S6K1 - kinase 1 of the ribosomal protein S6 functions as one of the key components of the PI3K / mTOR / S6K1 signaling path. This signaling pathway regulates various cellular processes - protein synthesis, cell growth, proliferation, homeostasis and others. It has also been shown that dysregulation of S6K1 is associated with pathologies such as cancer, obesity, aging, and diabetes. So far, the isoforms p85, p70 and p60, which are produced using the first three translation initiation starts, have been described, as well as an alternatively spliced p31 isoform, which is considered as proto-oncogenic.
Earlier, on the MCF-7 cell line, using CRISPR/Cas9 technology, 2 model cell lines were obtained with different expression of the studied isoforms: clone F1 with blocked expression of the p85 isoform (p85- / p70 + / p60 +) and clone F3 where expression of all isoforms was blocked (p85- / p70- / p60-). To study the contribution of the p85 isoform to signaling, mRNA of the full-length S6K1 sequence was transfected into the cells of clone F3, and stable cell line with p85 overexpression was obtained (F3/p85).
To study the activity of isoforms, the following cell treatments were carried out: 24 hours of starvation, 24 hours of serum starvation + 3 hours of restimulation (15% FBS) and 24 hours of starvation + 3 hours of restimulation with rapamycin, an inhibitor of the mTORC1 complex. The following antibodies were used for Western blot analysis: C-terminal polyclonal affinity purified antibodies [Savinska L, 2015], pThr389 S6K1 antibodies (Cell Signaling), pSer 235/236 S6 (Cell Signaling), beta-actin (Sigma).
The results showed that there was no significant change in the F1 clone compared to the wild type. After restimulation, a high level of rapamycin-sensitive phosphorylation of protein S6 is observed. An analysis of the activity of various isoforms showed that the p70 isoform is most responsible for the phosphorylation of the S6 protein.
In clone F3/p85, in addition to a high level of expression of the p85 form in the presence of serum in the medium, the p60 isoform is also expressed, which probably uses an alternative, third start of translation, whereas neither the level of p70 expression nor its phosphorylation is increased in comparison with clone F3. Both expressed forms, p60 and p85, had a high level of phosphorylation. Importantly, starvation did not decrease the level of Thr389 phosphorylation of all S6K1 and Ser 235/236 isoforms in S6. In this way, overexpression of the p85 isoform may contribute to cell survival, which does not contradict the latest literature data [1].