BIOCHEMICAL MECHANISMS OF AN RB-ASSOCIATED SP1 INHIBITOR

视网膜母细胞瘤易感基因产物p105Rb(Rb)通常是 被认为是控制细胞的重要调节因子 在细胞增殖和调节细胞周期中起重要作用。生物化学 RB的行动机制仍不清楚。变换性质 几种DNA肿瘤病毒癌蛋白至少部分地依赖于， 关于它们与Rb结合的能力，并可能将Rb从其 细胞对应的细胞。最近的报道表明，RB直接 与三个重要的转录因子E2F、ATF-2和ELF-2相互作用 1.有了这些，很明显Rb结合了许多细胞蛋白， 它可能直接或间接地参与转录 对控制细胞生长所需的一组基因的调节。 事实上，六个细胞基因已经被确定为靶点 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，我们发现Rb可以激活c-jun的转录。 基因通过c-jun启动子内的SPL结合部位。这个 Rb刺激SPL介导的反式激活的机制是 将SPL从抑制剂SPL-I中释放出来。这一令人兴奋的观察结果 需要进一步调查。这项建议的具体目标 L)分离和鉴定介导细胞因子的细胞因子(S 抑制SPL结合活性。2.)研究生化机制 通过产生针对SPL抑制物(SPL-I)的抗血清。3.)要确定 导致SP L刺激的分子事件 视网膜母细胞瘤基因产物的反式激活。4.)为了研究SPL是否- I的表达或其与RB的结合依赖于细胞周期。这些 目标可以通过使用以下方法纯化蛋白质来实现 肝素-琼脂糖凝胶过滤和亲和层析，过氧化氢酶检测， 凝胶滞留分析、缺失、接头扫描突变体和位点- 定向突变，细菌表达的GST融合蛋白，细胞- 自由转录-翻译系统，免疫沉淀，32P- 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是至关重要的。这种SPL抑制剂的克隆很可能 提供对其身份、功能和监管的洞察。我们相信 这项提议不仅将确定RB的生化功能， 但也将对我们理解一个 两类不同的癌蛋白Rb和c-之间的功能联系- Jun，它们参与了细胞生长的控制以及定义 一种调控c-jun表达的新机制。

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.