Abstract
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.
Highlights
Growth and division of cells depending on the availability of nutrients, energy resources, as well as responding to internal and external stimuli are coordinated by signaling system based on a multilevel cascade of serine-threonine protein kinases
A total RNA preparation was isolated from A. thaliana, and reverse transcription was performed using ‘AtS6K2-rev-3UTR’ primer, complementary to 3 ́UTR of AtRPS6K2 mRNA, but not AtRPS6K1 mRNA, allowing to discriminate between молекулярная и клеточная биология / molecular and cell biology 235
Tracks: М – protein markers; N – negative control; L – lysate of bacteria synthesizing recombinant proteins; LF – proteins of wash-through fractions after loading of bacterial lysates onto Ni-NTA agarose; W – proteins eluted from Ni-NTA agarose with His-buffer containing 20 мМ of imidazole; E1, E2, E3 – proteins eluted from Ni-NTA agarose with His-buffer containing 250 мМ of imidazole
Summary
Growth and division of cells depending on the availability of nutrients, energy resources, as well as responding to internal and external stimuli are coordinated by signaling system based on a multilevel cascade of serine-threonine protein kinases. These kinases transmit signals from internal and external events to the protein synthesis apparatus, causing inhibition or enhancement of protein synthesis (Turck et al, 2004; Wol ters, Jürgens, 2009; Henriques et al, 2014; Rexin et al, 2015; Roustan et al, 2016). The fully activated RPS6K in turn phosphorylates the S6 ribosomal protein (RPS6) (Williams et al, 2003)
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