Advertisement

Russian Journal of Bioorganic Chemistry

, Volume 45, Issue 6, pp 758–765 | Cite as

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

  • A. E. Vasilyeva
  • D. D. Yanshina
  • G. G. Karpova
  • A. A. MalyginEmail author
Article

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.

Keywords:

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

Notes

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.

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.

REFERENCES

  1. 1.
    Khatter, H., Myasnikov, A.G., Natchiar, S.K., and Klaholz, B.P., Nature, 2015, vol. 520, pp. 640–645.CrossRefGoogle Scholar
  2. 2.
    Graifer, D. and Karpova, G., Biochimie, 2015, vol. 109, pp. 1–17.CrossRefGoogle Scholar
  3. 3.
    Peña, C., Hurt, E., and Panse, V.G., Nat. Struct. Mol. Biol., 2017, vol. 24, pp. 689–699.CrossRefGoogle Scholar
  4. 4.
    Vladimirov, S.N., Ivanov, A.V., Karpova, G.G., Musolyamov, A.K., Egorov, T.A., Thiede, B., Wittmann-Liebold, B., et al., Eur. J. Biochem., 1996, vol. 239, pp. 144–149.CrossRefGoogle Scholar
  5. 5.
    Odintsova, T.I., Muller, E.C., Ivanov, A.V., Egorov, T.A., Bienert, R., Vladimirov, S.N., Kostka, S., et al., J. Protein Chem., 2003, vol. 22, pp. 249–258.CrossRefGoogle Scholar
  6. 6.
    Hornbeck, P.V., Kornhauser, J.M., Latham, V., Murray, B., Nandhikonda, V., Nord, A., Skrzypek, E., et al., Nucleic Acids Res., 2019, vol. 47, pp. D433–D441.Google Scholar
  7. 7.
    Warner, J.R. and McIntosh, K.B., Mol. Cell, 2009, vol. 23, pp. 3–11.CrossRefGoogle Scholar
  8. 8.
    Wang, W., Nag, S., Zhang, X., Wang, M.H., Wang, H., Zhou, J., and Zhang, R., Med. Res. Rev., 2015, vol. 35, pp. 225–285.CrossRefGoogle Scholar
  9. 9.
    Ban, N., Beckmann, R., Cate, J.H., Dinman, J.D., Dragon, F., Ellis, S.R., Lafontaine, D.L., et al., Curr. Opin. Struct. Biol., 2014, vol. 24, pp. 165–169.CrossRefGoogle Scholar
  10. 10.
    Martin, I., Kim, J.W., Lee, B.D., Kang, H.C., Xu, J.C., Jia, H., Stankowski, J., et al., Cell, 2014, vol. 157, pp. 472–485.CrossRefGoogle Scholar
  11. 11.
    Ruvinsky, I. and Meyuhas, O., Trends Biochem. Sci., 2006, vol. 31, pp. 342–348.CrossRefGoogle Scholar
  12. 12.
    Sharifulin, D.E., Grosheva, A.S., Bartuli, Y.S., Malygin, A.A., Meschaninova, M.I., Ven’yaminova, A.G., Stahl, J., et al., Biochim. Biophys. Acta, 2015, vol. 1849, pp. 930–939.CrossRefGoogle Scholar
  13. 13.
    Kim, T.S., Kim, H.D., and Kim, J., Biochim. Biophys. Acta, 2009, vol. 1793, pp. 395–405.CrossRefGoogle Scholar
  14. 14.
    Kim, T.S., Kim, H.D., Shin, H.S., and Kim, J., J. Biol. Chem., 2009, vol. 284, pp. 21 201–21 208.CrossRefGoogle Scholar
  15. 15.
    Lee, S.B., Kwon, I.S., Park, J., Lee, K.H., Ahn, Y., Lee, C., Kim, J., et al., J. Biol. Chem., 2010, vol. 285, pp. 29 457–29 468.CrossRefGoogle Scholar
