BIOCHEMICAL MECHANISMS OF AN RB-ASSOCIATED SP1 INHIBITOR

RB 相关 SP1 抑制剂的生化机制

• 批准号: 2110203
• 负责人: Robert Chiu
• 金额: $ 19.7万
• 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/2110203
• 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; 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p105 Rb（RB）通常是 被认为是一个重要的调节器，在控制细胞 增殖和调节细胞周期。生化 RB的作用机制尚不清楚。变换性质 的几种DNA肿瘤病毒癌蛋白依赖，至少部分， 它们与RB结合的能力，并可能将RB从其 细胞对应物。最近的报告表明，RB直接 与三种重要的转录因子E2 F、ATF-2和E1 F相互作用。 1.有了这些，很明显RB结合了许多细胞蛋白， 它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，六个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的机制 RB通过其抑制细胞增殖。 最近，我们已经证明RB激活c-jun的转录， 通过c-jun启动子内的Spl结合位点将基因导入。的 RB刺激Sl介导的反式激活的机制是 从抑制剂Spl-I释放Spl。这个令人兴奋的观察 需要进一步调查本提案的具体目标 1）分离和表征介导细胞因子的细胞因子， 抑制Spl结合活性。2.）的情况。研究生化机制 通过产生针对Spl抑制剂（Spl-1）的抗血清。3.）第三章以确定 导致刺激Spl介导的细胞凋亡的分子事件 成视网膜细胞瘤基因产物的反式激活。4.）为了研究Spl- I的表达或其与RB的结合是细胞周期依赖性的。这些 这些目的可以通过使用纯化蛋白质来实现， 肝素-琼脂糖，凝胶过滤和亲和层析，CAT测定， 凝胶阻滞试验、缺失、接头扫描突变体和位点- 定向诱变，细菌表达GST融合蛋白，细胞- 自由转录-翻译系统，免疫沉淀，32 p- 正磷酸标记细胞和同步化细胞。 Spl-I作为细胞靶点的分子机制研究 RB非常重要。这种Spl抑制剂的克隆将可能 深入了解其身份、功能和监管。我们认为 这一建议不仅将确定RB的生化功能， 也将对我们理解 两类不同的癌蛋白RB和c- Jun，参与细胞生长的控制，以及定义 c-jun表达调控的新机制。