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Abstract：
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
摘要：
■T细胞急性淋巴细胞白血病（T-ALL）是一种以未成熟T淋巴细胞增殖为特征的白血病。NOTCH1是T-ALL中最常见的突变基因之一。NOTCH1在T细胞发育中的表达取决于植物同源结构域指蛋白6（PHF6），在T-ALL中起肿瘤抑制作用。一些研究表明,PHF6表达对于NOTCH1表达是必需的。因此,本文研究了PHF6的翻译后修饰是否在调节NOTCH1表达和T-ALL细胞系增殖中起作用。
■分析了PHF6的氨基酸序列，发现推定的蛋白激酶A（PKA）磷酸化基序RDRS199在几种脊椎动物物种中是保守的，并且根据PhosphoSite数据库，预计S199位点将被磷酸化。因此,构建了人PHF6的真核表达载体,并且通过定点诱变将密码子199更改为编码非磷酸化丙氨酸和磷酸化模拟天冬氨酸的密码子。通过蛋白质印迹分析确认PHF6载体的异位表达后，使用蛋白质印迹分析鉴定了这些蛋白质对NOTCH1表达的影响，使用MTT测定白血病细胞增殖，和使用流式细胞术的T细胞的细胞表面标志物的表达。
■野生型PHF6的异位表达刺激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，这种效应在PHF6的丙氨酸和天冬氨酸突变体中均减弱。此外,两种突变体似乎也对NOTCH1表达产生负面影响，尽管丙氨酸突变体的作用更严重。
■放在一起，本研究中显示的保守S199磷酸化位点突变体发挥的不同生物学活性暗示,导致该残基差异磷酸化的信号传导途径可能对PHF6的活性具有实质性影响,因此可能构成T-ALL的潜在治疗靶标.
■分析了PHF6的氨基酸序列，发现推定的蛋白激酶A（PKA）磷酸化基序RDRS199在几种脊椎动物物种中是保守的，并且根据PhosphoSite数据库，预计S199位点将被磷酸化。因此,构建了人PHF6的真核表达载体,并且通过定点诱变将密码子199更改为编码非磷酸化丙氨酸和磷酸化模拟天冬氨酸的密码子。通过蛋白质印迹分析确认PHF6载体的异位表达后，使用蛋白质印迹分析鉴定了这些蛋白质对NOTCH1表达的影响，使用MTT测定白血病细胞增殖，和使用流式细胞术的T细胞的细胞表面标志物的表达。
■野生型PHF6的异位表达刺激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，这种效应在PHF6的丙氨酸和天冬氨酸突变体中均减弱。此外,两种突变体似乎也对NOTCH1表达产生负面影响，尽管丙氨酸突变体的作用更严重。
■放在一起，本研究中显示的保守S199磷酸化位点突变体发挥的不同生物学活性暗示,导致该残基差异磷酸化的信号传导途径可能对PHF6的活性具有实质性影响,因此可能构成T-ALL的潜在治疗靶标.
