蛋白激酶CyclinE/CDK2调控去甲基化酶PHF8的机制及生物学意义

蛋白激酶CyclinE/CDK2在细胞周期调控中发挥重要作用。我们通过串联亲和纯化技术筛选到与CyclinE/CDK2 互作的新蛋白-组蛋白去甲基化酶PHF8；初步研究表明它能被CyclinE/CDK2磷酸化。为了阐明CyclinE/CDK2磷酸化PHF8在细胞周期调控中的作用及其生物学意义，我们拟进行以下研究：1、通过质谱分析，定位CyclinE/CDK2磷酸化PHF8的位点；然后分析CyclinE/CDK2对于PHF8的磷酸化是否影响其去甲基化酶活性；2、通过流式细胞术等方法，研究PHF8是否协同CyclinE/CDK2参与细胞周期的调节，以及PHF8的磷酸化是否影响细胞周期G1/S期的转换；3、通过RNA深度测序技术, 比较PHF8 及其磷酸化对细胞周期相关基因转录的影响，探究PHF8调控基因表达的机制；4、通过小鼠模型以及临床样本分析，研究PHF8及其磷酸化与肿瘤发生的相关性。

Protein kinase cyclinE/CDK2 plays an important role in G1/S transition during cell cycle. By taking the approach of TAP-tagged purification technique, we identified a novel cyclinE/CDK2 substrate called PHF8 which is a histone demethylase. Our preliminary data showed that PHF8 can interact with cyclinE/CDK2 and be phosphorylated by cyclinE/CDK2. In order to further elucidate the biological significance of PFH8 phosphorylation by cyclinE/CDK2 and its effect on cell cycle progression, we propose to study the following aspects: 1. To identify the phosphorylation site of PHF8 by cyclinE/CDK2 using mass spectrometry; to generate PHF8 mutants and analyze whether phosphorylation of PHF8 by cyclinE/CDK2 effects its localization and demethylase activity; 2. To examine whether the phosphorylation status of PHF8 influences the cell cycle progression by using flow cytometry analysis; to investigate whether PHF8 coordinates with CyclinE/CDK2 in regulating DNA replication and G1/S transition; 3. To generate the inducible cells for PHF8 or its mutants, and examine whether the phosphorylation of PHF8 affects cell cycle related gene transcription; 4. To analyze if there is any correlation between the expression level and the phosphosphorylation of PHF8 and the tumorigenesis.