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

Abstract

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 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.