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...

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Datum:	2011
Hauptverfasser:	Gudkova, D.O., Panasyuk, G.G., Nemazanyy, I.O., Filonenko, V.V.
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Sprache:	English
Veröffentlicht:	Інститут молекулярної біології і генетики НАН України 2011
Schriftenreihe:	Вiopolymers and Cell
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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
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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. 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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

PI3K/mTOR-dependent signaling pathway as a possible regulator of processing body assembly

Institution

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