CELLULAR FUNCTION OF THE RETINOBLASTOMA GENE PRODUCT

视网膜母细胞瘤基因Rb最初被认为是 人类视网膜母细胞瘤的抑制因子。Rb基因在许多组织中表达 在整个发育过程中，细胞类型和Rb蛋白起着重要的作用 在调节细胞分裂和细胞分化中的作用。这个 目前的Rb功能模型认为它与转录因子结合， 如E2F，以抑制其活性。Rb的磷酸化 细胞周期蛋白依赖的蛋白激酶使蛋白结合功能失活， 并导致对细胞分裂至关重要的基因的激活。虽然 这个模型原则上可能是正确的，它是建立在一个相当肤浅的基础上的 对Rb蛋白的理解，并不能解释 Rb纯合子突变小鼠。这个模型有两个方面过于简单化，因为 从我们最近的结果可以看出。首先，我们发现RB包含两个 蛋白质结合域。除了所谓的“A/B口袋”， 结合病毒癌蛋白和E2F，Rb含有一个C末端蛋白结合 域(CPBD)。我们可以看到，CPBD和A/B口袋在功能上是 独立自主。其次，我们定位了Rb中的八个磷酸化位点。 使用磷酸化位点突变体的初步结果表明 不同的结合部位可能调节Rb的不同结合域。这 研究结果表明，Rb可以以几种功能状态之一存在 这取决于哪些位点是磷酸化的。我们将致力于实现这些目标 关于Rb蛋白的两个新见解，我们的近期目标包括 对Rb蛋白作为分子功能的假设的检验 “媒人”促进特定蛋白质复合体在体内的组装 原子核。我们建议RB通过它的两个绑定将 结构域，否则可能不会相互作用的蛋白质。每个人 Rb介导的蛋白复合体可由磷酸化的激酶调节 Rb，并且这些络合物的组成可以通过 特定位点的磷酸化。为了检验这一假设，我们将重点关注 关于以下四个具体目标； (1)进一步鉴定C端蛋白结合域(CPBD) RB的。我们将定义该属性域的最小序列，确定其 用核磁共振研究溶液的结构并研究它是否与其他核结合 酪氨酸激酶。(2)。以确定CPBD是否可以干扰 野生型Rb蛋白的功能。“相亲”假说 预测两个蛋白质结合域中的任何一个的破坏将 停用RB。如果是这样的话，RB的CPBD生产过剩应该竞争 结合细胞蛋白，中和Rb功能。(3)。至 确定C末端磷酸化的特定调节作用 RB的遗址。将准备特定的磷酸化位点突变并 它们对A/B袋和CPBD的活动的影响将是 测量以演示差异调节。(4)。为了研究 丙氨酸和谷氨酸特异性突变对磷酸化的生物学效应 网站。我们将调查不同的磷酸化位点 对Rb的生物学功能有不同的调节作用。

The retinoblastoma gene, RB, was initially identified as the putative suppressor of retinoblastoma in human. The RB gene is expressed in many cell types throughout development and the RB protein plays an important role in the regulation of cell division and cellular differentiation. The current model of RB function proposes that it binds transcription factors, such as E2F, to inhibit their activity. Phosphorylation of RB by the cyclin-dependent protein kinases inactivates the protein binding function, and leads to the activation of genes important to cell division. Although this model may in principle be correct, it is based on a rather superficial understanding of the RB protein and does not explain the phenotypes of the RB homozygous mutant mice. Two aspects of this model are too simplistic as indicated by our recent results. First, we have found that RB contains two protein binding domains. In addition to the so-called "A/B pocket" that binds viral oncoproteins and E2F, RB contains a C-terminal protein binding domain (CPBD). The CPBD and the A/B pocket, we can show, are functionally independent. Second, we have mapped eight phosphorylation sites in RB. Preliminary results using phosphorylation site mutants have indicated that the different sites may regulate the different binding domains of RB. This finding suggests that RB can exist in one of several functional states depending on which of the sites are phosphorylated. We will pursue these two new insights on the RB protein and our immediate goal includes the testing of a hypothesis that the RB protein functions as a molecular "matchmaker" to promoter the assembly of specific protein complexes in the nucleus. We propose that RB brings together, through its two binding domains, proteins that otherwise may not interact with one another. Each RB-mediated protein complex can be regulated by kinases that phosphorylate RB, and the composition of those complexes can be altered by the phosphorylation of specific sites. To test this hypothesis we will focus on the following four specific aims; (1) To further characterize the C-terminal protein binding domain (CPBD) of RB. We will define the minimal sequences of this domain, determine its solution structure by NMR and investigate whether it binds other nuclear tyrosine kinases. (2). To determine if the CPBD can interfere with the function of the wild type RB protein. The "matchmaking" hypothesis predicts that disruption of either of the two protein binding domains will inactivate RB. If so, overproduction of the CPBD of RB should compete for the binding of cellular proteins and neutralize the RB function. (3). To determine the specific regulatory roles of the C-terminal phosphorylation sites of RB. Specific phosphorylation site mutations will be prepared and their effect on the activity of the A/B pocket and the CPBD will be measured to demonstrate the differential regulation. (4). To study the biological effects of specific Ala and Glu mutations at the phosphorylation sites. We will investigate whether the different phosphorylation sites have different regulatory roles on the biological function of RB.