DETAILED ANALYSIS OF P21/CDK2 INTERACTION

这是一个为研究项目提供部分支持的香农奖 低于指定研究所的资助范围，但在 卓越边际。香农奖旨在提供支持 测试该方法的可行性；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； 或进行独立的项目，以证明PI的研究 能力或为已经功成名就的 申请。下面的摘要摘自原始文件 由首席调查员提交。 这项提议的目的是对蛋白质进行详细分析- P21的蛋白质相互作用及氨基酸残基的测定 这种蛋白质和CDK2对于这种相互作用是至关重要的。我们也 希望找出可能在很小程度上改变这种相互作用的突变体 但在功能上意义重大。这将由 “双标记”试验在正文中描述。我们在第一部中展示了 该检测方法能够检测到 CDK2和p21之间的相互作用。此外，我们已经本地化了 P21的CDK2结合区与近35个氨基酸残基结合 该基因的5‘端与另一种CDK p27Kip1有同源性 由转化生长因子β诱导的抑制作用。使用一种“修饰”的p21基因 在保守区域引入了沉默突变，以产生 独特的，每30-40个碱基6个碱基的限制性内切酶，我们将创建一个 突变的p21基因的“库”，其中的每一个氨基酸都会发生变化。 然后将对这些突变的p21基因进行结合能力测试。 CDK2采用“双标记”分析。噬菌体编码突变体p21 保留其与CDK2结合能力的分子将被取回， 对斑块进行纯化和测序。通过这种方式，我们应该能够识别 P21上的残基是与CDK2结合的关键。我们会 通过进行体外和体内结合来确认这些结果 实验和蛋白激酶抑制试验。我们将执行类似的操作 P21与cdk2结合位点定位实验。我们也会 使用“双重标记”分析试图识别“补偿性” CDK2上的突变，可以恢复P21突变体的结合 它们的一种或多种“关键”氨基酸发生了突变。就这样， 我们可以确定两种蛋白质之间的接触点，并将它们联系起来 与已发表的CDK2的晶体结构进行了比较。最后，我们会 进行“竞争”实验，在这些实验中我们将尝试生成 P21突变体对CDK2的亲和力高于野生型 P21，如正文所述。他们还将接受测试，以确定他们的 抑制CDK激酶的能力，与其结合的亲和力 量过了。在正文中描述了附加的实验， 包括p21 CDK选择性突变体的筛选。总的来说， 这些数据应该提供分子工具，可以用来探索 P21诱导的抑制和促进作用的性质和机制 基于CDK抑制的抗癌新策略研究进展 肿瘤细胞的数量。

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.