精氨酸甲基化对p16功能的影响及其分子机制研究

项目名称： 精氨酸甲基化对p16功能的影响及其分子机制研究

项目编号： No.30800557

项目类型： 青年科学基金项目

立项/批准年度： 2009

项目学科： 轻工业、手工业

项目作者： 王秀莉

作者单位： 东北师范大学

项目金额： 18万元

中文摘要： 肿瘤抑制子p16INK4A（p16蛋白）是周期依赖激酶的抑制子，它通过与CDK4/CDK6形成复合体来阻止CDK4/CDK6与cyclinD的结合，从而抑制RB蛋白的磷酸化进而阻断细胞周期的正常进行。在我们的研究中发现p16蛋白的翻译后修饰对它的功能有重要影响。首先，p16蛋白的甲基化存在于很多种细胞系中。其次，p16蛋白的22位、131位、138位精氨酸受到了甲基化修饰，因为将这几个位点突变成赖氨酸以后，p16蛋白的甲基化程度降低。Western Blotting和TUNEL分析表明，与野生型p16蛋白相比，22位、131位、138位精氨酸同时突变的p16蛋白（p16KKK）可以诱导更高的凋亡比率。不仅如此，免疫共沉淀实验结果显示，p16蛋白精氨酸甲基化水平下降会促进p16与CDK4的结合。与此同时，PRMT6参与了p16蛋白的精氨酸甲基化过程。流式细胞术结果证明，在A549细胞中，过表达PRMT6可以拮抗野生型的p16对G1期的周期停滞。而且，PRMT6可以与p16相互结合，从而抑制了p16与CDK4的结合。所以，PRMT6可以通过增加p16精氨酸甲基化情况来抑制细胞凋亡

中文关键词： p16；精氨酸甲基化；PRMT6；CDK4；细胞周期

英文摘要： As a cyclin-dependent kinase inhibitor, the tumor suppressor p16INK4A (p16) forms a complex with CDK4 and CDK6 to prevent them from interacting with cyclin D, thus inhibiting the phosphorylation of the retinoblastoma protein and blocking the cell cycle progression. We describe here a novel aspect of the posttranslational control that has an important functional consequence on p16 protein. We first discovered that the p16 protein was methylated in various cell lineages. We then determined that the arginine 22, 131 and 138 of p16 were the methylation sites, because mutations of these arginine residues to lysine resulted in hypomethylation of p16. Western blotting and TUNEL analyses revealed that the p16 protein bearing these point mutations (p16KKK) induced a higher apoptosis ratio than wild-type p16 in A549 cells. Furthermore, co-immunoprecipitation assays suggested that decrease of the p16 arginine methylation level promoted the association of p16 with CDK4. Additionally, we determined that the protein arginine methyltransferase 6 (PRMT6) was responsible for the p16 arginine methylation. Results from flow cytometric analysis (FACS) demonstrated that PRMT6 overexpression counteracted the cell cycle arrest at G1 phase induced by wild-type p16 in A549 cells. We also showed that PRMT6 was able to interact with p16, and that the intensity of p16-CDK4 association was reduced upon PRMT6 overexpression. Consequently, PRMT6 inhibited cell apoptosis via increase of p16 arginine methylation. Together, data presented in this report establish that methylation at specific arginine residues of p16 protein by PRMT6 may be critical for the activity of p16.

英文关键词： p16； arginine methylation； PRMT6； CDK4； cell cycle