Fig 2: Loss of eEF-2K up-regulates the Akt-mTOR signaling and HSP90 in CD8+ T cells.(A) Western blot analysis of up-regulation of Akt–phospho-Akt proteins followed by quantification using ImageJ software of independent experiments. (B) Phospho-RPS6kb was determined by Western blots and validated by phospho-flow cytometric analysis. (C) Western blot analysis confirms that loss of eEF-2K up-regulates the mTOR signaling pathway. Independent blots were quantified with ImageJ software and normalized to β-actin. (D) Seahorse metabolic profile shows that the dysfunctional metabolism in eEF-2K KO CD8+ T cells is partially restored to WT levels on treatment with rapamycin. (E) Survival of eEF-2K KO CD8+ T cells was partially restored to WT levels on treatment with rapamycin. (F) The LC-MS/MS spectra indicated that all major heat shock proteins (HSPs) were up-regulated in eEF-2K KO CD8+ T cells compared to their WT counterparts with significant up-regulation of HSP90-aa1 and HSP90-ab1. mTORi = rapamycin. Data shown are representative values derived from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.005, unpaired t test.

Fig 3: The effects of eEF-2K on CD8+ T cells are mediated through the Akt-mTOR-S6K pathway.The illustration shows that Akt-mTOR-S6K signaling is hyperregulated in CD8+ T cells lacking eEF-2K. The up-regulation of HSP90/NF-κB induced by eEF-2K ablation may be responsible for the activation of Akt-mTOR-S6kb signaling, leading to an early enhanced metabolic state of eEF-2K KO CD8+ T cells.

Fig 4: C220 is required for the suppression of raptor ubiquitination, and reorganize mTOR complex assembly.a–c Cells were transduced with empty control lentivirus or the various UCH-L1 mutants and extracts were subjected to immunoprecipitation for mTOR (a), raptor (b), or rictor (c), and were probed as indicated. Note how the expression of wild-type or S18Y UCH-L1 reduced the association of raptor with mTOR while increasing that of rictor while C220S, along with the catalytically inactive D30K and C90A constructs, are unable to do so. d Cells as in (A-C) were further transduced to express FLAG-tagged ubiquitin (FLAG-Ub) and SDS-extracts were subjected to immunoprecipitation for raptor. All blots are representative of the results in 3–5 independent experiments.

Fig 5: Loss of eEF-2K alters the survival, proliferation, and function of CD8+ T cells.CD8+ T cells from WT or eEF-2K KO C57BL/6 mice were activated with anti-CD3/CD28 antibodies. The percent survival, cytokine production, and proliferation were assessed. (A) Percent survival. (B) Proliferation by CFSE-based flow cytometric staining and mass spectrometric proteomics analysis of Ki-67. (C) ELISA of IL-2 production on days 1 and 3 after stimulation. (D) Comparative SABG staining analysis. The SABG-stained (blue colored) cells were counted, and the mean value from three independent fields was represented graphically. **P < 0.01. High SABG staining and p53 and p21 expression are indicative of senescent phenotype of CD8+ T cells. (E) CEACAM-1 expression as assessed by confocal microscopy (dual expression of red and green) and IL-6 expression by flow cytometric analysis on WT versus eEF-2K KO CD8 T cells, indicating senescence. ns, not significant. **P < 0.01. (F) Graphical representation of CEACAM-1–expressing cells per field and independent replicates of flow cytometric analysis of IL-6 cytokine. (G) Western blot analysis of cell cycle arrest senescence-associated p53 and p21 used with mTOR inhibitor rapamycin. (H) Flow cytometric histogram analysis of CD27 costimulatory marker. (I) Graphical analysis of independent replicates of CD27 expression derived from flow cytometric dot-plot analysis. (J) Flow cytometric histogram representation of CD28 costimulatory marker. (K) Graphical analysis of independent replicates of CD28 expression derived from flow cytometric dot-plot analysis. Data shown are representative values derived from three identical and independent experiments. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.0001, unpaired t test.