація мРНК, mTOR, сигнальні шляхи. Цель. Исследовать роль PI3K/mTOR-зависимого сигнального пути в регуляции сборки процессивных телец. Методы. Использо - ваны методы иммуноцитохимии и иммуноблоттинга. Результаты. Показано, что обработка клеток специфическими ингибиторами PI3K/mTOR-сигнального пути приводит к изменениям в количестве и размерах процессивных телец в клетке, а также протеасомной деградации основного белка процессивных телец RCD-8. Выводы. Мы предположили, что PI3K/mTOR-сигнальный путь регулирует динамику образования процессивных телец, обеспечивая стабильность скеффолдного белка процессивных телец RCD-8, и, как следствие, нормализует метаболизм мРНК Ключевые слова: процессивные тельца, иммуноцитохимия, деградация мРНК, mTOR, сигнальные пути. 2011 Article PI3K/mTOR-dependent signaling pathway as a possible regulator of processing body assembly / D.O. Gudkova, G.G. Panasyuk, I.O. Nemazanyy, V.V. Filonenko // Вiopolymers and Cell. — 2011. — Т. 27, № 5. — С. 369-372. — Бібліогр.: 14 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.000125 https://nasplib.isofts.kiev.ua/handle/123456789/156374 576.311.348 en Вiopolymers and Cell application/pdf Інститут молекулярної біології і генетики НАН України |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| collection |
DSpace DC |
| language |
English |
| description |
Aim. To study the role of PI3K/mTOR signaling pathway in regulation of processing body (PB) assembly.
Methods. During this study we employed cell imaging technique and Western blot analysis. Results. It was
shown that treatment of cells with the specific inhibitors of PI3K/mTOR pathway leads to changes of PBs’
number and size within cells as well as proteasomal degradation of their scaffold protein RCD-8. Conclusions.
We speculate that mTOR/PI3K pathway may regulate in part the dynamic of PB formation in the cell by affecting the stability of RCD-8 protein and therefore controle mRNA metabolism.
Keywords: processing bodies, immunocytochemistry, mRNA degradation, mTOR, signaling pathway. |
| format |
Article |
| author |
Gudkova, D.O. Panasyuk, G.G. Nemazanyy, I.O. Filonenko, V.V. |
| spellingShingle |
Gudkova, D.O. Panasyuk, G.G. Nemazanyy, I.O. Filonenko, V.V. PI3K/mTOR-dependent signaling pathway as a possible regulator of processing body assembly Вiopolymers and Cell |
| author_facet |
Gudkova, D.O. Panasyuk, G.G. Nemazanyy, I.O. Filonenko, V.V. |
| author_sort |
Gudkova, D.O. |
| title |
PI3K/mTOR-dependent signaling pathway as a possible regulator of processing body assembly |
| title_short |
PI3K/mTOR-dependent signaling pathway as a possible regulator of processing body assembly |
| title_full |
PI3K/mTOR-dependent signaling pathway as a possible regulator of processing body assembly |
| title_fullStr |
PI3K/mTOR-dependent signaling pathway as a possible regulator of processing body assembly |
| title_full_unstemmed |
PI3K/mTOR-dependent signaling pathway as a possible regulator of processing body assembly |
| title_sort |
pi3k/mtor-dependent signaling pathway as a possible regulator of processing body assembly |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| publishDate |
2011 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/156374 |
| citation_txt |
PI3K/mTOR-dependent signaling pathway as a possible regulator of processing body assembly / D.O. Gudkova, G.G. Panasyuk, I.O. Nemazanyy, V.V. Filonenko // Вiopolymers and Cell. — 2011. — Т. 27, № 5. — С. 369-372. — Бібліогр.: 14 назв. — англ. |
| series |
Вiopolymers and Cell |
| work_keys_str_mv |
