Ukr.Biochem.J. 2013; Volume 85, Issue 6, Nov-Dec, pp. 46-52


Identification of Tudor domain containing 7 protein as a novel partner and a substrate for ribosomal protein S6 kinaseS – S6K1 and S6K2

O. Skorokhod1,3, G. Panasyuk1, I. Nemazanyy1, I. Gout1,2, V. Filonenko1,3

1Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kyiv;
2University College London, United Kingdom;
3The State Key Laboratory of Molecular and Cellular Biology, Kyiv, Ukraine;

Ribosomal protein S6 kinases (S6Ks) are principal regulators of cell size, growth and metabolism. Signaling via the PI3K/mTOR pathway mediates the activation of S6Ks in response to various mitogenic stimuli, nutrients and stresses. To date, the regulation and cellular functions of S6Ks are not fully understood. Our aim was to investigate and characterize the interaction of S6Ks with the novel binding partner of S6Ks, Tudor domain containing 7 protein (TDRD7), which is a scaffold protein detected in complexes involved in the regulation of cytoskeleton dynamics, mRNA transport and translation, non-coding piRNAs processing and transposons silen­cing. This interaction was initially detected in the yeast two-hybrid screening of HeLa cDNA library and further confirmed by pull-down and co-immunoprecipitation assays. In addition we demonstrated that TDRD7 can form a complex with other isoform of S6K – S6K2. Notably, both isoforms of S6K were found to phosphorylate TDRD7 in vitro at multiple phosphorylation sites. Altogether, these findings demonstrate that TDRD7 is a novel substrate of S6Ks, suggesting the involvement of S6K signaling in the regulation of TDRD7 cellular functions.

