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

DOI: 10.1038/s41598-019-39410-z

Authors: Abdullah R. Ahmed (Central Laser Facility, Research Complex at Harwell) , Raymond J. Owens (Protein Production UK, Research Complex at Harwell; The Wellcome Centre for Human Genetics) , Christopher D. Stubbs (Central Laser Facility, Research Complex at Harwell) , Anthony W. Parker (Central Laser Facility, Research Complex at Harwell) , Richard Hitchman (Evotec (UK) Ltd) , Rahul B. Yadav (Evotec (UK) Ltd) , Maud Dumoux (Diamond Light Source) , Chris Hawes (Oxford Brookes University) , Stanley W. Botchway (Central Laser Facility, Research Complex at Harwell)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Scientific Reports , VOL 9

State: Published (Approved)
Published: March 2019

Abstract: 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.

Journal Keywords: Biophysics; Nutrient signalling

Subject Areas: Biology and Bio-materials, Medicine


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