  16. 16.
    Kim, H.D., Lee, J.Y., and Kim, J., Biochem. Biophys. Res. Commun., 2005, vol. 333, pp. 110–115.CrossRefGoogle Scholar
  17. 17.
    Jang, C.Y., Kim, H.D., and Kim, J., Biochem. Biophys. Res. Commun., 2012, vol. 421, pp. 474–478.CrossRefGoogle Scholar
  18. 18.
    Arif, A., Yao, P., Terenzi, F., Jia, J., Ray, P.S., and Fox, P.L., Wiley Interdiscip. Rev. RNA, 2018, vol. 9, p. e1441.Google Scholar
  19. 19.
    Matragkou, Ch., Papachristou, H., Karetsou, Z., Papadopoulos, G., Papamarcaki, T., Vizirianakis, I.S., Tsiftsoglou, A.S., et al., J. Mol. Biol., 2009, vol. 392, pp. 1192–1204.CrossRefGoogle Scholar
  20. 20.
    Ivanov, A.V., Malygin, A.A., and Karpova, G.G., Mol. Biol. (Moscow), 2011, vol. 45, pp. 959–966.CrossRefGoogle Scholar
  21. 21.
    Ivanov, A.V., Malygin, A.A., and Karpova, G.G., Mol. Biol. (Moscow), 2013, vol. 47, pp. 140–148.CrossRefGoogle Scholar
  22. 22.
    Parakhnevich, N.M., Ivanov, A.V., Malygin, A.A, and Karpova, G.G., Mol. Biol. (Moscow), 2007, vol. 41, pp. 4–51.Google Scholar
  23. 23.
    Malygin, A.A., Parakhnevitch, N.M., Ivanov, A.V., Eperon, I.C., and Karpova, G.G., Nucleic Acids Res., 2007, vol. 35, pp. 6414–6423.CrossRefGoogle Scholar
  24. 24.
    O’Donohue, M.F., Choesmel, V., Faubladier, M., Fichant, G., and Gleizes, P.E., J. Cell Biol., 2010, vol. 190, pp. 853–866.CrossRefGoogle Scholar
  25. 25.
    Kubota, S., Copeland, T.D., and Pomerantz, R.J., Oncogene, 1999, vol. 18, pp. 1503–1514.CrossRefGoogle Scholar
  26. 26.
    Da, CostaL., Tchernia, G., Gascard, P., Lo, A., Meerpohl, J., Niemeyer, C., Chasis, J.A., et al., Blood, 2003, vol. 101, pp. 5039–5045.CrossRefGoogle Scholar
  27. 27.
    Malygin, A., Parakhnevitch, N., and Karpova, G., Biochim. Biophys. Acta, 2005, vol. 1747, pp. 93–97.CrossRefGoogle Scholar
  28. 28.
    Schmidt, C., Lipsius, E., and Kruppa, J., Mol. Biol. Cell, 1995, vol. 6, pp. 1875–1885.CrossRefGoogle Scholar
  29. 29.
    Parakhnevitch, N.M., Malygin, A.A., and Karpova, G.G., Biochemistry (Moscow), 2005, vol. 70, pp. 777–781.PubMedGoogle Scholar
  30. 30.
    Lam, Y.W., Lamond, A.I., Mann, M., and Andersen, J.S., Curr. Biol., 2007, vol. 17, pp. 749–760.CrossRefGoogle Scholar
  31. 31.
    Laemmli, U.K., Nature, 1970, vol. 227, pp. 680–685.CrossRefGoogle Scholar
  32. 32.
    Birnboim, H.C. and Doly, J., Nucleic Acids Res., 1979, vol. 7, pp. 1513–1523.CrossRefGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2019

Authors and Affiliations

  • A. E. Vasilyeva
    • 1
    • 2
  • D. D. Yanshina
    • 1
  • G. G. Karpova
    • 1
    • 2
  • A. A. Malygin
    • 1
    • 2
    Email author
  1. 1.Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of ScienceNovosibirskRussia
  2. 2.Novosibirsk State UniversityNovosibirskRussia

Personalised recommendations