Constructing the constitutively active ribosomal protein S6 kinase 2 from <i>Arabidopsis thaliana</i> (AtRPS6K2) and testing its activity <i>in vitro</i>
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 translation...
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| Format: | Article |
| Language: | English |
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Siberian Branch of the Russian Academy of Sciences, Federal Research Center Institute of Cytology and Genetics, The Vavilov Society of Geneticists and Breeders
2020-05-01
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| Series: | Вавиловский журнал генетики и селекции |
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| Online Access: | https://vavilov.elpub.ru/jour/article/view/2429 |
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| author | A. V. Zhigailov G. E. Stanbekova D. K. Beisenov A. S. Nizkorodova N. S. Polimbetova B. K. Iskakov |
| author_facet | A. V. Zhigailov G. E. Stanbekova D. K. Beisenov A. S. Nizkorodova N. S. Polimbetova B. K. Iskakov |
| author_sort | A. V. Zhigailov |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-07T16:05:32Z |
| format | Article |
| id | doaj.art-dce77cc4c9e249759f05a9885f2a1669 |
| institution | Directory Open Access Journal |
| issn | 2500-3259 |
| language | English |
| last_indexed | 2024-04-24T11:07:52Z |
| publishDate | 2020-05-01 |
| publisher | Siberian Branch of the Russian Academy of Sciences, Federal Research Center Institute of Cytology and Genetics, The Vavilov Society of Geneticists and Breeders |
| record_format | Article |
| series | Вавиловский журнал генетики и селекции |
| spelling | doaj.art-dce77cc4c9e249759f05a9885f2a16692024-04-11T15:31:02ZengSiberian Branch of the Russian Academy of Sciences, Federal Research Center Institute of Cytology and Genetics, The Vavilov Society of Geneticists and BreedersВавиловский журнал генетики и селекции2500-32592020-05-0124323323810.18699/VJ20.39-o1014Constructing the constitutively active ribosomal protein S6 kinase 2 from <i>Arabidopsis thaliana</i> (AtRPS6K2) and testing its activity <i>in vitro</i>A. V. Zhigailov0G. E. Stanbekova1D. K. Beisenov2A. S. Nizkorodova3N. S. Polimbetova4B. K. Iskakov5M.A. Aitkhozhin Institute of Molecular Biology and BiochemistryM.A. Aitkhozhin Institute of Molecular Biology and BiochemistryM.A. Aitkhozhin Institute of Molecular Biology and Biochemistry; Institute of Plant Biology and BiotechnologyM.A. Aitkhozhin Institute of Molecular Biology and BiochemistryM.A. Aitkhozhin Institute of Molecular Biology and BiochemistryM.A. Aitkhozhin Institute of Molecular Biology and Biochemistry; Institute of Plant Biology and BiotechnologyRibosomal 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.https://vavilov.elpub.ru/jour/article/view/2429wheat (triticum aestivum)s6 protein (tarps6) of 40s ribosomal subunitsarabidopsis thalianarps6-kinase 2 (atrps6k2)phosphomimetic mutationta rps6 phosphorylation |
| spellingShingle | A. V. Zhigailov G. E. Stanbekova D. K. Beisenov A. S. Nizkorodova N. S. Polimbetova B. K. Iskakov Constructing the constitutively active ribosomal protein S6 kinase 2 from <i>Arabidopsis thaliana</i> (AtRPS6K2) and testing its activity <i>in vitro</i> Вавиловский журнал генетики и селекции wheat (triticum aestivum) s6 protein (tarps6) of 40s ribosomal subunits arabidopsis thaliana rps6-kinase 2 (atrps6k2) phosphomimetic mutation ta rps6 phosphorylation |
| title | Constructing the constitutively active ribosomal protein S6 kinase 2 from <i>Arabidopsis thaliana</i> (AtRPS6K2) and testing its activity <i>in vitro</i> |
| title_full | Constructing the constitutively active ribosomal protein S6 kinase 2 from <i>Arabidopsis thaliana</i> (AtRPS6K2) and testing its activity <i>in vitro</i> |
| title_fullStr | Constructing the constitutively active ribosomal protein S6 kinase 2 from <i>Arabidopsis thaliana</i> (AtRPS6K2) and testing its activity <i>in vitro</i> |
| title_full_unstemmed | Constructing the constitutively active ribosomal protein S6 kinase 2 from <i>Arabidopsis thaliana</i> (AtRPS6K2) and testing its activity <i>in vitro</i> |
| title_short | Constructing the constitutively active ribosomal protein S6 kinase 2 from <i>Arabidopsis thaliana</i> (AtRPS6K2) and testing its activity <i>in vitro</i> |
| title_sort | constructing the constitutively active ribosomal protein s6 kinase 2 from i arabidopsis thaliana i atrps6k2 and testing its activity i in vitro i |
| topic | wheat (triticum aestivum) s6 protein (tarps6) of 40s ribosomal subunits arabidopsis thaliana rps6-kinase 2 (atrps6k2) phosphomimetic mutation ta rps6 phosphorylation |
| url | https://vavilov.elpub.ru/jour/article/view/2429 |
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