Panasyuk G. Functional interaction of Ribosomal S6 kinase and Protein Kinase 2

At first stage of the work, highty efficient protocol for the large-scale transformation of yeast in a semi-solid agarose medium that does not require plating has been developed. The protocol allows for the growth of transformed cells as separate colonies in the volume of semi-solid medium.Using activated form of S6K1 as a bait and HeLa cDNA library as a pool of pray clones eight novel binding partners of S6K1 were identified. Among them the interaction between S6K1 and regulatory subunit of casein kinase 2 has been discovered. The interaction between S6K1 and CK2 beta regulatory subunit was initially identified in a yeast two-hybrid screen and further confirmed in a pull-down assay and by co-immunoprecipitation of transiently expressed and endogenous proteins. Also, monoclonal antibodies (MAbs) against the CK2 subunit were produced and characterized. Furthermore, it was shown that endogenous S6K1 is in complex not only with CK2 subunit, but also with catalitic CK2a and CK2a' subunits.Further studies confirmed that both fullength S6K1 and S6K2 are phosphorylated by CK2 in conditions of in vitro kinase reaction. In addition, the localization of the CK2 phosphorylation site was narrowed down to Ser17 in S6K1. Taken together, this study establishes a functional link between S6K1 and CK2 signalling, which involves the regulation of S6K1 nuclear export by CK2-mediated phosphorylation of Ser 17. As a conclusion the model of nuclear-cytoplasmic CRM1-dependent shutling of S6K1 in response to various mitogenic stimula has been proposed.