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

Indolo[2,3- d ]benzazepines (indololatonduines) are rarely discussed in the literature. In this project, we prepared a series of novel indololatonduine derivatives and their Ru II and Os II complexes and investigated their microtubule-targeting properties in comparison with paclitaxel and colchicine. Compounds were fully characterized by spectroscopic techniques ( 1 H NMR and UV–vis), ESI mass-spectrometry, and X-ray crystallography, and their purity was confirmed by elemental analysis. The stabilities of the compounds in DMSO and water were confirmed by 1 H and 13 C NMR and UV–vis spectroscopy. Novel indololatonduines demonstrated anticancer activity in vitro in a low micromolar concentration range, while their coordination to metal centers resulted in a decrease of cytotoxicity. The preliminary in vivo activity of the Ru II complex was investigated. Fluorescence staining and in vitro tubulin polymerization assays revealed the prepared compounds to have excellent microtubule-destabilizing activities, even more potent than the well-known microtubule-destabilizing agent colchicine.

To generate HEK-293 cells with disrupted expression of S6K1 isoforms: p85, p70 and p60. Methods. CRISPR/Cas9 gene editing, Western blotting, immunofluorescent analysis, RT-PCR analysis, MTT assay, scratch assay. Results. Several clones of HEK-293 cells with a complete loss of p85/p70/p60-S6K1 protein expression were generated. The effects of p85/ p70/p60-S6K1 knockout on Akt/mTORC1/S6K1 signaling and cell proliferation and migration were assessed. Conclusions. The generated cell lines can be used to study a role played by S6K1 in cell physiology and to gain more detailed insight into cellular functions of the S6K1 isoforms. The HEK-293 cells exhibit downregulation of Akt phosphorylation on Ser473 and subsequent attenuation of cell growth rate, as well as inhibition of cell motility. K e y w o r d s: mTOR/S6K1 signaling, CRISPR/Cas9, S6K1 isoforms. К л ю ч е в ы е с л о в а: mTOR/S6K1 сигналинг, CRISPR/Cas9, изоформы S6K1.

the phosphatidylinositol-3-kinase (PI3k)/mechanistic target of rapamycin complex 1 (mtorc1) signaling pathway controls plenty of cellular functions regulating phosphorylation one of its mediators ribosomal protein S6 kinase 1 (S6k1). alternative translation of the common S6k1 transcript can generate three protein kinase isoforms, including p85-S6k1, p70-S6k1 and p60-S6k1. the catalytic activity of S6k1 is modulated by mitogens and growth factors via phosphorylation at three critical sites such as the activation loop (T-loop site), turn motif (TM site), and hydrophobic motif (HM site). Both members of the PI3K/mTORC1 pathway, PDk1 and mtorc1, directly phosphorylate the t-loop site and Hm site, respectively. Indeed, most studies aimed at elucidating S6k1 regulation have focused on p70-and p85-S6k1. meanwhile, however, the activity of p60-S6K1 and its regulation have not been elucidated so far. To test whether p60-S6K1 was an active kinase isoform that was regulated similar to p70/p85-S6k1, we employed previously generated p85 -/p70 -/ p60 + HEK-293 cells. First, an in vitro kinase assay confirmed the ability of p60-S6K1 to phosphorylate ribosomal protein S6 (rpS6), a well-known S6K1 substrate. Next, analysis of p60-S6K1 phosphorylation under different cell growth conditions showed that p60-S6K1 does not have detectable levels of phosphorylation at PDk1-and mtorc1-regulated sites, yet this isoform undergoes phosphorylation at the tm site. Finally, we found that activity of p60-S6K1 was not sensitive to mitogenic stimulation and cell treatment by potent inhibitor of the PI3K1/mTORC1-dependent signaling pathway rapamycin suggesting the existence of a PI3K/ mtorc1-independent mechanism of p60-S6k1 regulation in Hek-293. the data of the current study suggest that the p60-S6k1 isoform possesses intrinsic kinase activity that is independent of PI3k/mtorc1 signaling regulation in Hek-293 cells. What is more, modulation of p60-S6k1 activity via the PI3k/mtorc1 signaling pathway seems to be cell-type specific, since the p60-S6K1 isoform undergoes PDK1-and mTORC1-mediated phosphorylation in breast cancer cell line MCF-7. K e y w o r d s: p60-S6 kinase 1 (p60-S6K1), PI3K/mTORC1 signaling pathway, kinase activity, regulation by protein phosphorylation. R ibosomal protein S6 kinase 1 (S6K1) controls a number of cellular processes, including protein synthesis, lipid metabolism, cell growth, proliferation and migration, through its kinase activity towards plenty of downstream mediators. The S6K1 gene, referred to as RPS6KB1 in humans, encodes two major isoforms, p70-S6K1 and p85-S6K1 that differs by 23aa at N-terminus, which promote all mentioned cellular functions [1,2].Diverse environmental cues (i.e., serum, epidermal growth factor, insulin-like growth factor/ insulin, and phorbol esters) activate p70/p85 S6K1, mainly via the PI3K/mTORC1 signaling pathway. Signaling by growth factors and mitogens initiates phosphorylation of S6K1 on at least eight sites, where three of them, T229/T252, S371/S394 and T389/T412, are critical for ...