The Identification and characterization of the methylation of Retinablastoma

描述(申请人提供)：视网膜母细胞瘤(RB)蛋白是细胞周期进程、衰老和分化的关键调节因子。直到最近，Rb功能的调控一直被认为是通过细胞周期蛋白依赖性蛋白激酶的磷酸化来实现的。然而，最近还发现了Rb蛋白的其他几个翻译后修饰，包括乙酰化和总甲基化。Rb的这些修饰可能会影响其在成年细胞中的功能，确保发育和老化生物体细胞中的动态平衡。我们的长期目标是更好地了解哺乳动物细胞中控制Rb功能的细胞机制，重点是Rb的修饰及其功能相互作用。组蛋白甲基化是控制染色质结构和基因表达的关键细胞过程。新出现的证据表明，非组蛋白蛋白的甲基化也起着重要的细胞作用。在这里，我们建议研究哺乳动物细胞中Rb甲基化及其调控。我们发现Rb在特定的赖氨酸残基上被SMYD2甲基转移酶甲基化。我们推测该甲基化事件参与了Rb功能的调控。我们的第一个具体目标是检查这一甲基化事件的调节。为此，我们将在不同的细胞环境中、在培养中和在小鼠中研究Rb甲基化模式。特别是，我们将研究细胞周期阶段、分化状态、衰老和细胞应激的存在如何影响SMYD2的Rb甲基化。我们的第二个具体目标是确定SMYD2基因Rb甲基化的后果。基于先前关于甲基化如何影响另一种细胞周期和细胞死亡调节因子p53功能的观察，我们将结合生化、分子和细胞方法来分析不同条件下Rb的功能，包括DNA损伤反应和细胞周期进展。总之，这些实验将为RB在细胞中的作用模式提供新的见解。Rb是多种细胞过程的中央调节因子。更好地了解哺乳动物细胞中控制Rb功能的机制，最终将为控制癌症等年龄相关疾病的Rb功能提供新的方法。

DESCRIPTION (provided by applicant): The Retinoblastoma (RB) protein is a critical regulator of cell cycle progression, senescence, and differentiation. Until recently, the control of RB function was thought to be achieved through its phosphorylation by cyclin-dependent kinases. However, several other post-translational modifications of the RB protein have recently been uncovered, including acetylation and sumoylation. These modifications of RB may affect its functions in adult cells, ensuring homeostasis in cells of developing and aging organisms. Our long-term goal is to better understand the cellular mechanisms controlling RB function in mammalian cells, focusing on the modifications of RB and their functional interactions. Methylation of histones is a key cellular process to control chromatin structure and gene expression. Emerging evidence suggests that methylation of non-histone proteins also plays important cellular roles. Here, we propose to investigate RB methylation and its regulation in mammalian cells. We have found that RB is methylated at a specific lysine residue by the SMYD2 methyltransferase. We hypothesize that this methylation event participates in the control of RB function. Our first specific goal is to examine the regulation of this methylation event. To this end, we will study RB methylation pattern in various cellular contexts, in culture and in mice. In particular, we will investigate how the cell cycle stage, the differentiation status, senescence, and the presence of cellular stress may affect RB methylation by SMYD2. Our second specific aim is to determine the consequences of RB methylation by SMYD2. Based on previous observations regarding how methylation affects the function of p53, another cell cycle and cell death regulator, we will use a combination of biochemical, molecular, and cellular approaches to assay RB function under different conditions, including DNA damage response and cell cycle progression. Together, these experiments will provide novel insights into the mode of action of RB in cells. RB is a central regulator of multiple cellular processes. A better understanding of the mechanisms that control RB function in mammalian cells will ultimately provide novel ways to control RB function in age- related diseases such as cancer.