CELLULAR FUNCTION OF THE RETINOBLASTOMA GENE PRODUCT

视网膜母细胞瘤基因 RB 最初被确定为假定的 人类视网膜母细胞瘤的抑制剂。 RB基因在多种细胞中表达 细胞类型在整个发育过程中起着重要作用 在细胞分裂和细胞分化的调节中发挥作用。 的 当前的 RB 功能模型表明它结合转录因子， 例如E2F，以抑制其活性。 RB 的磷酸化 细胞周期蛋白依赖性蛋白激酶使蛋白结合功能失活， 并导致对细胞分裂重要的基因的激活。 虽然 这个模型原则上可能是正确的，它是基于相当肤浅的 对 RB 蛋白的理解，并没有解释 RB 蛋白的表型 RB纯合突变小鼠。 该模型的两个方面过于简单化： 我们最近的结果表明。 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”之外 结合病毒癌蛋白和 E2F，RB 包含 C 端蛋白结合 域（CPBD）。 我们可以证明，CPBD 和 A/B 口袋在功能上是 独立。 其次，我们绘制了 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 不同的位点可能调节RB的不同结合域。 这个 研究结果表明 RB 可以以几种功能状态之一存在 取决于哪些位点被磷酸化。 我们将追求这些 关于 RB 蛋白的两个新见解，我们的近期目标包括 检验 RB 蛋白作为分子的假设 “媒人”促进特定蛋白质复合物的组装 核。 我们建议 RB 通过其两个绑定将 域，否则可能不会彼此相互作用的蛋白质。 每个 RB 介导的蛋白质复合物可以通过磷酸化激酶进行调节 RB，这些复合物的组成可以通过 特定位点的磷酸化。 为了检验这个假设，我们将重点关注 以下四个具体目标； (1) 进一步表征C端蛋白结合域(CPBD) RB。 我们将定义该域的最小序列，确定其 通过 NMR 检测溶液结构并研究其是否与其他核结合 酪氨酸激酶。 （2）。 确定 CPBD 是否会干扰 野生型RB蛋白的功能。 “牵线搭桥”假说 预测两个蛋白质结合域中的任何一个的破坏都会 使 RB 失活。 如果是这样，RB 的 CPBD 生产过剩应该竞争 细胞蛋白的结合并中和 RB 功能。 （3）。 至 确定C端磷酸化的具体调节作用 RB 站点。 将制备特定的磷酸化位点突变并 它们对 A/B 袋和 CPBD 活性的影响将是 测量以证明差异调节。 （4）。 为了研究 磷酸化时特定 Ala 和 Glu 突变的生物学效应 网站。 我们将研究不同的磷酸化位点是否 对RB的生物学功能具有不同的调节作用。

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.