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Mutations Preventing the Phosphorylation of Human Ribosomal Protein uS15 at Y38 and S48 Reduce the Efficiency of its Transfer into the Nucleolus

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Abstract

The ribosomal protein uS15 is one of the key proteins forming the small (40S) ribosomal subunit structure around the central domain of 18S rRNA. According to a number of proteomic studies based on mass-spectrometry analysis, there are many phosphorylation sites in this protein. However, when the protein uS15 is contained in the 40S subunit, it does not carry such posttranslational modification. In this study, it was found that the cytoplasmic protein kinases of HEK293T cells are able to phosphorylate the human recombinant ribosomal protein uS15 with significantly greater efficiency than that of the nucleus protein kinases. The effect of the amino acid substitutions Y38A and S48A preventing the phosphorylation of the ribosomal protein uS15 at the corresponding sites, on the transport of the recombinant protein uS15 fused to the green fluorescent protein in the nucleolus was studied. It was shown that single mutations at the above sites have little effect on the transport of this protein, whereas double mutation reduces the efficiency of this process by more than a quarter. The findings suggest the importance of phosphorylation of the ribosomal protein uS15 by cytoplasmic protein kinases at several sites, including Y38A and S48A, for its efficient transfer into the nucleolus, where pre-ribosomal subunits are assembled.

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ACKNOWLEDGMENTS

The authors are grateful to I.A. Zaporozhchenko for conducting microscopic studies of cells producing chimeric proteins.

FUNDING

This work was financially supported by the Russian Foundation for Basic Research (grant no. 17-04-00528), as well as the Basic Budget Financing of the Institute of Chemical Biology and Fundamental Medicine of the Siberian Branch of the Russian Academy of Sciences (project АААА-А17-117020210022-4) and financing under the program 5-100 of the Ministry of Education and Science.

Author information

Correspondence to A. A. Malygin.

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COMPLIANCE WITH ETHICAL STANDARDS

The study was performed according to all ethical standards.

Conflict of Interests

The authors declare that they do not have a conflict of interest.

Additional information

Translated by P. Kuchina

Corresponding author: phone: +7 (383) 363-51-39; fax: +7 (383) 363-51-53; e-mail: malygin@niboch.nsc.ru.

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Vasilyeva, A.E., Yanshina, D.D., Karpova, G.G. et al. Mutations Preventing the Phosphorylation of Human Ribosomal Protein uS15 at Y38 and S48 Reduce the Efficiency of its Transfer into the Nucleolus. Russ J Bioorg Chem 45, 758–765 (2019). https://doi.org/10.1134/S1068162019060372

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Keywords:

  • human ribosomal protein uS15
  • phosphorylation
  • protein kinases
  • site-directed mutagenesis
  • intracellular transport
  • nucleolus