Keywords: , , , ,


  1. Grove JR, Banerjee P, Balasubramanyam A, Coffer PJ, Price DJ, Avruch J, Woodgett JR. Cloning and expression of two human p70 S6 kinase polypeptides differing only at their amino termini. Mol Cell Biol. 1991 Nov;11(11):5541-50. PubMedPubMedCentralCrossRef
  2. Saitoh M, ten Dijke P, Miyazono K, Ichijo H. Cloning and characterization of p70(S6K beta) defines a novel family of p70 S6 kinases. Biochem Biophys Res Commun. 1998 Dec 18;253(2):470-6. PubMed, CrossRef
  3. Jenö P, Ballou LM, Novak-Hofer I, Thomas G. Identification and characterization of a mitogen-activated S6 kinase. Proc Natl Acad Sci USA. 1988 Jan;85(2):406-10. PubMed, PubMedCentral, CrossRef
  4. Gout I, Minami T, Hara K, Tsujishita Y, Filonenko V, Waterfield MD, Yonezawa K. Molecular cloning and characterization of a novel p70 S6 kinase, p70 S6 kinase beta containing a proline-rich region. J Biol Chem. 1998 Nov 13;273(46):30061-4. PubMed, CrossRef
  5. Koh H, Jee K, Lee B, Kim J, Kim D, Yun YH, Kim JW, Choi HS, Chung J. Cloning and characterization of a nuclear S6 kinase, S6 kinase-related kinase (SRK); a novel nuclear target of Akt. Oncogene. 1999 Sep 9;18(36):5115-9. PubMed, CrossRef
  6. Pende M, Um SH, Mieulet V, Sticker M, Goss VL, Mestan J, Mueller M, Fumagalli S, Kozma SC, Thomas G. S6K1(-/-)/S6K2(-/-) mice exhibit perinatal lethality and rapamycin-sensitive 5′-terminal oligopyrimidine mRNA translation and reveal a mitogen-activated protein kinase-dependent S6 kinase pathway. Mol Cell Biol. 2004 Apr;24(8):3112-24. PubMed, PubMedCentral, CrossRef
  7. Shima H, Pende M, Chen Y, Fumagalli S, Thomas G, Kozma SC. Disruption of the p70(s6k)/p85(s6k) gene reveals a small mouse phenotype and a new functional S6 kinase. EMBO J. 1998 Nov 16;17(22):6649-59. PubMed, PubMedCentral, CrossRef
  8. Andres JL, Johansen JW, Maller JL. Identification of protein phosphatases 1 and 2B as ribosomal protein S6 phosphatases in vitro and in vivo. J Biol Chem. 1987 Oct 25;262(30):14389-93. PubMed
  9. Avruch J, Belham C, Weng Q, Hara K, Yonezawa K. The p70 S6 kinase integrates nutrient and growth signals to control translational capacity. Prog Mol Subcell Biol. 2001;26:115-54. Review. PubMedCrossRef
  10. Ruvinsky I, Meyuhas O. Ribosomal protein S6 phosphorylation: from protein synthesis to cell size. Trends Biochem Sci. 2006 Jun;31(6):342-8. Review. PubMed, CrossRef
  11. Giraud J, Leshan R, Lee YH, White MF. Nutrient-dependent and insulin-stimulated phosphorylation of insulin receptor substrate-1 on serine 302 correlates with increased insulin signaling. J Biol Chem. 2004 Jan 30;279(5):3447-54. PubMed, CrossRef
  12. Fenton TR, Gout IT. Functions and regulation of the 70kDa ribosomal S6 kinases. Int J Biochem Cell Biol. 2011 Jan;43(1):47-59. Review. PubMed, CrossRef
  13. Thomas G. The S6 kinase signaling pathway in the control of development and growth. Biol Res. 2002;35(2):305-13. Review. PubMed, CrossRef
  14. Valovka T, Verdier F, Cramer R, Zhyvoloup A, Fenton T, Rebholz H, Wang ML, Gzhegotsky M, Lutsyk A, Matsuka G, Filonenko V, Wang L, Proud CG, Parker PJ, Gout IT. Protein kinase C phosphorylates ribosomal protein S6 kinase betaII and regulates its subcellular localization. Mol Cell Biol. 2003 Feb;23(3):852-63. PubMed, PubMedCentral, CrossRef
  15. Hirose T, Kawabuchi M, Tamaru T, Okumura N, Nagai K, Okada M. Identification of tudor repeat associator with PCTAIRE 2 (Trap). A novel protein that interacts with the N-terminal domain of PCTAIRE 2 in rat brain. Eur J Biochem. 2000 Apr;267(7):2113-21. PubMed, CrossRef
  16. Yamochi T, Nishimoto I, Okuda T, Matsuoka M. ik3-1/Cables is associated with Trap and Pctaire2. Biochem Biophys Res Commun. 2001 Sep 7;286(5):1045-50. PubMedCrossRef
  17. Chuma S, Hosokawa M, Kitamura K, Kasai S, Fujioka M, Hiyoshi M, Takamune K, Noce T, Nakatsuji N. Tdrd1/Mtr-1, a tudor-related gene, is essential for male germ-cell differentiation and nuage/germinal granule formation in mice. Proc Natl Acad Sci USA. 2006 Oct 24;103(43):15894-9. PubMed, PubMedCentral, CrossRef
