PIKFYVE/PtdIns(3,5)P2 activates kinases during Th17 differentiation. (A) The workflow for the proteomic PtdIns(3,5)P2 binding screen using murine CD4⁺ T cell lysates is presented. (B) Pathway analysis using the Ingenuity software package was performed using proteins that were associated with PtdIns(3,5)P2 beads as input based on three biological replicates. (C) The phosphoproteomics workflow is outlined. (D) Murine 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). The relative abundance of phosphopeptides observed in CD4⁺ T cells activated with different stimuli is depicted as a heatmap. (E) The number of phosphorylation sites with reduced abundance because of rapamycin and apilimod treatment using a twofold cutoff is depicted. Venn diagrams were constructed to compare the common and unique phosphopeptides between CD4⁺ T cells activated in the presence of rapamycin or apilimod. A twofold cutoff using the quantitative proteomics data was utilized to classify a phosphopeptide as belonging to the indicated group. (F) The apilimod-regulated phosphorylation sites were utilized as input into the Ingenuity software package to identify biological pathways regulated by PIKFYVE generation of PtdIns(3,5)P2.