AT gudkovado pi3kmtordependentsignalingpathwayasapossibleregulatorofprocessingbodyassembly AT panasyukgg pi3kmtordependentsignalingpathwayasapossibleregulatorofprocessingbodyassembly AT nemazanyyio pi3kmtordependentsignalingpathwayasapossibleregulatorofprocessingbodyassembly AT filonenkovv pi3kmtordependentsignalingpathwayasapossibleregulatorofprocessingbodyassembly AT gudkovado pi3kmtorzaležnijsignalʹnijšlâhâkmožlivijregulâtorutvorennâprocesivnihtílecʹ AT panasyukgg pi3kmtorzaležnijsignalʹnijšlâhâkmožlivijregulâtorutvorennâprocesivnihtílecʹ AT nemazanyyio pi3kmtorzaležnijsignalʹnijšlâhâkmožlivijregulâtorutvorennâprocesivnihtílecʹ AT filonenkovv pi3kmtorzaležnijsignalʹnijšlâhâkmožlivijregulâtorutvorennâprocesivnihtílecʹ AT gudkovado pi3kmtorzavisimyjsignalʹnyjputʹkakvozmožnyjregulâtorobrazovaniâprocessivnyhtelec AT panasyukgg pi3kmtorzavisimyjsignalʹnyjputʹkakvozmožnyjregulâtorobrazovaniâprocessivnyhtelec AT nemazanyyio pi3kmtorzavisimyjsignalʹnyjputʹkakvozmožnyjregulâtorobrazovaniâprocessivnyhtelec AT filonenkovv pi3kmtorzavisimyjsignalʹnyjputʹkakvozmožnyjregulâtorobrazovaniâprocessivnyhtelec |
| first_indexed |
2025-11-26T23:49:19Z |
| last_indexed |
2025-11-26T23:49:19Z |
| _version_ |
1849898788253073408 |
| fulltext |
PI3K/mTOR-dependent signaling pathway as
a possible regulator of processing body assembly
D. O. Gudkova1, 2, G. G. Panasyuk1, I. O. Nemazanyy1, V. V. Filonenko1
1Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
2Taras Shevchenko Kyiv National University
64, Volodymyrska Str., Kyiv, Ukraine, 01033
filonenko@imbg.org.ua
Aim. To study the role of PI3K/mTOR signaling pathway in regulation of processing body (PB) assembly.
Methods. During this study we employed cell imaging technique and Western blot analysis. Results. It was
shown that treatment of cells with the specific inhibitors of PI3K/mTOR pathway leads to changes of PBs’
number and size within cells as well as proteasomal degradation of their scaffold protein RCD-8. Conclusions.
We speculate that mTOR/PI3K pathway may regulate in part the dynamic of PB formation in the cell by affec-
ting the stability of RCD-8 protein and therefore controle mRNA metabolism.
Keywords: processing bodies, immunocytochemistry, mRNA degradation, mTOR, signaling pathway.
Introduction. It is well known that phosphorylation of
S6Ks and 4E-BPs by mTOR regulates the rate of trans-
lation initiation in response to diverse extracellular sti-
muli, such as growth factors, mitogenes and nutrients
[1–3]. According to the model of steric competition for
binding of protein complexes to the cap on mRNA it has
been proposed that initial formation of the cap-de-pen-
dent translation initiation complex may affect in part
the total rate of mRNA translation via preventing the de-
capping of these targets. However there is no evidence
that PI3K/mTOR signaling pathway may directly affect
the activity of decaping machinery and as a consequen-
ce RNA degradation that occurs in specific sites within
the cell. Recently it was shown that non translating
mRNAs can be accumulated in cytoplasmic RNP gra-
nules [4–8]. At present the best-characterized mRNP
granules in the somatic cell cytoplasm are processing
bodies (PBs) and stress granules (SGs) [5]. They are
ubiquitotus highly dynamic membraneless structures,
which contain pool of untranslated mRNAs. PBs conta-
in enzymes and their co-activators for mRNA degrada-
tion and translational repression [6, 8], whereas SGs re-
present mRNPs with number of proteins involved in
the translation initiation including 40S ribosomal subu-
nit [4, 5]. RCD-8 is known as one of scaffold proteins
of PBs, which is crucial for their assembly [9, 10].
Previously, we have generated mouse polyclonal
antibodies against bacterially expressed recombinant N-
terminal region of RCD-8 [11]. The specificity of anti-
bodies was analyzed by immunofluorescence approach.
