BIOCHEMICAL MECHANISMS OF AN RB-ASSOCIATED SP1 INHIBITOR

RB 相关 SP1 抑制剂的生化机制

• 批准号: 2654167
• 负责人: Robert Chiu
• 金额: $ 22.16万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-01 至 2000-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2654167
• 关键词: affinity chromatography; antiserum; cell cycle; cell free system; chemical binding; fusion gene; gel filtration chromatography; gel mobility shift assay; gene deletion mutation; gene expression; genetic promoter element; genetic transcription; immunoprecipitation; laboratory rabbit; molecular site; oncoproteins; protein purification; protooncogene; retinoblastoma protein; site directed mutagenesis; synchronous cell division; transcription factor; tumor suppressor genes

The retinoblastoma susceptibility gene product p105Rb (RB) is generally believed to be an important regulator in the control of cellular proliferation and in regulating the cell cycle. The biochemical mechanisms for RB's action remain unclear. The transforming properties of several DNA tumor virus oncoproteins are dependent, at least in part, on their ability to bind to RB and presumably to sequester RB from its cellular counterparts. Recent reports have suggested that RB directly interacts with three important transcription factors, E2F, ATF-2 and ElF- 1. With these, it is clear that RB binds a number of cellular proteins, which may be directly or indirectly involved in transcriptional regulation of a set of genes required for controlling cell growth. Indeed, Six cellular genes have been identified as targets of transcriptional regulation by RB. These results reveal a new mechanism by which RB constrains cellular proliferation. Recently, we have demonstrated that RB activates transcription of c-jun gene through the Spl binding site within the c-jun promoter. The mechanism by which RB stimulates Spl-mediated transactivation is liberation of Spl from an inhibitor, Spl-I. This exciting observation warrants further investigation. The specific objectives of this proposal are: l) To isolate and characterize the cellular factor(s) that mediate inhibition of Spl binding activity. 2.) To study biochemical mechanisms by generation of antisera against Spl inhibitor (Spl-I). 3.) To determine the molecular events leading to stimulation of Sp l -mediated transactivation by retinoblastoma gene product. 4.) To study whether Spl- I expression or its binding to RB is cell cycle dependent. These objectives can be accomplished by purification of protein by using heparin-agarose, gel filtration and affinity chromatography, CAT assay, gel retardation assays, deletion, linker scanning mutant and site- directed mutagenesis, bacterially expressed GST fusion proteins, cell- free transcription-translation system, immunoprecipitation, 32p- orthophosphate labeled cells and synchronized cells. The study of the molecular mechanisms of Spl-I as a cellular target for RB is critically important. The cloning of this Spl inhibitor will likely provide insight into its identity, function and regulation. We believe that this proposal will not only identify a biochemical function for RB, but also will have a significant impact on our understanding of a functional link between two distinct classes of oncoproteins, RB and c- Jun, that are involved in the control of cell growth, as well as defining a novel mechanism for the regulation of c-jun expression.

视网膜母细胞瘤易感基因产物p105Rb（RB）一般为 被认为是细胞控制的重要调节因子 增殖和调节细胞周期。生化 RB 的作用机制仍不清楚。转化属性 几种 DNA 肿瘤病毒癌蛋白至少部分依赖于 关于它们与 RB 结合并可能将 RB 与其隔离的能力 细胞对应物。最近的报告表明 RB 直接 与三个重要的转录因子 E2F、ATF-2 和 ElF 相互作用 1. 通过这些，很明显 RB 结合了许多细胞蛋白， 可能直接或间接参与转录 控制细胞生长所需的一组基因的调节。 事实上，六种细胞基因已被确定为 RB 的转录调控。这些结果揭示了一种新机制 RB 通过其限制细胞增殖。 最近，我们证明RB激活c-jun的转录 基因通过 c-jun 启动子内的 Spl 结合位点。这 RB 刺激 Spl 介导的反式激活的机制是 从抑制剂 Spl-I 中释放 Spl。这个令人兴奋的观察 值得进一步调查。本提案的具体目标 是： l) 分离和表征介导的细胞因子 Spl结合活性的抑制。 2.) 研究生化机制 通过产生针对 Spl 抑制剂 (Spl-I) 的抗血清。 3.) 确定 导致 Sp l 介导的刺激的分子事件 视网膜母细胞瘤基因产物的反式激活。 4.) 研究是否Spl- I 表达或其与 RB 的结合是细胞周期依赖性的。这些 可以通过使用纯化蛋白质来实现目标 肝素琼脂糖、凝胶过滤和亲和层析、CAT 测定、 凝胶阻滞测定、缺失、接头扫描突变体和位点 定向诱变、细菌表达的 GST 融合蛋白、细胞- 游离转录-翻译系统，免疫沉淀，32p- 正磷酸盐标记的细胞和同步细胞。 Spl-I作为细胞靶点的分子机制研究 RB 至关重要。这种 Spl 抑制剂的克隆可能会 深入了解其身份、功能和监管。我们相信 该提案不仅会确定 RB 的生化功能， 也将对我们的理解产生重大影响 两种不同类别的癌蛋白 RB 和 c 之间的功能联系 Jun，参与细胞生长的控制，以及定义 调节c-jun表达的新机制。