Deciphering the code of RB phosphorylation

项目摘要 为了简单起见，我们倾向于想象大多数蛋白质具有单一的作用机制。视网膜母细胞瘤 肿瘤抑制因子是一种不符合这种简单分类的蛋白质。RB1在功能上 在大多数人类癌症中失活，并对其蛋白产物（RB）的分子特性进行了研究 强烈地。尽管有这些研究，RB的作用机制仍然是一个谜。已报告RB 与数百种蛋白质发生物理联系，已经提出了许多不同的相互作用， 有助于其肿瘤抑制特性。RB研究社区面临着一个难题： 这些相互作用中的一种是真实的，而另一种则不是，一种蛋白质如何协调它对如此多的相互作用的影响呢 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。这个概念是，正常细胞不包含单一形式的 RB，但是差异磷酸化产生具有不同结合的RB的多种亚型， 属性，并且可能执行不同的角色。本质上，RB的作用是通过磷酸化来定制的。 已知RB具有14个CDK磷酸化位点。我们最近开发了一种方法， 使用基于质谱的蛋白质组学来分析RB复合物。我们还生成了一组同基因的 在细胞培养中，我们可以用突变的RB蛋白代替内源性RB蛋白， 单个CDK磷酸化位点或单个磷酸化模拟突变。在本申请中，我们建议使用 这些工具来破译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， 定义RB的每种单磷酸化同种型的结合特性。在目标2中，我们将确定 通过识别它们控制的转录程序， 通过它们所针对的基因组位点。使用这种结合信息和转录谱，我们将确定 分子间的相互作用，使特定的单磷酸化亚型的RB控制不同的程序， 转录。这些实验将共同产生一个分子信息框架， 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.