We have detected some specific foci in the cytoplasm of
HEK293 cells. To check whether these structures cor-
respond to PBs or SGs (another related kind of mRNP
granules) we overexpressed the markers of PBs and
SGs fused with fluorescent proteins in HEK293 cells.
Using IF microscopy we confirmed the specificity of pu-
rified antibodies with respect to PB.
In view of the absence of published data regarding
regulation of PB assembly in mammalian cells by sig-
naling pathways we decided to investigate the role of
known mTOR/PI3K signaling pathway inhibitors in dy-
namic of PB formation.
Materials and methods. Plasmids and antibodies.
Rabbit anti-RCD-8 antibodies were obtained from «Be-
369
ISSN 0233–7657. Biopolymers and Cell. 2011. Vol. 27. N 5. P. 369–372
Institute of Molecular Biology and Genetics, NAS of Ukraine, 2011
thyl Laboratories» (USA). Mouse anti-RCD-8 antibo-
dies were described previously [11]. Anti-mouse FITC-
conjugated antibodies were from Jackson ImmunoRe-
search Laboratories. Anti-mouse Texas Red-conjuga-
ted antibodies were from «Invitrogen» (USA). Anti-
phospho-p70 S6K1 (Thr 389), phospho-4E-BPs and β-
actin antibodies were obtained from «Millipore» (USA).
Secondary HRP-labelled anti-mouse and anti-rabbit
antibodies were from «Cell Signaling» (USA).
Cell culture and immunocytochemistry. HEK293
cells were obtained from the American Type Culture Col-
lection and maintained in DMEM supplemented with
10 % fetal bovine serum («HyClone», UK), penicillin
(200 U/ml) and streptomycin (200 mg/ml). To induce
PB assembly/disassembly HEK293 cells were expo-
sed to sodium arsenite (III) (0.5 mM), cycloheximide
(CHX, 50 mg/ml), LY294008 (50 µM), rapamycin
(200 nM) and 2-deoxyglucose (2-DG, 25 mM) for
30 min at 37 oC. All reagents were obtained from «Sig-
ma» (USA). For inhibition of proteasomal degradation
MG132 («Merck», Germany) was added to cells for 6 h
at final concentration 10 µM. For immunofluorescent
staining HEK293 cells were grown in tissue culture
chambers («Nunc», Denmark), fixed with 3.7 % para-
formaldehyde in PBS, and permeabilized with PBS-T
(0.2 % Tween-20) three times for 5 min. Unspecific
binding was blocked by 45 min incubation with 5 %
FBS diluted in PBS-T. Afterwards staining with prima-
ry and secondary antisera was performed as described
previously [12, 13].
Cell nuclei were stained with Hoechst 33258 dye
(«Invitrogen», USA). Fluorescently labelled cells were
analyzed with a Zeiss LSM Meta 510 confocal micro-
scope, and the images were edited using the LSM510
image browser software. All pictures were acquired and
processed using the same settings.
SDS-PAGE, Western blotting. HEK293 cell protein
extracts were resolved in SDS-polyacrylamide gels and
transferred to PVDF membrane («Millipore»). Memb-
ranes were incubated in blocking solution and probed
with primary rabbit antibodies, diluted in TBST (25 mM
Tris-HCl (pH 7.4), 0.8 % NaCl, 0.02 % KCl, and 0.05 %
Tween). After washing three times with TBST the mem-
brane was incubated with HRP-conjugated goat anti-
rabbit IgG («Promega», USA). Finally, the membrane
was developed using an ECL kit («GE Healthcare»,
UK) and then exposed to Agfa X-ray film.
