Fig 2: ERK2 phosphorylation of PPARγ AF-1 domain.(A) In vitro ERK2 phosphorylation sites within the AF-1 (pS112 and pT75) identified by mass spectrometry. A site that undergoes cis-trans isomerization that was previously identified (W39-P40) is also annotated. (B) ERK2 phosphorylation of 15N-labeled AF-1 followed by time-course 2D [1H,15N]-HSQC NMR. (C) ERK2 phosphorylation of 15N-labeled AF-1 P76A mutant followed by time-course 2D [1H,15N]-HSQC NMR.

Fig 3: Two sites in the PPARγ AF-1 domain are phosphorylated by ERK2. (A) In vitro ERK2 phosphorylation sites within the AF-1 (pS112 and pT75) identified by mass spectrometry. A site that undergoes cis–trans isomerization that was previously identified (W39-P40) is also annotated. (B) ERK2 phosphorylation of 15N-labeled AF-1 followed by time-course 2D [1H,15N]-HSQC NMR confirms both phosphorylation sites (pS112 and pT75). (C) ERK2 phosphorylation of 15N-labeled AF-1 P76A mutant followed by time-course 2D [1H,15N]-HSQC NMR reveals only a single phosphorylation site (pS112). The gray dashed box and arrow indicate the shift of the peak corresponding to T75 which occurs due to mutation of the adjacent proline.

Fig 4: Phosphorylation and stabilization of VISTA, induced by high glucose and mediated by ERK2, facilitate tumor immune evasion and enhance tumor growth. (A-D) VISTA-depleted CT26 cells were subcutaneously injected into BALB/c mice (n = 6 per group). Tumor volumes were calculated (A). Representative images of tumors (left) and tumor weight (right) for indicated groups (B). Flow cytometric analysis was performed to quantify CD8+ T cell infiltration in shCon and shVISTA CT26 tumors (C). Tumor-infiltrating T cells were stimulated with PMA, ionomycin, and brefeldin A for 3 h, followed by detection of IFN-γ+ and GzmB+ cells via flow cytometry (D). (E-G) The impact of VISTA T245 phosphorylation on tumor growth in nondiabetic and diabetic mice (n = 6 per group). CT26 homologous tumor model schematic (E), tumor weight (F), and tumor volumes (G) are shown. (H-J) Combined VISTA depletion and PD-1 treatment synergistically inhibited CT26 allograft growth in diabetic mice (n = 6 per group). Representative tumor images (H), tumor weight (I), and tumor volumes (J) are displayed. (K-M) The combination of S02 and PD-1 therapy demonstrated synergistic tumor growth inhibition effects in a CT26 allograft model of diabetic mice (n = 6 per group). Representative tumor images (K), tumor weight (L), and tumor volumes (M) are shown. Data are expressed as mean ± SD. N.S. indicates no significant difference for the specified comparison. *p < 0.05, **p < 0.01, ***p < 0.001

Fig 5: ERK2 binds to VISTA and phosphorylates it at S248, thereby upregulating its stability by reducing the ubiquitination of VISTA. (A) SW480 cells with or without ERK1/2 knockdown were incubated in media containing various glucose concentrations for 4 h, followed by treatment with MG132 (10 µM) for 6 h. (B) SW480 cells were transfected with Flag-Vector or Flag-VISTA for 48 h, followed by treatment with different concentrations of glucose medium for 4 h. (C) HEK293T cells were transiently transfected with WT Flag-VISTA or the indicated VISTA mutants and cultured in media with different glucose concentrations for 4 h. (D) In vitro kinase assays were conducted by incubating GST or GST-ERK2 with His-VISTA or His-VISTA S248A proteins in the presence of ATP-γ-S, followed by alkylation with PNBM. (E) SW480 cells were cultured in media containing the indicated glucose concentrations for 4 h. (F) HEK293T cells were transiently transfected with WT Flag-VISTA or the indicated VISTA mutants and cultured in media with the specified glucose concentrations for 4 h. (G) SW480 cells were incubated for 4 h in media with either 1 mM or 10 mM glucose, in the presence of DMSO or U0126 (10 µM). (H) SW480 cells were cultured in media containing 1 mM or 10 mM glucose for 4 h, followed by treatment with or without MG132 (10 µM) for 6 h. (I) SW480 cells with VISTA depletion were reconstituted with either WT Flag-rVISTA or the Flag-rVISTA S248A mutant, transfected with HA-Ub, and then cultured in media with the indicated glucose concentrations for 4 h before treatment with MG132 (10 µM) for 6 h. (J) HEK293T cells were co-transfected with ERK2 WT or ERK2 CA, Flag-VISTA, and HA-Ub and cultured in media with low glucose concentrations for 4 h. (K) SW480 and SW1116 cells depleted of VISTA were reconstituted with either WT Flag-rVISTA or the Flag-rVISTA S248A mutant, co-cultured with activated CD8+ T cells, and Ki67, GzmB, and IFN-γ levels in CD8+ T cells were assessed by flow cytometry. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. (A, B, E-G, I, J) Immunoprecipitation and immunoblot analyses were performed using the indicated antibodies. (C, D, H) Immunoblotting was carried out with the specified antibodies