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Функціональна взаємодія кінази S6 рибосомного білка S6K1 та протеїнкінази 2 / сторінка 13

Функціональна взаємодія кінази S6 рибосомного білка S6K1 та протеїнкінази 2
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It also allows yeast library propagation with a significantly higher colony density. The protocol was developed on the basis of several published techniques and requires fewer reagents when compared to other approaches. Using this protocol, we managed to achieve transformation efficiencies of 0.25–1x106/µg of library DNA or 2–8 million independent colonies/400 mL of the semi-solid agarose 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.
CK2 is one of the most conserved protein kinases in evolution. It is naturally found in tetrameric complexes consisting of two catalytic (alfa and/or alfaґ) subunits and two regulatory beta subunits. The kinase is constitutively active serine/threonine protein kinase and does not require second messengers, such as phospholipids, cyclic nucleotides, and calcium for full activation. CK2 is known to phosphorylate a plethora of substrates, found in various cellular compartments.
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. Furthermore, it was shown that endogenous S6K1 is in complex not only with CK2 subunit, but also with catalitic CK2б and CK2б’ subunits.
Analysis of CK2 and S6K1 precipitated complexes from HEK293 cells demonstrates that both enzymes are present in complex in active form.
Bioinformatic analysis of primary structure of S6K1 has shown the presence of several potential CK2 phosphorylation sites, were the Ser17 was the most preferential. 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. Mutational analysis of S17A mutant indicates that Ser17 is a major in vitro phosphorylation site for CK2. However, the presence of additional CK2 phosphorylation sites on S6K1 has not been exluded.
Furthermore, the functional importance of CK2/S6K1 interaction and phosphorylation of Ser17 has been revealed. It was shown for the first time that the phosphorylation-mimicking mutant of S6K1 (S17E) does not translocate to the nucleus in serum stimulated cells. Treatment of cells with the nuclear export inhibitor Leptomycin B indicated that the S17E mutant accumulates in the nucleus to the same extent as S6K1wt. These results indicate that nuclear import of the S17E mutant is not affected while the export is significantly enhanced. We also provide evidence that nuclear export of S6K1 is mediated by a CRM1-dependent mechanism. Additionaly, acumulation of endogenous S6K1 in the nucleus in time and douse dependent maner was demonstrated after HEK 293 cells were treated with direct inhibitor of CK2 – TBB.
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
Also, monoclonal antibodies (MAbs) against the CK2 subunit were produced and characterized. For this purpose bacterially expressed CK2-6His-GST recombinant protein has been used as an antigen. A total of 10 IgG-producing cell lines were selected and further tested for their reactivity with the CK2 subunit using ELISA, Western blots, immunoprecipitation, and immunostaining of formaldehyde-fixed paraffin-embedded tissue sections. In summary, the produced antibodies will be useful for researchers investigating signaling pathways involving CK2 kinase and their deregulation in human pathologies.
Key words: ribosomal S6 kinase (S6K1), yeast two hybrid system.

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Реферат на тему: Функціональна взаємодія кінази S6 рибосомного білка S6K1 та протеїнкінази 2

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