Results and discussion. In the beginning we loo-
ked for possible changes in PBs’ number and/or size un-
der different experimental conditions. For this purpose
we treated cells with signal transduction and metabolic
inhibitors, targeting the mTOR/PI3K pathway, namely
LY294002 (an inhibitor of PI-3 kinase), rapamycin (an
inhibitor of mTORC1) and 2-DG (an inhibitor of gly-
colysis, which activates AMPK and therefore leads to
mTORC1 inactivation) [14]. After the treatment for
30 min cells were stained with anti-RCD-8 affinity pu-
rified antibodies. As shown on Fig. 1 (see inset) we ob-
served that treatment of HEK293 cells with LY294002
and 2-DG leads to the enrichment of PBs within the
cytoplasm. Moreover, we could observe changes not on-
ly in number of PBs but in their size as well. Notably,
observed changes differed from typical induction of PBs
with sodium arsenite (a known inductor of PBs assemb-
ly) [4, 5]. Thus, treatment with LY294002 resulted in the
production of predominantly small processing bodies in
contrast to 2-DG treatment, which led to the appearance
of PBs similary to the effect of arsenite. It is known that
PB assembly occurs through formation of small partic-
les with further fusion into the larger PBs [4]. We be-
lieve that LY294002 targeting PI3K signaling initiates
formation of small PBs but their fusion may require
additional impacts. Unexpectedly treatment of cells
with direct mTOR inhibitor rapamycin (which inhibits
only mTORC1 complex, but not mTORC2) (Fig. 1, b,
see inset) led to the opposite effect, namely to the disap-
pearance of PBs, like treatment with known inhibitor of
translation – CHX [4, 5] (Fig. 1, e, see inset). This fin-
ding is very intriguing, because treatment with
LY294002 indirectly inhibits the activity of mTOR ki-
nase in both mTORC1 and mTORC2 complexes in
mammalian cells.
These data indicate that the inhibition of mTOR-ki-
nase via different mechanisms and in different comple-
xes has led to opposite effects in respect to PB assemb-
ly. In addition such effect may be facilitated by additio-
nal mTOR substrates other than S6K1 and 4E-BPs since
the mTOR dependent phosphorylation of both of them is
down regulated by LY294002 and rapamycin (Fig. 2, A).
Despite the similar effect on PB disassembly in-
duced by CHX and rapamycin, we show that they exhi-
bit different effects on mTOR kinase substrates – S6K
and 4E-BPs. As it is demonstrated on Fig. 2, A, in con-
trast to rapamycin CHX treatment elevates S6K1 phos-
370
GUDKOVA D. O. ET AL.
ISSN 0233–7657. Biopolymers and Cell. 2011. Vol. 27. N 5
14
16
18
20
22
24
26
28
x102
#
#
a
b
c
d
e
A B
1st 2nd
period treatment
Fig. 2. The influence of Allopurinol on the cell number at day 12th.
magnification 60 × 1.25 × 20. The first group of the cells was trea-
ted during the entire period of the culturing, from day 1st until the
day 12th (b, c), whereas the second group was treated during the
second period, which was starting from day 6th until day 12th (d, e).
Also, it was used t-student test to calculate the significance of the
results. Results were considered significant, if p < 0.05. The results
related with the low concentration of Allopurinol in comparison
with each other and Control (a) were statistically significant
0
1
2
3
4
5
Control (1)
Allopurinol low conc (2) Allopurinol high conc (3)
Allopurinol low conc (4) Allopurinol high conc (5)
Early period treatment
Late period treatment
1 2 3 4 5
x103
A B
Fig. 3. The number of dead cells in the field, stained with the Trypan
Blue c on 12th day after seeding human embryonic neuronal cells. Mag-
nification 60 × 1.25 × 20. The pictures were taken on day 12th (A). There
were calculated results of the observation of 3 different fields from the
plates for every group (B). The first column represents the control gro-
up. The second column represents the cells treated with low, the third –
with high concentrations of Allopurinol reflecting the conditions when
the cells in the culture were receiving the Allopurinol from day 1st to
day 12th. 4th and 5th columns represent the group of the cells receiving
the low and hight concentrations of Allopurinol from day 6th to day 12th.
The results were accepted as a statistically significant when p < 0.05
a b c
d e f
10 :m
Fig..1. HEK293 cells were treated with rapa-
mycin (b), LY294002 (c), 2-deoxyglucose (d),
CHX (e) and sodium arsenite (f) during 30 min.
