CELLULAR FUNCTION OF THE RETINOBLASTOMA GENE PRODUCT

视网膜母细胞瘤基因产物的细胞功能

• 批准号: 2099023
• 负责人: JEAN Y.J. WANG
• 金额: $ 19.66万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-03 至 1997-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2099023
• 关键词: binding proteins; cell cycle; cell differentiation; gel filtration chromatography; gene deletion mutation; growth inhibitors; ion exchange chromatography; laboratory mouse; laboratory rabbit; microinjections; molecular cloning; nuclear magnetic resonance spectroscopy; oncoproteins; phosphorylation; protein sequence; protein structure function; protein tyrosine kinase; transfection; tumor suppressor genes

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.

视网膜母细胞瘤基因RB最初被认为是 人视网膜母细胞瘤抑制因子。 RB基因在许多细胞中表达， 在整个发育过程中，RB蛋白在细胞类型中起着重要的作用。 在调节细胞分裂和细胞分化中的作用。 的 RB功能的当前模型提出它结合转录因子， 例如E2 F，以抑制它们活性。 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 这个模型在原则上可能是正确的，它是基于一个相当肤浅的 RB蛋白的理解，并没有解释的表型， RB纯合子突变小鼠。 该模型的两个方面过于简单， 从我们最近的结果来看。 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E2 F，RB含有C-末端蛋白结合 域（CPBD）。 我们可以证明，CPBD和A/B口袋在功能上是 独立的 其次，我们已经绘制了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， 不同的位点可能调控RB的不同结合域。 这 研究结果表明，RB可以存在于几种功能状态之一， 这取决于哪个位点被磷酸化。 我们将继续努力， RB蛋白的两个新的见解，我们的近期目标包括 RB蛋白作为分子功能的假设的检验 “媒人”，以促进特定蛋白质复合物的组装， 原子核 我们建议RB通过其两个约束力 结构域，蛋白质，否则可能不会相互作用。 每个 RB介导的蛋白质复合物可由磷酸化 RB，并且这些复合物的组成可以通过 特定位点的磷酸化。 为了验证这一假设，我们将重点 四个具体目标： (1)进一步表征C-末端蛋白结合结构域（CPBD） 的RB。 我们将定义这个域的极小序列，确定它的 溶液结构，并研究它是否结合其他核 酪氨酸激酶 （二）、 为了确定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结构域中的任何一个的破坏将 别理他 如果是这样的话，RB的CPBD的过度生产应该竞争 结合细胞蛋白并中和RB功能。 （三）、 到 确定C-末端磷酸化的特定调节作用 RB的位置。 将制备特异性磷酸化位点突变， 它们对A/B口袋和CPBD活性的影响将是 测量以证明差分调节。 （四）、 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 对RB的生物学功能有不同的调节作用。