Fig. 4: The persistent phosphorylation of cytoplasmic mTORC1 substrates in cells with non-lysosomal mTOR is not due to impaired or slower substrate dephosphorylation.
From: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources
a,b, mTOR/LAMP2 colocalization (a) and its quantification (b). mTOR delocalizes away from lysosomes already after 2 h of BafA1 treatment. Time course of BafA1 treatment (100 nM, 2–8 h) to block lysosomal function in HEK293FT cells. Magnified insets shown to the right (a). Scale bars, 25 μm and insets, 5 μm. n = 49–50 individual cells from five independent fields per condition (see also Methods). c–e, Dephosphorylation kinetics of lysosomal (TFEB) and cytoplasmic (S6K and 4E-BP1) substrates of mTORC1 upon BafA1 treatment (100 nM, 1–8 h) in HEK293FT cells showing a rapid drop in TFEB phosphorylation, whereas that of S6K/4E-BP1 remains largely unaffected even at much later timepoints (c). Quantification of TFEB phosphorylation in (d) and S6K phosphorylation in (e). f,g, The rapamycin time course (20 nM, 1–30 min) in control (WT) and RagA/B KO cells, assessing S6K dephosphorylation kinetics (f) and the quantification of S6K phosphorylation (g). The rate of S6K dephosphorylation is similar between Rag-proficient and Rag-deficient cells. Arrowheads indicate bands corresponding to different protein forms when multiple bands are present. P, phosphorylated form. Data in graphs shown as mean ± s.e.m. **P < 0.01, ***P < 0.001, ****P < 0.0001. n.s., non-significant. Source numerical data and unprocessed blots are available in Source data.
