Transcription factor CCAAT/Enhancer binding protein alpha (C/EBPα) is a key regulator of myeloid differentiation, granulopoiesis in particular. Although CEBPA mutations are found in more than 10% in AML, functional inhibition of C/EBPα protein is also widely observed in AML. Here, we sought to examine if SKP2, an aberrantly enhanced E3 ubiquitin ligase in primary AMLs inhibits C/EBPα stability to induce differentiation block. Here we employed cell based assays such as transfections, immunoblotting, co-immunoprecipitation, luciferase and gel shift assays along with differentiation assays to investigate SKP2 regulated C/EBPα protein stability in acute myeloid leukemia. Here we discovered that oncogenic E3 ubiquitin ligase SCF ubiquitinates and destabilizes C/EBPα in a proteasome-dependent manner. Our data demonstrates that SKP2 physically interacts with C-terminal of C/EBPα and promotes its K48-linked ubiquitination-mediated degradation leading to its reduced transactivation potential, DNA binding ability and cellular functions. We further show that while overexpression of SKP2 inhibits both ectopic as well as endogenous C/EBPα in heterologous (HEK293T) as well as myeloid leukemia cells respectively, SKP2 depletion restores endogenous C/EBPα leading to reduced colony formation and enhanced myeloid differentiation of myeloid leukemia cells. Using Estradiol-inducible K562-C/EBPα-ER cells as yet another model of granulocytic differentiation, we further confirmed that SKP2 overexpression indeed inhibits granulocytic differentiation by mitigating C/EBPα stability. Our findings identify SKP2 as a potential negative regulator of C/EBPα stability and function in AML which suggests that SKP2 can be potentially targeted in AML to restore C/EBPα and overcome differentiation block.

中文翻译：

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E3 连接酶 SCFSKP2 泛素化并降解急性髓系白血病中的肿瘤抑制因子 C/EBPα

转录因子 CCAAT/增强子结合蛋白 α (C/EBPα) 是骨髓分化，特别是粒细胞生成的关键调节因子。尽管CEBPA突变在AML中发现率超过10%，但C/EBPα蛋白的功能抑制在AML中也广泛观察到。在这里，我们试图检查 SKP2（一种在原发性 AML 中异常增强的 E3 泛素连接酶）是否会抑制 C/EBPα 稳定性以诱导分化阻断。在这里，我们采用基于细胞的测定，例如转染、免疫印迹、免疫共沉淀、荧光素酶和凝胶转移测定以及分化测定来研究急性髓系白血病中 SKP2 调节的 C/EBPα 蛋白稳定性。在这里，我们发现致癌 E3 泛素连接酶 SCF 以蛋白酶体依赖性方式泛素化 C/EBPα 并使其不稳定。我们的数据表明，SKP2 与 C/EBPα 的 C 端发生物理相互作用，并促进其 K48 连接的泛素化介导的降解，导致其反式激活潜力、DNA 结合能力和细胞功能降低。我们进一步表明，虽然 SKP2 的过表达分别抑制异源 (HEK293T) 和髓系白血病细胞中的异位和内源性 C/EBPα，但 SKP2 耗竭会恢复内源性 C/EBPα，导致髓系白血病的集落形成减少并增强髓系分化。细胞。使用雌二醇诱导的 K562-C/EBPα-ER 细胞作为粒细胞分化的另一种模型，我们进一步证实 SKP2 过表达确实通过减轻 C/EBPα 稳定性来抑制粒细胞分化。我们的研究结果确定 SKP2 是 AML 中 C/EBPα 稳定性和功能的潜在负调节因子，这表明 SKP2 可能成为 AML 中的潜在靶点，以恢复 C/EBPα 并克服分化障碍。