Fig. 4: EPRS1 binds specifically to PI(3)P and Rab5 to coordinate early endosomal anti-inflammatory AKT signaling.

a PIP strips precoated with indicated lipids were incubated with purified Strep-EPRS1 and probed with an anti-Strep antibody. b Sequence alignment of EPRS1 with PTEN to show the PI(3)P-binding consensus motif. The last row shows mutations engineered to neutralize early endosomal localization. c PIP strips were incubated with purified Strep-EPRS1 WT or its mutant (MT) and probed with an anti-Strep antibody for the same exposure time. d HeLa cells were transfected with a mCherry-tagged EV, Rab5, Rab7, LAMP1, or LC3 (red), in combination with EPRS1-YFP (green). Confocal microscopy analysis showing colocalization of EPRS1 and organelle-specific proteins in transfected cells following LPS (500 ng/ml) stimulation for 30 min. White arrowheads indicate colocalized early endosomes, and the magnified images are displayed in the right panel. The colocalization index of EPRS1 and specific organelle markers was quantified using Pearson’s correlation (bottom, n = 5 cells for EV, n = 9 cells for Rab5, n = 7 cells for other markers). Error bars indicate the mean ± SEM. Data were analyzed using Student’s two-tailed t-test. e HeLa cells were transfected with YFP-tagged EPRS1 (green) WT or non-endosomal associated MT in combination with Rab5(Q79L)-CFP (cyan). After 16 h, cells were fixed following LPS stimulation for 30 min and then analyzed by immunofluorescence with an antibody specific for p-AKT (red). Scale bars, 10 μm. f Confocal microscopy analysis of colocalization of EPRS1 Linker-GFP (green) and Rab5(Q79L)-mCherry (red) in live HeLa cells. MT, K974A/K977A/P978A/K980A mutant. Colocalized images in the white dotted boxes. Scale bars, 10 μm. g LPS (100 ng/ml) treatment of U937 cells induces translocation of Ser999-phosphorylated EPRS1 and AKTs to Rab5-positive early endosomes. An anti-GAPDH antibody was used as a cytosolic marker. h Schematic depicting the interaction between EPRS1 and AKT in the Rab5-positive early endosome to regulate downstream GSK3β signaling. The conserved region within the linker domain (aa 974–980), located in the spacer between the third WHEP repeat domain and the PARS1 domain, is important for PI(3)P binding. Data are representative of three independent experiments, each with similar results. Source data are provided as a Source Data file.