Department of Medical Biology and Genetics, Faculty of Medicine, Akdeniz University, Antalya, Turkiye.
Department of Pediatrics, Faculty of Medicine, Akdeniz University, Antalya, Turkiye.
Turk J Med Sci. 2023 Aug 11;53(5):1234-1243. doi: 10.55730/1300-0144.5689. eCollection 2023.
BACKGROUND/AIM: T-cell acute lymphoblastic leukemia (T-ALL) is a form of leukemia characterized by the proliferation of immature T lymphocytes. NOTCH1 is one of the most frequently mutated genes in T-ALL. NOTCH1 expression in T-cell development depends on plant homeodomain finger protein 6 (PHF6), which plays a tumor suppressor role in T-ALL. Several studies have shown that PHF6 expression is essential for NOTCH1 expression. Therefore, whether posttranslational modification of PHF6 plays a role in the regulation of NOTCH1 expression and T-ALL cell line proliferation was investigated herein.
The amino acid sequence of PHF6 was analyzed and it was found that a putative protein kinase A (PKA) phosphorylation motif RDRS199 was conserved in several vertebrate species and the S199 site was expected to be phosphorylated according to the PhosphoSite database. Therefore, an eukaryotic expression vector of human PHF6 was constructed, and the codon 199 was changed to the codon encoding the nonphosphorylatable alanine and the phosphorylation-mimicking aspartic acid via site-directed mutagenesis. After confirming the ectopic expressions of the PHF6 vectors by western blot analysis, the effects of these proteins were identified on the NOTCH1 expression using western blot analysis, leukemic cell proliferation using MTT assay, and expressions of the cell surface markers of T-cells using flow cytometry.
The ectopic expression of wild-type PHF6 stimulated the formation of CD4 + T-cells. While the expression of the wild-type PHF6 suppressed the growth of the leukemic cell line, this effect was diminished in both the alanine and aspartic acid mutants of PHF6. In addition, both mutants also seemed to negatively affect the NOTCH1 expression, although the effect of the alanine mutant was more severe.
Taken together, the different biological activities exerted by the conserved S199 phosphorylation-site mutants shown in this study implicate that signaling pathway(s) leading to differential phosphorylation of this residue may have a substantial effect on the activity of PHF6, and thus may constitute a potential therapeutic target in T-ALL.
背景/目的：T 细胞急性淋巴细胞白血病（T-ALL）是一种以不成熟 T 淋巴细胞增殖为特征的白血病。NOTCH1 是 T-ALL 中最常突变的基因之一。NOTCH1 在 T 细胞发育中的表达依赖于植物同源结构域蛋白 6（PHF6），PHF6 在 T-ALL 中发挥肿瘤抑制作用。几项研究表明，PHF6 的表达对于 NOTCH1 的表达是必需的。因此，本文研究了 PHF6 的翻译后修饰是否在 NOTCH1 表达和 T-ALL 细胞系增殖的调节中发挥作用。
分析 PHF6 的氨基酸序列，发现几个脊椎动物物种中存在一个假定的蛋白激酶 A（PKA）磷酸化模体 RDRS199，根据 PhosphoSite 数据库，S199 位点预计被磷酸化。因此，构建了人 PHF6 的真核表达载体，并通过定点突变将 199 位密码子突变为编码非磷酸化丙氨酸和磷酸化天冬氨酸的密码子。通过 Western blot 分析确认 PHF6 载体的异位表达后，通过 Western blot 分析确定这些蛋白对 NOTCH1 表达的影响、MTT 测定法确定对白血病细胞增殖的影响，以及通过流式细胞术确定 T 细胞表面标志物的表达。
野生型 PHF6 的异位表达刺激了 CD4+T 细胞的形成。虽然野生型 PHF6 的表达抑制了白血病细胞系的生长，但在 PHF6 的丙氨酸和天冬氨酸突变体中，这种作用都减弱了。此外，尽管丙氨酸突变体的影响更为严重，但这两种突变体似乎也对 NOTCH1 的表达产生负面影响。
综上所述，本研究中显示的保守 S199 磷酸化位点突变体表现出的不同生物学活性表明，导致该残基差异磷酸化的信号通路可能对 PHF6 的活性有实质性影响，因此可能成为 T-ALL 的潜在治疗靶点。