Picture a represent cells treated with DMSO.
After treatment cells were fixed with 3.7 % FA,
and stained with anti-RCD-8 antibodies. As se-
condary were used FITC-conjugated antibo-
dies. Cell nuclei were stained with Hoechst
33258. Magnification × 100
Figure to article D. O. Gudkova et al.
Figures to article K. E. Danielyan, G. A. Kevorkian
phorylation and slightly up-regulates 4E-BPs phospho-
rylation. Such effect may represent some compensato-
ry mechanism after translation inhibition.
Next, to determine the dependence of PB assemb-
ly on the availability of growth factors, we starved
HEK293 cells for 24 h and then stimulated with 20 %
fetal bovine serum for 0.5 or 3 h. We observed reduc-
tion in the number of processing bodies upon starva-
tion, while serum stimulation induced their formation
(data not shown). This observation clearly indicates
that the assembly of PBs is dependent on the presence
of growth factors, which can activate major signal trans-
duction pathways within cell.
We have also examined the level of endogenous
RCD-8 in cells upon the above treatments. Unexpec-
tedly, we found that the amount of RCD-8 protein was
reduced in response to all tested inhibitors. The stron-
gest effect was observed with 2-DG treatment (up to
50 % reduction) (Fig. 2, B). To study it in more details,
we carried out a time-course treatment with 2-DG,
which included 15 min and 30 min time points. Signi-
ficant decrease of RCD-8 was observed after 15 min of
treatment (Fig. 3, A). Introducing proteasomal inhibitor
MG132 before 2-DG treatment (Fig. 3, A) we have
found that the level of endogenous RCD-8 is regulated
by proteasome-mediated degradation and the changes
are observed even after 15 min upon treatment with 2-
DG (Fig. 3, A).
To study the half-life of RCD-8 in mammalian
cells under normal conditions we carried out the expe-
riment in the presence of CHX, which prevents bio-
synthesis of novel proteins in cells. The results clearly
indicated that RCD-8 is a quite stable protein with a
half-life of more than 24 h (Fig. 3, B). So, decreasing of
endogenous level of RCD-8 after treatment with diffe-
rent reagents could be explained by degradation of
RCD-8 protein via proteasomal pathway. At the same
time, the decreasing in endogenous RCD-8 does not
correlate with observed increasing number of proces-
sing bodies within the cytoplasm after the treatment.
The most reasonable explanation of observed effect co-
uld be as follow. Basically, the process of mRNA degra-
dation occurs in PB, but due to the presence of decap-
ping machinery and co-activators in the cytoplasm it
can take place there as well but significantly slowly.
Number of previous work [4, 6] described the dynamic
shuttling of main PBs components between PBs and cy-
toplasm, and PBs represent sites with much more hi-
gher concentration of those proteins than in the cyto-
plasm. Therefore we assume we were able to detect
RCD-8 only in PBs as intracellular sites with elevated
local level of RCD-8 but not others where its content is
much lower.
According to our data we speculate that PI3K/
mTOR pathway may affect the processes of PB as-
sembly. As mentioned above, PBs represent cellular
structures for storing the whole pool of non-translating
mRNAs which can undergo either degradation or stora-
ge in those foci. At the same time there is no evidence
about precise mechanism, which may regulate a fate of
stored mRNAs. The appearance of huge PBs within
cell can indicate a total increased level of mRNAs’ de-
capping as well as accumulation of silenced mRNAs.
Thus, we speculate that PI3K/mTOR signaling path-
way can be one of possible mechanisms, which keep in
balance the ratio between translated and non-translated
mRNAs.
Conclusions. In summary, we showed that the regu-
lation of processing body assembly might be media-
ted through the PI3K/mTOR signaling pathway. At the
same time regulation of PB assembly in part occurs
through the proteasomal degradation of PBs main com-
ponent – scaffold protein RCD8.
