The Р60-S6K1 isoform of ribosomal protein S6 kinase 1 is a product of alternative mRNA translation

Zaiets, I. V.; Sivchenko, A. S.; Хоруженко, А. И.; Savinska, L. O.; Filonenko, Valeriy

doi:10.15407/ubj90.04.025

Cited by 8 publications

(16 citation statements)

References 29 publications

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“…We recently reported that HEK-293 cells expressing only the p60 isoform (p85 -/p70 -/ p60 + HEK-293 cells) display higher rates of cell proliferation and cell migration compared to HEK-293 with knockout of all isoforms [6]. These observations suggest that p60-S6K1 could be involved in the control of cell proliferation and motility.…”

Section: Resultsmentioning

confidence: 98%

“…HEK-293 and p85 -/p70 -/p60 + HEK-293 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) (GIBCO) containing 10% fetal bovine serum (FBS) (GIBCO), 100 units/ml penicillin, and 100 µg/ml streptomycin. Development of cell clones modified with CRISPR/Cas9 targeting p85-and p70-S6K1 was described in [6]. For experiments involving serum stimulation, rapamycin and LY294002 inhibition, cells were washed twice with phosphatebuffered saline (PBS) and after that serum-starved for 24 h in DMEM deprived of serum.…”

Section: Methodsmentioning

confidence: 99%

“…Plasmids and recombinant proteins. Generation of the pSpCas9(BB)-2A-Puro (PX459) V2.0 DNA vector containing anti-p85/p70 was described previously [6]. Purified recombinant 6X-His-tagged rpS6 was a gift from Garifulin O. M. (Department of cell signalling, IMBG of NASU, Kyiv, Ukraine).…”

Section: Methodsmentioning

confidence: 99%

“…However, our knowledge about p60-S6K1 expression, regulation and activity remains quite limited. It has been suggested that p60-S6K1 originates from an alternative translation event [5,6] and represents the N-terminally truncated S6K1 isoform (53 amino acids from the N-terminus of p85-S6K1, ΔN53-p85) lacking the mTORC1 binding site, called the TOS motif [5]. Although p60-S6K1 does not possess the TOS motif, it was reported in [5] that the S6K1 isoform is modulated by phosphorylation at the mTORC1-regulated site (T359 in p60-S6K1) in MCF-7 cells, as well as it is subjected to S371 (S341 in p60-S6K1) and T421/S424 (T391/S394 in p60-S6K1) phosphorylation similar to the major p70-S6K1 isoforms in the same cells.…”

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confidence: 99%

“…Recently, applying the CRISPR/Cas9 approach, we generated HEK-293 cells with knockout of p70and p85-S6K1 (p85 -/p70 -/p60 + HEK-293) allowing the cells to exclusively express the p60-S6K1 isoform [6]. Expression of p60-S6K1 in these cells was shown to rescue a rate of cell proliferation and migration as compared to HEK-293 with knockout of all three isoforms (p85 -/p70 -/p60 -HEK-293).…”

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p60-S6K1 represents a novel kinase active isoform with the mode of regulation distinct from p70/p85-S6K1 isoforms

Zaiets¹,

Holiar²,

Sivchenko³

et al. 2019

Ukr.Biochem.J

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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 ...

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Section: Resultsmentioning

confidence: 98%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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