Deciphering the code of RB phosphorylation

项目总结 为了简单起见，我们倾向于认为大多数蛋白质都有单一的作用机制。视网膜母细胞瘤 肿瘤抑制因子就是一种挑战这种简单分类的蛋白质。Rb1在功能上 大多数人类癌症中的灭活及其蛋白产物(Rb)的分子性质已经被研究 紧锣密鼓。尽管进行了这项研究，RB的作用机制仍然是一个谜。据报道，RB 以物理方式与数百种蛋白质结合，许多不同的相互作用已被提出 有助于其抑制肿瘤的特性。RB研究界面临着一个难题： 在这些相互作用中，有些是真实的，有些不是，一种蛋白质如何协调其对如此多的 潜在目标？最近几个实验室的研究表明，这些问题的答案 问题在于Rb磷酸化的密码。其概念是正常细胞不包含单一形式的 Rb，但这种差异磷酸化产生具有不同结合的多种Rb亚型 属性，并且可能扮演不同的角色。本质上，Rb的作用是通过磷酸化来调节的。 已知Rb有14个CDK磷酸化位点。我们最近开发了一些方法，使我们能够 使用基于质谱学的蛋白质组学来分析Rb复合体。我们还生成了等基因的电池板 在细胞培养中，我们可以用突变的Rb蛋白取代内源性的Rb蛋白，这种突变的Rb蛋白只包含一个 单一的cdk磷酸化位点，或单一的模拟磷酸化突变。在本应用程序中，我们建议使用 这些工具可以破译Rb磷酸化的密码。在目标1中，我们将使用最先进的蛋白质组学来 定义RB的每个单磷酸化异构体的结合特性。在目标2中，我们将确定 这些相互作用的功能后果通过识别它们控制的转录程序和 通过它们所针对的基因组位置。使用此结合信息和转录配置文件，我们将识别 允许RB的特定单磷酸化异构体控制不同程序的分子相互作用 抄写。这些实验将共同产生一个分子信息框架，该框架对 能够理解RB的作用机制。

Project summary For simplicity we tend to imagine that most proteins have a single mechanism of action. The retinoblastoma tumor suppressor is an example of a protein that defies this simple classification. RB1 is functionally inactivated in most human cancers and the molecular properties of its protein product (RB) have been studied intensively. Despite this research, RB's mechanism of action has remained an enigma. RB has been reported to physically associate with hundreds of proteins and many different interactions have been proposed to contribute to its tumor suppressive properties. The RB research community is faced with a conundrum: which of these interactions are real, which are not, and how could one protein co-ordinate its effects on so many potential targets? Recent studies from several laboratories have suggested that the answers to these questions lie in a code of RB phosphorylation. The concept is that normal cells do not contain a single form of RB, but that differential phosphorylation generates multiple isoforms of RB that have different binding properties and, presumably, perform different roles. In essence, the action of RB is tailored by phosphorylation. RB is known to have 14 sites of CDK phosphorylation. We have recently developed methods that allow us to use mass spectrometry-based proteomics to profile RB complexes. We have also generated panels of isogenic cell cultures in which we can replace the endogenous RB protein with mutant RB proteins that contain just a single cdk phosphorylation site, or a single phospho-mimicking mutation. In this application we propose to use these tools to decipher this code of RB phosphorylation. In Aim 1 we will use state-of-the-art proteomics to define the binding properties of each of the mono-phosphorylated isoforms of RB. In Aim 2 we will identify the functional consequences of these interactions by identifying the transcriptional programs that they control and by genomic loci that they target. Using this binding information and transcription profiles we will identify the molecular interactions that allow specific mono-phosphorylated isoforms of RB to control distinct programs of transcription. Together these experiments will generate a framework of molecular information that is critical to be able to understand RB's mechanism of action.