371
PI3K/mTOR-DEPENDENT SIGNALING PATHWAY
As LY Rap CHX Control
A
$-actin
p4E-BPs
pS6K1
Control LY Rap 2-DG
a-RCD
Na3VO4
$-actin
B
Fig. 2. A – cycloheximide (CHX) treatment does not affect mTOR acti-
vity; B – inhibition of PI3K/mTOR pathway reduces endogenous level
of RCD-8. HEK293 were treated with arsenite (As), LY294002 (LY),
rapamycin (Rap), CHX, Na3VO4 and 2-deoxyglucose (2-DG) as descri-
bed in Materials and methods. Then cells were lyzed in Laemmli buf-
fer and analyzed in immunoblot using specific antibodies to RCD-8,
pS6K, p4E-BPs and beta-actin
Acknowledgments. We thank Dr. V. Gorchev and
Dr. S. Karakhim for help with confocal studies. This work
was supported by the FEBS Collaborative Experimen-
tal Scholarships for Central and Eastern Europe.
Д. О. Гуд ко ва, Г. Г. Па на сюк, І. О. Не ма за ний, В. В. Філо нен ко
PI3K/mTOR-за леж ний сиг наль ний шлях як мож ли вий ре гу ля тор
утво рен ня про це сив них тілець.
Ре зю ме
Мета. Дослідити роль PI3K/mTOR-за леж но го сиг наль но го шля ху
в ре гу ляції утво рен ня про це сив них тілець. Ме то ди. Ви ко рис та но
ме то ди іму но ци тохімії та іму ноб ло тин гу. Ре зуль та ти. По ка за -
но, що об роб ка клітин спе цифічни ми інгібіто ра ми PI3K/mTOR-
сиг наль но го шля ху при зво дить до змін у кількості та розмірах
про це сив них тілець та про те а сом ної дег ра дації од но го з основ -
них білків про це сив них тілець RCD-8. Вис нов ки. Ми при пус ти ли,
що PI3K/mTOR-сиг наль ний шлях ре гу лює ди наміку утво рен ня про-
це сив них тілець у клітині, за без пе чу ю чи стабільність ске фол дно-
го білка про це сив них тілець RCD-8, і, як наслідок, нор малізує ме-
та болізм РНК у цілому.
Клю чові сло ва: про це сивні тільця, іму но ци тохімія, дег ра дація
мРНК, mTOR, сиг нальні шля хи.
Д. О. Гуд ко ва, А. Г. Па на сюк, И. А. Не ма за ный, В. В. Фи ло нен ко
PI3K/mTOR-за ви си мый сиг наль ный путь как воз мож ный
ре гу ля тор об ра зо ва ния про цес сив ных те лец
Ре зю ме
Цель. Иссле до вать роль PI3K/mTOR-за ви си мо го сиг наль но го пу-
ти в ре гу ля ции сбор ки про цес сив ных те лец. Ме то ды. Исполь зо -
ва ны ме то ды им му но ци то хи мии и им му ноб лот тин га. Ре зуль-
таты. По ка за но, что об ра бот ка кле ток спе ци фи чес ки ми ин ги би-
то ра ми PI3K/mTOR-сиг наль но го пути при во дит к из ме не ни ям в
ко ли чес тве и раз ме рах про цес сив ных те лец в клет ке, а так же
про те а сом ной дег ра да ции основ но го бел ка про цес сив ных те лец
RCD-8. Вы во ды. Мы пред по ло жи ли, что PI3K/mTOR-сиг наль ный
путь ре гу ли ру ет ди на ми ку об ра зо ва ния про цес сив ных те лец, обе-
спе чи вая ста биль ность скеф фол дно го бел ка про цес сив ных те лец
RCD-8, и, как сле дствие, нор ма ли зу ет ме та бо лизм мРНК
Клю че вые сло ва: про цес сив ные тель ца, им му но ци то хи мия,
дег ра да ция мРНК, mTOR, сиг наль ные пути.
REFERENCES
1. Averous J., Proud C. G. When translation meets transformation:
the mTOR story // Oncogene.–2006.–25, N 48.–P. 6423–6435.
2. Sun S. Y., Rosenberg L. M., Wang X., Zhou Z., Yue P., Fu H.,
Khuri F. R. Activation of Akt and eIF4E survival pathways by
rapamycin-mediated mammalian target of rapamycin inhibition
// Cancer Res.–2005.–65, N 16.–P. 7052–7058.
