DETAILED ANALYSIS OF P21/CDK2 INTERACTION

P21/CDK2 相互作用的详细分析

• 批准号: 2008946
• 负责人: F J GERMINO
• 金额: $ 11.75万
• 依托单位: UNIV OF MED/DENT NJ-R W JOHNSON MED SCH
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-06-01 至 2002-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2008946
• 关键词: cell cycle; chemical binding; cyclins; enzyme linked immunosorbent assay; flow cytometry; gene mutation; guanine nucleotide binding protein; intermolecular interaction; molecular site; oncoproteins; protein kinase; protein structure function; transfection; yeasts

This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. The abstract below is taken from the original document submitted by the principal investigator. The aim of this proposal to perform a detailed analysis of the protein- protein interactions of p21 and to determine the amino acid residues on this protein and CDK2 which are critical for this interaction. We also hope to identify mutants which alter this interaction in perhaps small but functionally significant ways. This will be accomplished by the "double-tagging" assay described in the text. We show in the first section of this grant proposal that this assay is capable of detecting the interaction between CDK2 and p21. Furthermore, we have localized the CDK2-binding domain of p21 to an approximately 35 amino acid region near the gene's 5'end which shares homology with p27Kip1, another CDK inhibitor induced by TGFbeta. Using a "modified" p21 gene in which silent mutations have been introduced in the conserved region to generate unique, 6 bp restriction enzyme sites every 30-40 bp, we will create a "library" of mutant p21 genes in which every amino acid will be altered. These mutant p21 genes will then be tested for their ability to bind to CDK2 using the "double-tagging" assay. Phage encoding mutant p21 molecules which retain their ability to bind to CDK2 will be retrieved, plaque purified and sequenced. In this way we should be able to identify the residues on p21 which are critical for binding to CDK2. We will confirm these results by performing in vitro and in vivo binding experiments and kinase inhibition assays. We will perform similar experiments to localize the binding sites of p21 on CDK2. We also will use the "double-tagging" assay to attempt to identify "compensatory" mutations on CDK2 which can restore the binding of P21 mutants which have had one or more of their "critical" amino acids mutated. In this way, we can identify the points of contact between the two proteins and relate them to the published crystal structure of CDK2. Finally, we will perform "competition" experiments in which we will attempt to generate mutants of P21 which exhibit higher affinity for CDK2 then does wild-type P21, as described in the text. They also will be tested for their ability to inhibit CDK kinase, and their binding affinity will be measured. Additional experiments are described within the text, including the screening for CDK-selective mutants of p21. In total, these data should provide molecule tools which can be employed to explore the nature and mechanism of p21-induced inhibition and perhaps facilitate the development of novel anti-cancer strategies based upon CDK inhibition of tumor cells.

这是香农奖，为研究项目提供部分支持 低于指定机构的资助范围但在 卓越边际。 香农奖旨在提供支持 测试该方法的可行性；进行进一步的测试并完善 研究技术；对可用数据集进行二次分析； 或开展可以展示 PI 研究成果的离散项目 能力或对已经有功绩的人给予额外的重视 应用。 以下摘要摘自原始文件 由主要研究者提交。 该提案的目的是对蛋白质进行详细分析 p21 的蛋白质相互作用并确定其氨基酸残基 这种蛋白质和 CDK2 对于这种相互作用至关重要。 我们也 希望找到能够在很小的范围内改变这种相互作用的突变体 但功能上重要的方式。 这将通过 文中描述了“双标记”测定。 我们在第一个展示 该拨款提案的一部分表明该测定能够检测 CDK2 和 p21 之间的相互作用。 此外，我们还本地化了 p21 的 CDK2 结合域位于附近约 35 个氨基酸区域 该基因的 5' 末端与另一个 CDK p27Kip1 具有同源性 TGFbeta 诱导的抑制剂。 使用“修饰的”p21 基因，其中 沉默突变已被引入保守区域以产生 每 30-40 bp 有一个独特的 6 bp 限制性内切酶位点，我们将创建一个 突变p21基因的“文库”，其中每个氨基酸都会被改变。 然后将测试这些突变 p21 基因结合的能力 CDK2 使用“双标记”测定法。 编码突变体 p21 的噬菌体 保留与 CDK2 结合能力的分子将被回收， 噬菌斑纯化并测序。 这样我们应该能够识别 p21 上的残基对于与 CDK2 的结合至关重要。 我们将 通过进行体外和体内结合证实这些结果 实验和激酶抑制测定。 我们也会进行类似的操作 定位 p21 在 CDK2 上的结合位点的实验。 我们也会 使用“双标记”测定来尝试识别“补偿” CDK2 上的突变可以恢复 P21 突变体的结合， 他们的一个或多个“关键”氨基酸发生了突变。 这样， 我们可以识别两种蛋白质之间的接触点并将其关联起来 将它们与已发布的 CDK2 晶体结构联系起来。 最后，我们将 进行“竞争”实验，我们将尝试生成 P21 突变体对 CDK2 的亲和力高于野生型 P21，如文中所述。 他们还将接受测试 抑制 CDK 激酶的能力，其结合亲和力为 测量。 文本中描述了其他实验， 包括筛选 p21 的 CDK 选择性突变体。 总共， 这些数据应该提供可用于探索的分子工具 p21 诱导抑制的性质和机制，或许有助于 基于CDK抑制的新型抗癌策略的开发 肿瘤细胞。