  18. Hosokawa M, Shoji M, Kitamura K, Tanaka T, Noce T, Chuma S, Nakatsuji N. Tudor-related proteins TDRD1/MTR-1, TDRD6 and TDRD7/TRAP: domain composition, intracellular localization, and function in male germ cells in mice. Dev Biol. 2007 Jan 1;301(1):38-52. PubMed, CrossRef
  19. Kotaja N, Bhattacharyya SN, Jaskiewicz L, Kimmins S, Parvinen M, Filipowicz W, Sassone-Corsi P. The chromatoid body of male germ cells: similarity with processing bodies and presence of Dicer and microRNA pathway components. Proc Natl Acad Sci USA. 2006 Feb 21;103(8):2647-52. PubMed, PubMedCentral, CrossRef
  20. Conte N, Delaval B, Ginestier C, Ferrand A, Isnardon D, Larroque C, Prigent C, Séraphin B, Jacquemier J, Birnbaum D. TACC1-chTOG-Aurora A protein complex in breast cancer. Oncogene. 2003 Nov 6;22(50):8102-16. PubMed, CrossRef
  21. Callebaut I, Mornon JP. LOTUS, a new domain associated with small RNA pathways in the germline. Bioinformatics. 2010 May 1;26(9):1140-4. PubMed, CrossRef
  22. Selenko P, Sprangers R, Stier G, Bühler D, Fischer U, Sattler M. SMN tudor domain structure and its interaction with the Sm proteins. Nat Struct Biol. 2001 Jan;8(1):27-31.
  23. Amikura R, Hanyu K, Kashikawa M, Kobayashi S. Tudor protein is essential for the localization of mitochondrial RNAs in polar granules of Drosophila embryos. Mech Dev. 2001 Sep;107(1-2):97-104. PubMed, CrossRef
  24. Huyen Y, Zgheib O, Ditullio RA Jr, Gorgoulis VG, Zacharatos P, Petty TJ, Sheston EA, Mellert HS, Stavridi ES, Halazonetis TD. Methylated lysine 79 of histone H3 targets 53BP1 to DNA double-strand breaks. Nature. 2004 Nov 18;432(7015):406-11. PubMed, CrossRef
  25. Jin J, Xie X, Chen C, Park JG, Stark C, James DA, Olhovsky M, Linding R, Mao Y, Pawson T. Eukaryotic protein domains as functional units of cellular evolution. Sci Signal. 2009 Nov 24;2(98):ra76. PubMed, CrossRef
  26. Côté J, Richard S. Tudor domains bind symmetrical dimethylated arginines. J Biol Chem. 2005 Aug 5;280(31):28476-83. PubMed, CrossRef
  27. Thomson T, Lasko P. Tudor and its domains: germ cell formation from a Tudor perspective. Cell Res. 2005 Apr;15(4):281-91. Review. PubMed, CrossRef
  28. Skorokhod O, Nemazanyy I, Breus O, Filonenko V, Panasyuk G. Generation and characterization of monoclonal antibodies to TDRD7 protein. Hybridoma (Larchmt). 2008 Jun;27(3):211-6. PubMed, CrossRef
  29. Val’ovka TI, Filonenko VV, Velyky MM, Drobot LB, Voterfill M, Matsuka HKh, Hut IT. Features of fibronectin-dependent activation of ribosomal protein S6 kinase (S6K1 and S6K2). Ukr Biokhim Zhurn. 2000 May-Jun;72(3):31-7. Ukrainian. PubMed
  30. Savinska L, Skorokhod O, Klipa O, Gout I, Filonenko V. Development of monoclonal antibodies specific to ribosomal protein S6 kinase 2. Hybridoma (Larchmt). 2012 Aug;31(4):289-94. PubMed, PubMedCentral, CrossRef
  31. Panasyuk G, Nemazanyy I, Zhyvoloup A, Bretner M, Litchfield DW, Filonenko V, Gout IT. Nuclear export of S6K1 II is regulated by protein kinase CK2 phosphorylation at Ser-17. J Biol Chem. 2006 Oct 20;281(42):31188-201. PubMed, CrossRef
  32. PhosphoNET: Human phosphosite knowledge base (PhosphoNET ID IPI00478741).
  33. Chevalier D, Allen BG. Purification of myelin basic protein from bovine brain. Protein Expr Purif. 2000 Mar;18(2):229-34. PubMed, CrossRef
  34. Panasyuk GG, Nemzanyy IO, Zhyvoloup AM, Filonenko VV, Gout IT. The beta subunit of casein kinase 2 as a novel binding partner of the ribosomal protein S6 kinase 1. Biopolym Cell. 2005; 21(5):407-412.  CrossRef
  35. HPRD: Human protein reference database (HPRD ID 11626).
  36. PPSP: Prediction of PK-Specific Phosphorylation Site.
  37. PSP: PhosphoSitePlus (PhosphoSitePlus ID Q8NHU6).

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