p16蛋白的磷酸化与精氨酸甲基化修饰影响其功能的相互关系及其分子机制研究

p16 has been known as a tumor suppressor gene, which suppresses proliferation of cells via direct inhibition of cell cycle progression. The p16 protein binds specifically to CDK4 and CDK6 to prevent them from interacting with cyclin D, which prevents G1/S cell cycle progression and cell proliferation. Accordingly, p16 is a potential target for cancer gene therapy. Previous studies have demonstrated that the phosphorylation of Ser152 of p16 protein promotes the interaction of p16 with CDK4,however,the phosphorylation of Ser8 abolishes its CDK4-inhibitory activity, suggesting that the post-translational modification of p16 participates in the regulation of p16 function. Whether other modification of p16 protein participates in its functionis unclear. In our previous study, we found 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. Furthermore, decrease of the p16 arginine methylation level promoted the association of p16 with CDK4 and exhibited a reinforced function in preventing cell proliferation. In this study, we plan to explore which pathway or kinase participate in phosphorylation of p16, the crosstalk between phosphorylation and arginine methylation and how the post-translational modification affects the senesence and apoptosis induced by p16 as an extension of our previous study. This study may be crucial to provide the theoretical and experimental basis for new therapeutic strategies for p16-related gene therapy.

p16是目前已知的抑癌基因中唯一通过直接抑制细胞周期而抑制细胞生长的基因，在肿瘤基因治疗中极具潜力。有研究表明p16蛋白的152位丝氨酸的磷酸化能促进p16与CDK4的结合，而8位丝氨酸磷酸化作用刚好相反，p16蛋白是否还存在其他翻译后修饰参与其功能的调控未见报道。我们的前期研究证实p16蛋白的甲基化存在于多种细胞系中，p16蛋白的22位、131位、138位精氨酸受到甲基化修饰。此外，p16蛋白精氨酸甲基化水平下降会促进p16与CDK4的结合并有效地抑制细胞周期进行。本项目拟在现有工作的基础上，进一步确定哪个信号通路或激酶参与p16的磷酸化修饰，探索p16的磷酸化和精氨酸甲基化修饰之间的关系，阐明磷酸化修饰与精氨酸甲基化对p16诱导的细胞衰老与细胞凋亡的影响及其分子机制，为p16抑制肿瘤提供新的理论依据，并为以后利用p16进行基因治疗肿瘤提供新的实验证据。

细胞周期依赖激酶抑制子p16INK4a (p16)蛋白抑制细胞周期依赖蛋白激酶CDK4或CDK6的活性，来抑制眼癌蛋白Rb的磷酸化促进低磷酸化Rb蛋白的积累导致细胞周期停滞，从而在细胞周期进程、细胞衰老和细胞凋亡中起重要作用。研究表明p16经常在很多种肿瘤（包括黑色素瘤，非小型肺癌，乳腺癌，结直肠癌，膀胱癌和头颈部鳞状细胞癌等）细胞中异常表达。多种机制参与p16的表达调控，包括转录调控、DNA缺失及启动子磷酸化等。本研究中，我们发现过氧化氢以剂量依赖的方式诱导293T细胞凋亡，也能诱导p16蛋白的磷酸化修饰。免疫共沉淀的实验结果表明过氧化氢诱导的p16磷酸化促进了p16与CDK4的相互作用。在p16突变体的过表达实验中我们发现相对于野生型的p16蛋白，第138位精氨酸突变为赖氨酸后的p16蛋白抑制细胞周期进行及诱导细胞凋亡的能力更强，而140位精氨酸突变为丙氨酸的p16蛋白的功能减弱。此外，我们发现140位丝氨酸突变后的p16精氨酸甲基化水平升高，而138位精氨酸突变的p16的丝氨酸磷酸化的水平升高。在WI-38细胞中的实验结果表明野生型的p16与过氧化氢均能促进细胞的衰老，并具有协同效果。总之，本研究的实验结果显示p16的138位精氨酸的甲基化修饰与140位丝氨酸的磷酸化修饰在调控细胞周期及细胞凋亡的功能方面具有相互拮抗的作用。p16是目前已知的抑癌基因中唯一通过直接抑制细胞周期而抑制细胞生长的基因，p16与人类多种肿瘤的发生、发展的关系非常密切，其致癌机制直接、清楚，作用对象单一，并且该基因小，易于操作，所以p16在肿瘤的基因治疗中极具潜力。本研究为p16相关的基因治疗提供了理论和实验基础。