Biopolymers and cell. 2002; 18 (2): 102 - 106

Use of yeast two-hybrid system in search of S6K1 and S6K2 binding partners

O. M. Zhyvoloup, I. O. Nemazanyy, N. V. Pobigailo, G. G. Panasyuk, S. S. Palchevskyy, O. P. Kuharenko, L. O. Savinska, G. V. Ovcharenko, M. I. Vudmaska, I. T. Gout, G. Kh. Matsuka, V. V. Filonenko

The kinase of the ribosomal protein S6 (S6K) is involved in the signalling pathways which regulate the cell proliferation, dif¬ferentiation and growth. The family of S6K consists of two forms (S6KJ and 2), which have cytoplasmic and nuclear splicing variants. The activity of S6K is regulated by phosphorylation/de-phosphorylation 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 S6KJ 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.