PIKFYVE signaling regulates STAT3 activation via mTORC1. (A) D10 CD4⁺ T cells were treated with vehicle, apilimod, or rapamycin for 1 h and activated under Th17 conditions (CD3/CD28⁺TGF-β⁺IL-6) for 10 min. A phosphoproteomic analysis measured the relative abundance of phosphopeptides for selected transcription factors by label-free quantitation (N = 4 biological replicates). (B) Primary naive murine CD4⁺ T cells isolated by negative selection were activated under Th17 polarization conditions. Immunoblotting was performed on cell lysates for p-STAT3 (S727) and total STAT3. Densitometry was performed on immunoblots and p-STAT3 (S727) was normalized to total STAT3. Data shown as mean ± SEM; N = 3 biological replicates. (C) Primary naive murine CD4⁺ T cells isolated by negative selection were activated under Th17 polarization conditions for 5 days in the presence of vehicle, apilimod, rapamycin, or iAKT. Immunoblotting was performed on cell lysates for p-STAT3 (S727). Densitometry was performed. A one-way ANOVA was used to calculate P values; N = 3 biological replicates. (D) Recombinant mTORC1 complex (mTOR/mLST8/Raptor) were reacted with ATP and recombinant STAT3. The phosphorylation of S727 on STAT3 by PIKFYVE was monitored by immunoblotting utilizing an antibody that recognizes p-STAT3 (S727). Densitometry was performed where p-STAT3 (S727) was normalized to total STAT3. Data are shown as mean ± SEM; N = 3 biological replicates. (E) Recombinant STAT3 was incubated with beads coated with different phosphatidylinositols. Immunoblotting was performed to determine the relative amount of STAT3 that was precipitated with each phosphatidylinositol bead. Data are shown as mean ± SEM (N = 3 biological replicates). (F) Recombinant MTOR complex (mTOR/mLST8/Raptor) or STAT3 was incubated with PtdIns(3)P or PtdIns(3,5)P2 polysomes. The amount of MTOR or STAT3 that precipitated with PtdIns(3)P or PtdIns(3,5)P2 polysomes was detected with immunoblotting. Data are shown as mean ± SEM; N = 3 biological replicates. P values were calculated with Student’s t tests. (G) Recombinant MTORC1 (mTOR/mLST8/Raptor) was reacted with recombinant STAT3 and ATP in the presence of no lipid, PI3P polysomes, or PI(3,5)P2 polysomes. Immunoblotting was performed for p-STAT3 (S727) and total STAT3. Data are shown as mean ± SEM; N = 3. (H) Primary naive murine CD4⁺ T cells isolated by negative selection (CD4⁺CD25⁻CD44^low) from wild type or STAT3 S727A mutant (STAT3^SA/SA) mice and cultured under Th17 conditions for 4 days. Flow cytometry was performed on gated CD4⁺ T cells to measure IL-17 and FoxP3. Data shown depict the mean ± SEM; N = 3 biological replicates. Source data are available for this figure: SourceData F5.