3. Liu L., Li F., Cardelli J. A., Martin K. A., Blenis J., Huang S. Ra-
pamycin inhibits cell motility by suppression of mTOR-me-
diated S6K1 and 4E-BP1 pathways // Oncogene.–2006.–25,
N 53.–P. 7029–7040.
4. Kedersha N., Stoecklin G., Ayodele M., Yacono P., Lykke-An-
dersen J., Fritzler M. J., Scheuner D., Kaufman R. J., Golan D.
E., Anderson P. Stress granules and processing bodies are dy-
namically linked sites of mRNP remodeling // J. Cell Biol.–
2005.–169, N 6.–P. 871–884.
5. Buchan J. R., Parker R. Eukaryotic stress granules: the ins and
outs of translation // Mol. Cell.–2009.–36, N 6.–P. 932–941.
6. Erickson S. L., Lykke-Andersen J. Cytoplasmic mRNP granules
at a glance // J. Cell Sci.–2011.–124, Pt 3.–P. 293–297.
7. Stinton L. M., Eystathioy T., Selak S., Chan E. K., Fritzler M. J.
Autoantibodies to protein transport and messenger RNA pro-
cessing pathways: endosomes, lysosomes, Golgi complex, pro-
teasomes, assemblyosomes, exosomes, and GW bodies // Clin.
Immunol.–2004.–110, N 1.–P. 30–44.
8. Parker R., Sheth U. P bodies and the control of mRNA transla-
tion and degradation // Mol. Cell.–2007.–25, N 5.–P. 635–646.
9. Garcia-Lozano J. R., Gonzalez-Escribano M. F., Wichmann I.,
Nunez-Roldan A. Cytoplasmic detection of a novel protein con-
taining a nuclear localization sequence by human autoanti-
bodies // Clin. Exp. Immunol.–1997.–107, N 3.–P. 501–506.
10. Yu J. H., Yang W. H., Gulick T., Bloch K. D., Bloch D. B. Ge-1 is
a central component of the mammalian cytoplasmic mRNA
processing body // RNA.–2005.–11, N 12.–P. 1795–1802.
11. Gudkova D. O., Panasyuk G. G., Nemazanyy I. O., Filonenko V.
V. Novel antibodies against RCD-8 as a tool to study processing
bodies // Biopolym. Cell.–2010.–26, N 6.–Р. 512–516.
12. Kedersha N., Anderson P. Mammalian stress granules and pro-
cessing bodies // Methods Enzymol.–2007.–431.–P. 61–81.
13. Balynska O. V., Baklaushev V. P., Areshkov P. O., Avdieiev S.
S., Boyko O. I., Chekhonin V. P., Kavsan V. M. Characterization
of new cell line stably expressing CHI3L1 oncogene // Biopo-
lym. Cell.–2011.–27, N 4.–Р. 285–290.
14. Yang Q., Inoki K., Kim E., Guan K. L. TSC1/TSC2 and Rheb ha-
ve different effects on TORC1 and TORC2 activity // Proc. Natl
Acad. Sci. USA.–2006.–103, N 18.–P. 6811–6816.
UDC 576.311.348
Received 16.06.11
372
GUDKOVA D. O. ET AL.
Control 5 15 30 60 min 2 3 6 12 18 24 h
RCD-8→
8-actin→
A B+ DMSO + MG132
RCD-8→
8-actin→
Control 15 30 15 30 min
Fig. 3. 2-Deoxyglucose (2-DG) treatment affects endogenous level of RCD-8 protein: A – HEK293 cells were pre-treated with MG132 or DMSO
for 6 h, then 2-DG was added for indicated time, after cells were lyzed in Laemmli buffer and total protein cell lysates were analyzed in
immunoblot with indicated antibodies; B – estimated period of RCD-8 half-life is more than 24 h. HEK293 cells were treated with cycloheximide
for indicated time points. Total protein lysates were prepared and analyzed as described for the above experiment
|