vavilovВавиловский журнал генетики и селекцииVavilov Journal of Genetics and Breeding2500-3259Institute of Cytology and Genetics of Siberian Branch of the RAS10.18699/VJ20.39-ovavilov-2429Research ArticleМОЛЕКУЛЯРНАЯ И КЛЕТОЧНАЯ БИОЛОГИЯMOLECULAR AND CELL BIOLOGYКонструирование постоянно активной киназы 2 рибосомного белка s6 из Arabidopsis thaliana (AtrPs6k2) и тестирование ее активности in vitroConstructing the constitutively active ribosomal protein S6 kinase 2 from Arabidopsis thaliana (AtRPS6K2) and testing its activity in vitrohttps://orcid.org/0000-0002-9646-033XЖигайловА. В.ZhigailovA. V.

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andrzhig@gmail.comhttps://orcid.org/0000-0002-7819-6475СтанбековаГ. Э.StanbekovaG. E.

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https://orcid.org/0000-0002-2116-7323БейсеновД. К.BeisenovD. K.

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https://orcid.org/0000-0002-1597-7207НизкородоваА. С.NizkorodovaA. S.

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https://orcid.org/0000-0002-2806-3009ПолимбетоваН. С.PolimbetovaN. S.

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https://orcid.org/0000-0002-5204-4377ИскаковБ. К.IskakovB. K.

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Институт молекулярной биологии и биохимии им. М.А. АйтхожинаКазахстанM.A. Aitkhozhin Institute of Molecular Biology and BiochemistryKazakhstanИнститут молекулярной биологии и биохимии им. М.А. Айтхожина; Институт биологии и биотехнологии растенийКазахстанM.A. Aitkhozhin Institute of Molecular Biology and Biochemistry; Institute of Plant Biology and BiotechnologyKazakhstan202028052020243233238Copyright © Жигайлов А.В., Станбекова Г.Э., Бейсенов Д.К., Низкородова А.С., Полимбетова Н.С., Искаков Б.К., 20202020Жигайлов А.В., Станбекова Г.Э., Бейсенов Д.К., Низкородова А.С., Полимбетова Н.С., Искаков Б.К.Zhigailov A.V., Stanbekova G.E., Beisenov D.K., Nizkorodova A.S., Polimbetova N.S., Iskakov B.K.This work is licensed under a Creative Commons Attribution 4.0 License.https://vavilov.elpub.ru/jour/article/view/2429

Рибосомный белок S6 (RPS6) – единственный белок 40S субчастиц эукариотических рибосом, способный фосфорилироваться. Рибосомы с фосфорилированным RPS6 могут селективно транслировать 5’TOP (5’-terminal oligopyrimidine)-содержащие мРНК, которые кодируют большинство белков трансляционного аппарата клеток. Исследование трансляционного контроля 5’TOP-мРНК, которые преимущественно транслируются, когда RPS6 фосфорилирован, и перестают транслироваться, когда RPS6 дефосфорилируется, является особенно важным. В клетках Arabidopsis thaliana AtRPS6 фосфорилируется киназой AtRPS6K2, для активации которой, в свою очередь, требуется ее фосфорилирование киназами верхнего уровня (AtPDK1 – по серину (S) 296, AtTOR – по треонину (T) 455 и также по S437). Мы клонировали кДНК-ген AtRPS6K2 и провели его мутагенез in vitro, заменив кодоны S296, S437 и T455 на триплеты, кодирующие фосфомиметическую глутаминовую кислоту (E). После экспрессии обеих кДНК в клетках Escherichia coli были выделены два рекомбинантных белка: немутированный вариант – AtRPS6K2 и мутированный вариант – AtRPS6K2(S296E, S437E, T455E), предположительно, находящийся в стабильно активном состоянии. Активность этих киназ была протестирована in vitro. Показано, что обе киназы способны фосфорилировать рибосомный белок TaRPS6 в составе 40S рибосомных субчастиц, выделенных из зародышей пшеницы (Triticum aestivum L.), но активность нативной киназы была ниже в сравнении с ее фосфомиметической формой. Способность рекомбинантной нативной киназы фосфорилировать TaRPS6 может быть объяснена ее фосфорилированием бактериальными киназами на стадиях экспрессии и выделения. Фосфомиметически мутированная киназа AtRPS6K2(S296E, S437E, T455E) может служить удобным средством для исследования избирательной трансляции 5’TOP-содержащих мРНК в бесклеточной системе из зародышей пшеницы, в которой большинство 40S рибосомных субчастиц имеет фосфорилированную форму TaRPS6. Кроме того, такой подход может найти биотехнологическое применение для создания генетически модифицированных растений с увеличенной биомассой и продуктивностью за счет стимуляции роста и деления клеток.

Ribosomal protein S6 (RPS6) is the only phosphorylatable protein of the eukaryotic 40S ribosomal subunit. Ribosomes with phosphorylated RPS6 can selectively translate 5’TOP-(5’-terminal oligopyrimidine)-containing mRNAs that encode most proteins of the translation apparatus. The study of translational control of 5’TOP-mRNAs, which are preferentially translated when RPS6 is phosphorylated and cease to be translated when RPS6 is de-phosphorylated, is particularly important. In Arabidopsis thaliana, AtRPS6 is phosphorylated by kinase AtRPS6K2, which should in turn be phosphorylated by upper level kinases (AtPDK1 – at serine (S) 296, AtTOR – at threonine (T) 455 and S437) for full activation. We have cloned AtRPS6K2 cDNA gene and carried out in vitro mutagenesis replacing codons encoding S296, S437 and T455 by triplets of phosphomimetic glutamic acid (E). After the expression of both natural and mutated cDNAs in Escherichia coli cells, two recombinant proteins were isolated: native AtRPS6K2 and presumably constitutively active AtRPS6K2(S296E, S437E, T455E). The activity of these variants was tested in vitro. Both kinases could phosphorylate wheat (Triticum aestivum L.) TaRPS6 as part of 40S ribosomal subunits isolated from wheat embryos, though the non-mutated variant had less activity than phosphomimetic one. The ability of recombinant non-mutated kinase to phosphorylate TaRPS6 can be explained by its phosphorylation by bacterial kinases during the expression and isolation steps. The phosphomimetically mutated AtRPS6K2(S296E, S437E, T455E) can serve as a tool to investigate preferential translation of 5’TOP-mRNAs in wheat germ cell-free system, in which most of 40S ribosomal subunits have phosphorylated TaRPS6. Besides, such an approach has a biotechnological application in producing genetically modified plants with increased biomass and productivity through stimulation of cell growth and division.

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The authors declare that there are no conflicts of interest present.