Direct imaging of the recruitment and phosphorylation of S6K1 in the mTORC1 pathway in living cells

• Published in: Scientific reports Vol. 9; no. 1; p. 3408
• Main Authors: Ahmed, Abdullah R., Owens, Raymond J., Stubbs, Christopher D., Parker, Anthony W., Hitchman, Richard, Yadav, Rahul B., Dumoux, Maud, Hawes, Chris, Botchway, Stanley W.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 04-03-2019; Nature Publishing Group
• Subjects: 14/35; 14/69; 38/109; 631/57; 631/80/86/2369; 82/83; 9/10; 96/106; Biosensors; Cytoplasm; Fluorescence resonance energy transfer; HEK293 Cells; Humanities and Social Sciences; Humans; Kinases; Mechanistic Target of Rapamycin Complex 1 - genetics; Mechanistic Target of Rapamycin Complex 1 - metabolism; multidisciplinary; Mutation; Phosphorylation; Protein Binding - drug effects; Ras Homolog Enriched in Brain Protein - genetics; Ras Homolog Enriched in Brain Protein - metabolism; Regulatory-Associated Protein of mTOR - genetics; Regulatory-Associated Protein of mTOR - metabolism; Ribosomal Protein S6 Kinases, 70-kDa - genetics; Ribosomal Protein S6 Kinases, 70-kDa - metabolism; Science; Science (multidisciplinary); Signal transduction; Sirolimus - pharmacology; TOR protein; TOR Serine-Threonine Kinases - genetics; TOR Serine-Threonine Kinases - metabolism
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-019-39410-z

Knowledge of protein signalling pathways in the working cell is seen as a primary route to identifying and developing targeted medicines. In recent years there has been a growing awareness of the importance of the mTOR pathway, making it an attractive target for therapeutic intervention in several diseases. Within this pathway we have focused on S6 kinase 1 (S6K1), the downstream phosphorylation substrate of mTORC1, and specifically identify its juxtaposition with mTORC1. When S6K1 is co-expressed with raptor we show that S6K1 is translocated from the nucleus to the cytoplasm. By developing a novel biosensor we demonstrate in real-time, that phosphorylation and de-phosphorylation of S6K1 occurs mainly in the cytoplasm of living cells. Furthermore, we show that the scaffold protein raptor, that typically recruits mTOR substrates, is not always involved in S6K1 phosphorylation. Overall, we demonstrate how FRET-FLIM imaging technology can be used to show localisation of S6K1 phosphorylation in living cells and hence a key site of action of inhibitors targeting mTOR phosphorylation.