Проаналізовано експресію а-та β-ізоформ кінази S6 рибосомного білка в різних тканинах щура. Виявлено, що експресія ізоформ кінази має чіткий тканиноспецифічний характер, при якому загальний рівень експресії S6Ka є дещо вищим у порівнянні з S6Κβ. Встановлено, що найвищі рівні експресії для а-ізоформи S6 кінази спостерігаються в мозку, а для β-ізоформи-в гонадах. При порівнянні з результатами аналізу експресії мРНК обох форм S6 кінази виявлено кореляцію з експресією S6K на білковому рівні лише в деяких випадках, що свідчить про різну ефективність трансляції мРНК даного ферменту в залежності від виду тканини.
Ribosomal protein S6 kinase 1 (S6K1) is a well-known downstream effector of mTORC1 (mechanistic target of rapamycin complex 1) participating primarily in the regulationA number of downstream effects of mTORC1, including protein biosynthesis, cell growth, proliferation and survival [4,5] are mediated via ribosomal protein S6 kinase 1 (S6K1), a well-studied mTORC1 substrate.The S6K1 gene (RPS6KB1) was shown to encode two well-known S6K1 isoforms, p85-S6K1 and p70-S6K1, that differ only by the presence of the Nterminal 23 a.a. extension in p85-S6K1 due to the use of alternative (the first and second ATG) translational start sites [6]. Recently, it has been discovered that the splicing factor SF2/ASF promotes the expression of the oncogenic and the only known S6K1 splice variant termed p31-S6K1 or S6K1-isoform 2 that is truncated from the C-terminus [7]. A mechanism underlying oncogenic properties of p31-S6K1 is unclear but it seems to be kinase-independent, since the kinase domain of the given isoform is severely truncated. e x p e r i m e n T a l w o r K S e x p e r i m e n T a l w o r K S
To generate and characterize MCF-7 cell lines with altered expression of p85, p70 and p60 S6K1 isoforms: p85-/p70-/p60-MCF-7 and p85-/p70-/p60+MCF-7. Methods. CRISPR/ Cas9 gene editing, western blot analysis, immunofluorescence analysis, scratch assay. Results. Modified MCF-7 cells with knocked down expression of p85, p70, p60 or only p85 and p70 S6K1 isoforms were generated. Selective inhibition of only p85 and p70 isoforms in p85-/p70-/p60+MCF-7 cells was accompanied by actin cytoskeleton rearrangements, appearance of fibroblast-like cell morphology and significantly increased cell locomotor activity. Downregulation of all three S6K1 isoforms in p85-/p70-/p60 -MCF-7 cells inhibited cell migration with no changes in the cell morphology. Alterations in had a different impact on the ribosomal protein S6 phosphorylation and Akt signaling. Conclusion. Analysis of the modified MCF-7 cell lines revealed different impact of expression of S6K1 isoforms on MCF7 cell locomotor activity and the S6K1-and Akt-dependent signaling. Our data suggest that p60-S6K1 could be involved in regulation of the cell migration. The generated cells can be used for further analysis of functional activity of the S6K1 isoforms. K e y w o r d s: mTOR/S6K1 signaling, MCF-7, S6K1, CRISPR/Cas9, breast cancer К л ю ч е в ы е с л о в а: mTOR/S6K1 сигналинг, MCF-7, S6K1, CRISPR/Cas9, рак молочной железы.
Generation of polyclonal antibodies specifi c to the ribosomal protein S6 kinase isoform-p85S6K1 and directed to the N-terminal (1-23 aa) extension of p85S6K1. Methods. Animal immunization with synthetic (1-23 aa) peptide, ELISA, western blot, immunoprecipitation, immunofl uorescent analysis. Results. Polyclonal antibodies have been generated, which specifi cally recognize only p85 but not p70 isoform of S6K1 in western blot, immunoprecipitation and immunofl uorescence analysis. Conclusions. The obtained antibodies can be recommended for studies on the p85S6K1 and other S6K1 isoforms possessing the N-terminal extension-the identifi cation of binding protein partners, analysis of subcellular localization under different physiological conditions, elucidation of the signal transduction pathways involving different S6K1 isoforms. K e y w o r d s: Ribosomal protein S6 kinase 1 (S6K1), S6K1 isoforms, p70 S6K1, p85 S6K1, polyclonal antibodies.
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