Plk1蛋白的甲基化修饰负调控自身激酶活性的机制研究

PLK1蛋白是一个高度保守的丝氨酸/苏氨酸激酶，它在有丝分裂的各个阶段、DNA复制、压力应急等多种细胞活动中发挥重要作用。PLK1蛋白在许多肿瘤中异常表达。虽然该蛋白的功能和作用底物有很多报道，但是PLK1激酶活性在细胞内的调控机制仍有待深入研究。我们前期研究发现，PLK1蛋白在K209位可发生甲基化修饰，该修饰与其紧邻的酶活性中心T210位点存在相互影响。且过表达K209M突变体引起有丝分裂期细胞数量减少。据此我们提出假说，PLK1蛋白甲基化修饰是调节其酶活性的一种重要方式。本课题拟从分子和细胞水平明确PLK1蛋白K209位的甲基化修饰酶；探究209甲基化对T210位磷酸化及其酶活性影响的分子机制；探究K209甲基化在细胞周期调控中的作用方式。本研究从非组蛋白翻译后修饰探讨重要蛋白激酶活性调控的分子机制，在理论层面为进一步开发针对PLK1蛋白靶点的安全的和高特异性的药物提供依据。

PLK1 protein is a highly conserved serine-threonine kinase that plays multiple intracellular roles including numerous stages of mitosis, DNA replication and stress response. PLK1 is overexpressed in many cancers. Although the biological roles and phosphorylated substrates of PLK1 have been extensively reported, the precise regulation of its kinase activity during cell cycle remained to be further investigated. Our preliminary data showed that PLK1 can be monomethylated at lysine 209 (K209me1), and K209me1 cross-talks with phosphorylated threonine 210 (T210, the core residue of PLK1 kinase activity). Overexpression of K209M mutation of PLK1, which mimics the methylated form, resulted in decreased numbers of mitotic cells, whereas overexpression of K209A mutation, which abolishes the methylation of PLK1, increased cell numbers in mitotic phase, compared with overexpression of wild-type PLK1 construct. The aims of this proposal are: 1) to identify the methyltransferase responsible for methylating K209 of PLK1; 2) to explore the molecular mechanism of how K209me1 attenuates T210 phosphorylation and kinase activity; 3) to determine the function of K209me1 in cell cycle progression at both molecular and cellular levels. This study investigates how post-translational modifications on a non-histone protein fine-tunes critical kinase activity of PLK1, which may shed light on the clinical drug development targeting on PLK1 with more safety and efficacy profiles.