Fig. 4: SCYL1 Ser754 is a novel mTORC1-regulated phosphosite.

a Schematic representation of SCYL1 protein structure. Known conserved domains and phosphorylation sites identified by MS are shown. CC = Coiled-coil domain. Black stars: phosphorylation events detected in independent AP-MS experiments; Red star: Ser754, identified reproducibly as regulated in in vitro mTOR kinase assays. Blue lollipop chart: number of citations referenced in Phosphosite Plus®⁵⁰ for indicated phosphosites. Pink lollipop chart: functional score for indicated phosphosites, as published in ref. 51. b SCYL1 Ser754 is rapidly dephosphorylated upon mTORC1 inhibition. Phosphoproteomic time-course of SCYL1 Ser754 phosphorylation levels in SILAC-labeled MCF-7 WT cells upon HBSS starvation or 100 nM rapamycin treatment. Average values of n = 2 biological replicates. c In vitro mTOR kinase assay qualifies SCYL1 Ser754 as direct mTOR target site. Purified mTOR was incubated with immunoprecipitated HA-SCYL1 in absence or presence of wortmannin (12 µM, 30 min). Two biological replicates were analyzed by LC-MS/MS. Results are displayed as a ratio of phosphosite intensities without/with wortmannin (Supplementary Data 3). d Time-course of SCYL1 Ser754 phosphorylation upon mTORC1 inhibition, detected by a phosphosite-specific antibody. WT MCF-7 cells were left untreated or treated with 100 nM rapamycin, starvation (HBSS), or 250 µM Torin1, for the indicated times. One representative time-course experiment out of three biological replicates is shown. Equal protein amounts were loaded. e Quantification of d. Phospho-Ser754-SCYL1 levels were normalized to total SCYL1 levels. Average of n = 4 biological replicates for DMEM/Rapa and DMEM/Starv measurements, and n = 3 biological replicates for DMEM/Torin1 measurements. Red line: rapamycin/DMEM 0 min; blue line: starvation/DMEM 0 min; green lines: Torin1/DMEM 0 min. All single values are indicated by colored dots. p-values: An unpaired two-tailed Student’s t-test was used to compare the indicated time points with the 0 min reference; Rapa10’/DMEM p = 0.0068(**); Rapa 30’/DMEM p = 0.017(*); Rapa 60’/DMEM p = 0.018(*); HBSS 60’/DMEM p = 0.038(*); Torin10’/DMEM p = 0.0029(**); Torin30’/DMEM p = 0.049(*); Torin60’/DMEM p = 0.043(*). Error bars, SEM. f SCYL1 Ser754 phosphorylation responds to mTORC1 reactivation. WT MCF-7 cells were starved in HBSS for 4 h, then released in complete DMEM supplemented with 50 µg/ml cycloheximide to increase the intracellular amino acid concentration for the indicated times. One representative time-course experiment out of three biological replicates is shown. Equal protein amounts were loaded. g Quantification of f. Phospho-Ser754-SCYL1 levels were normalized to total SCYL1 levels. Average of n = 3 biological replicates. p-values: An unpaired two-tailed Student’s t-test was used to compare the indicated time points with the starved cells reference time point (before release); 30’: p = 0.0028(**); 60’: p = 0.0018(**). Error bars, SEM. All source data are provided as Source Data file.