DETAILED ANALYSIS OF P21/CDK2 INTERACTION

P21/CDK2 相互作用的详细分析

• 批准号: 2712741
• 负责人: F J GERMINO
• 金额: $ 3.86万
• 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/2712741
• 关键词: cell cycle; chemical binding; cyclin dependent kinase; enzyme linked immunosorbent assay; flow cytometry; gene mutation; guanine nucleotide binding protein; intermolecular interaction; molecular site; oncoprotein p21; oncoproteins; 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.

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