DETAILED ANALYSIS OF P21/CDK2 INTERACTION

P21/CDK2 相互作用的详细分析

• 批准号: 2895348
• 负责人: F J GERMINO
• 金额: $ 11.93万
• 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/2895348
• 关键词: cell cycle; chemical binding; cyclin dependent kinase; enzyme linked immunosorbent assay; flow cytometry; gene mutation; guanine nucleotide binding protein; molecular site; oncoprotein p21; oncoproteins; protein protein interaction; 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.

这是一个香农奖，为研究项目提供部分支持 不属于指定研究所的资助范围，但在 卓越的边缘。 香农奖旨在提供支持 测试该方法的可行性;开发进一步的测试和改进 研究技术;对现有数据集进行二次分析; 或进行离散的项目，可以证明PI的研究 能力或增加已经有价值的 应用程序. 以下摘要摘自原始文件 由主要研究者提交。 这项提案的目的是对蛋白质进行详细分析- 蛋白质相互作用的p21，并确定氨基酸残基上 这种蛋白质和CDK 2对这种相互作用至关重要。 我们也 希望能鉴定出能在很小范围内改变这种相互作用突变体， 但功能上很重要 这将由 本文中描述的“双标记”测定。 我们首先展示 本拨款提案的一部分，该检测方法能够检测 CDK 2与p21的相互作用 此外，我们还将 p21的CDK 2结合结构域与附近的大约35个氨基酸区域 该基因的5 '端与另一个CDK p27 Kip 1具有同源性 TGF β诱导的抑制剂。 使用“修饰的”p21基因， 已经在保守区域中引入沉默突变以产生 独特的，每30-40 bp 6 bp限制性酶切位点，我们将创建一个 突变p21基因的“文库”，其中每个氨基酸都将被改变。 然后将测试这些突变的p21基因结合至 使用“双标记”测定法测定CDK 2。 噬菌体编码突变体p21 保留其与CDK 2结合能力的分子将被回收， 噬斑纯化并测序。 这样我们就能识别出 p21上对结合CDK 2至关重要的残基。 我们将 通过进行体外和体内结合来证实这些结果 实验和激酶抑制测定。 我们将执行类似 实验定位p21在CDK 2上的结合位点。 我们也将 使用“双标记”试验来尝试识别“补偿性” CDK 2上的突变，其可以恢复P21突变体的结合， 有一个或多个“关键”氨基酸发生了突变。 通过这种方式， 我们就能确定这两种蛋白质之间的接触点 它们与已公布的CDK 2晶体结构一致。 最后我们将 进行“竞争”实验，我们将尝试生成 对CDK 2表现出比野生型更高亲和力的P21突变体 P21，如文中所述。 他们也将接受测试， 抑制CDK激酶的能力，并且它们的结合亲和力将是 测定了 在文本中描述了另外的实验， 包括筛选p21的CDK选择性突变体。 总的来说， 这些数据应该提供分子工具， p21诱导抑制的性质和机制， 基于CDK抑制的新型抗癌策略的开发 肿瘤细胞。