Biopolym. Cell. 2002; 18(2):102-109.
Use of yeast two-hybrid system in search of S6K1 and S6K2 binding partners
1Zhyvoloup O. M., 1Nemazanyy I. O., 1Pobigailo N. V., 1Panasyuk G. G., 1Palchevskyy S. S., 1Kuharenko O. P., 1Savinska L. O., 1Ovcharenko G. V., 1Vudmaska M. I., 1, 2Gout I. T., 1Matsuka G. Kh., 1Filonenko V. V.
  1. Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
  2. Ludwig Institute for Cancer Research
    91 Riding house str., London, UK, WIP8BT


The kinase of the ribosomal protein S6 (S6K) is involved in the signalling pathways which regulate the cell proliferation, differentiation and growth. The family of S6K consists of two forms (S6K1 and 2), which have cytoplasmic and nuclear splicing variants. The activity of S6K is regulated by phosphorylation/dephosphorylation events and specific protein-protein interactions. We have employed yeast two-hybrid system in search of novel S6K binding partners. Among six created «bait» constructs, only the full length S6K1 meets the requiments of the yeast two-hybrid screening. The large-scale screening of mouse embryo cDNA library allowed us to identify 26 positive clones and 13 of them were confirmed in mating analysis. The restriction and sequence analysis of these clones showed that all of them contain cDNAs of approximetly 4000 bp with the same restriction pattern and sequence at 5' and 3' ends. Comparison of the sequences obtained with the genomic and protein databases has shown that identified cDNA codes a fragment of novel protein. The primary structure of this protein and specificity of its interaction with S6K. are currently under investigation.


[1] Phizicky EM, Fields S. Protein-protein interactions: methods for detection and analysis. Microbiol Rev. 1995;59(1):94-123.
[2] Gyuris J, Golemis E, Chertkov H, Brent R. Cdi1, a human G1 and S phase protein phosphatase that associates with Cdk2. Cell. 1993;75(4):791-803.
[3] Chien CT, Bartel PL, Sternglanz R, Fields S. The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc Natl Acad Sci U S A. 1991;88(21):9578-82.
[4] Vojtek AB, Hollenberg SM, Cooper JA. Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell. 1993;74(1):205-14.
[5] Wang T, Donahoe PK, Zervos AS. Specific interaction of type I receptors of the TGF-beta family with the immunophilin FKBP-12. Science. 1994;265(5172):674-6.
[6] Shih HM, Goldman PS, DeMaggio AJ, Hollenberg SM, Goodman RH, Hoekstra MF. A positive genetic selection for disrupting protein-protein interactions: identification of CREB mutations that prevent association with the coactivator CBP. Proc Natl Acad Sci U S A. 1996;93(24):13896-901.
[7] Dufner A, Thomas G. Ribosomal S6 kinase signaling and the control of translation. Exp Cell Res. 1999;253(1):100-9.
[8] Lane HA, Fernandez A, Lamb NJ, Thomas G. p70s6k function is essential for G1 progression. Nature. 1993;363(6425):170-2.
[9] 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;273(46):30061-4.
[10] Chou MM, Blenis J. The 70 kDa S6 kinase complexes with and is activated by the Rho family G proteins Cdc42 and Rac1. Cell. 1996;85(4):573-83.
[11] Peterson RT, Desai BN, Hardwick JS, Schreiber SL. Protein phosphatase 2A interacts with the 70-kDa S6 kinase and is activated by inhibition of FKBP12-rapamycinassociated protein. Proc Natl Acad Sci U S A. 1999;96(8):4438-42.
[12] Akimoto K, Nakaya M, Yamanaka T, Tanaka J, Matsuda S, Weng QP, Avruch J, Ohno S. Atypical protein kinase Clambda binds and regulates p70 S6 kinase. Biochem J. 1998;335 (Pt 2):417-24.
[13] Miller JH. Experiments in molecular genetics. Cold Spring Harbor Laboratory, 1972, 466 p.
[14] Estojak J, Brent R, Golemis EA. Correlation of two-hybrid affinity data with in vitro measurements. Mol Cell Biol. 1995;15(10):5820-9.
[15] Berry DR. The Biology of Yeasts. Hodder & Stoughton Educational. 1982; 64 p.