Fig 1: The schematic diagram illustrates the role of RNF128 in the IL3/STAT5 signaling pathway. The IL-3 signaling pathway relies on the IL-3 receptor complex, comprised of IL-3Rα, responsible for IL-3 binding specificity, and the common beta chain IL-3Rβ, shared with GM-CSF and IL-5 receptors. Upon IL-3 binding to its specific receptor, IL-3Rα initiates a cascade of downstream signaling events, involving the activation of JAK kinases, particularly JAK2. This activation subsequently leads to STAT5 phosphorylation, facilitating the transcription of genes like ID1, PIM1, and CD69. Notably, RNF128 demonstrates specific binding to IL-3 receptor alpha chain IL-3Rα, distinct from the common beta chain IL-3Rβ. The functional role of RNF128 is underscored as it promotes K27-linked polyubiquitination of IL-3Rα, culminating in its degradation through the lysosomal pathway. Consequently, RNF128 plays a crucial role in tempering excessive inflammatory responses
Fig 2: RNF128 interacts with IL-3 receptor IL-3Rα. (A) RNF128 expression levels significantly elevated upon IL-3 treatment. THP-1 cells were treated with IL-3 for 1 or 2 h, and the expression of RNF128 was evaluated using qPCR. (B) RNF128 interacts with IL-3Rα, not IL-3Rβ. 293T cells were transfected with HA-RNF128, along with Flag-tagged IL-3Rα and IL-3Rβ, and lysed after 24 h. The lysates subjected to immunoprecipitation using an anti-Flag antibody, followed by immunoblotting with the indicated antibodies. (C) The reverse interaction was confirmed by immunoprecipitation using an anti-Myc antibody in cells co-expressing Myc-RNF128 and Flag-IL-3Rα. (D, E) The N-terminal domain of RNF128 is primarily responsible for binding to IL-3Rα. 293T cells were transfected with Flag-IL-3Rα and HA-tagged truncated form of RNF128, and the immunoprecipitation analysis was conducted using an anti-Flag antibody. (F) Analysis of the colocalization between RNF128 and IL-3Rα. HeLa cells co-expressing RNF128 and IL-3Rα were subjected to immunostaining, with RNF128 detected using FITC and IL-3Rα using TRITC. Fluorescence signals were observed using confocal laser scanning microscopy. Scale bars: 10 μm. The data are presented as the mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001
Fig 3: RNF128 negatively regulates IL-3-triggered signaling. (A, B) Effects of Rnf128 on GM-CSF-induced phosphorylation of Stat5 and transcription of downstream genes. Wild-type and Rnf128-deficient BMDMs were treated with GM-CSF (10 ng/ml) or not for the indicated times before immunoblotting analysis and qPCR analysis. (C, D) Effects of Rnf128-deficient on Il-3-induced phosphorylation of Stat5. Wild-type and Rnf128-deficient BMDMs were either untreated or treated with Il-3 (25 ng/mL) for the specified durations. Subsequently, immunoblotting analysis was conducted to detect p-Stat5 levels, with quantification illustrating the ratio of p-Stat5 to Stat5 (D). (E) Effects of Rnf128-deficient on Il-3-induced Id1, Pim1 and Cd69 genes. (F, G) Effects of RNF128 knockdown on IL-3-induced phosphorylation of Stat5. RNF128 knockdown and control TF-1 cells were subjected to overnight starvation, followed by stimulation with IL-3 (20 ng/mL) for the indicated durations. Subsequently, immunoblotting analysis was conducted for p-STAT5, and the quantification of p-STAT5/STAT5 ratios is presented (G). (H) Effects of RNF128 knockdown on the transcription of the ID1, PIM1 AND CD69 genes induced by IL-3. * P < 0.05; ** P < 0.01; *** P < 0.001. Statistical analysis was performed using two-way ANOVA with Dunnett’s post-hoc test
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