PIKFYVE is a dominant regulator of mTORC1 during Th17 induction. D10 CD4⁺ T cells were activated under Th17 conditions (CD3/CD28⁺TGF-β⁺IL-6) for 10 min with vehicle, apilimod, or rapamycin. A phosphoproteomic analysis measured the relative abundance of phosphorylated peptides by label-free quantitation (N = 4 biological replicates). (A) Depicted is a schematic of T-cell receptor (TCR)-AKT/mammalian target of rapamycin signaling in T cells. Residues that had reduced phosphorylation with rapamycin or apilimod treatment relative to vehicle control are highlighted. (B) The relative abundance of phosphopeptides for proteins in T cell signaling is depicted in a heatmap (N = 4 biological replicates). (C) An internalization assay was performed to monitor the extracellular and intracellular levels of the TCR/CD3 complex 30 min after Th17 polarization ± apilimod treatment (N = 3 biological replicates). (D–H) Peak areas from the label-free quantitative proteomics data were calculated for (D) p-CD3γ (S148) and mTORC1 substrates including (E) p-PRAS40 (S184), (F) p-RAPTOR (S863), (G) p-S6K (S369), and (H) p-ULK (S637). Data shown depict the mean ± SEM. P values were calculated by a Student’s t test in panel C. ANOVA one-way followed by Tukey test was used to calculate P values in panels D–H.