Structure-Function Relationship of Tumor Suppressors

描述(由申请者提供)：在上一个资助期间，我们获得了重要的发现，表明四个重要的蛋白质Gankyrin、IkappaBalpha、Tax和SEI-1中的每一个都与细胞周期蛋白依赖性激酶4(CDK4)相互作用，可能有不同的机制。下一个授权期的主要目的是了解CDK4与三种结构性表达的人类蛋白--Gankyrin、IkappaBalpha和SEI-1相互作用的结构和生化机制和生物学功能，而病毒蛋白税不包括在这项修订的提案中。将使用三种主要方法：(A)使用核磁共振来确定溶液结构；(B)使用合理突变与活性分析相结合的方法来剖析特异性的结构基础；(C)使用基于荧光的结合分析来确定gankyrin、IkappaBalpha和SEI-1与CDK4的Kd。在特定的目标1中，我们计划使用定点突变来确定对其与CDK4结合至关重要的甘结蛋白的关键残基，并将使用基于荧光的分析来评估甘结蛋白及其突变体的Kd值。随后，通过合理的设计，将产生一组CDK4突变体，并将其用于定位参与甘草蛋白结合的CDK4残基。此外，将使用核磁共振和突变数据生成gankyrin/CDK4结构模型，与C.Weghorst合作，我们将表征从癌症组织中鉴定的gankyrin突变。在具体目标2中，我们计划解决IkappaBalpha(残基1-206)的CDK4结合域的溶液结构，它也是一个锚蛋白重复蛋白。与对gankyrin-CDK4相互作用所描述的相同的突变研究将被用于绘制IkappaBalpha和CDK4之间的相互作用。这些结果将允许比较gankyrin、IkappaBalpha和肿瘤抑制基因p16和p18的INK4家族。在具体目标3中，我们计划确定具有不同活性的SEI-1和/或其截短形式的结构，并使用定点突变来剖析对不同活性重要的残基，并鉴定对SEI-1与SEI-1的不同功能结构域结合至关重要的CDK4残基。

DESCRIPTION (provided by applicant): In the last funding period we obtained important findings suggesting that each of the four important proteins gankyrin, IkappaBalpha, Tax, and SEI-1 interacts with cyclin-dependent kinase 4 (CDK4), with potentially different mechanisms. The main thrust of the next granting period is to understand the structural and biochemical mechanisms and biological functions of the interactions between CDK4 and three constitutively expressed human proteins, gankyrin, IkappaBalpha, and SEI-1, while the viral protein Tax is not included in this revised proposal. Three major approaches will be used: (a) use of NMR to determine solution structures; (b) use of rational mutagenesis combined with activity assays to dissect the structural basis of specificity; (c) use of fluorescence-based binding assay to determine the Kd of gankyrin, IkappaBalpha, and SEI-1 with CDK4. In Specific Aim 1, we plan to use site-directed mutagenesis to identify key residues of gankyrin important for its binding to CDK4, and the Kd values of gankyrin and mutants will be evaluated using a fluorescence-based assay. Subsequently, a set of CDK4 mutants will be generated through rational design and will be used to map out the residues of CDK4 involved in the binding of gankyrin. Furthermore, a gankyrin/CDK4 structural model will be generated using both NMR and mutagenesis data, hi collaboration with C. Weghorst, we will characterize gankyrin mutants identified from cancer tissues. In Specific Aim 2, we plan to solve the solution structure of the CDK4-binding domain of IkappaBalpha (residues 1-206), which is also an ankyrin-repeat protein. The same mutagenesis studies as described for gankyrin-CDK4 interactions will be used to map out the interaction between IkappaBalpha and CDK4. These results will allow comparison between gankyrin, IkappaBalpha, and the INK4 family of tumor suppressors p16 and p18. In Specific Aim 3, we plan to determine the structures of SEI-1 and/or its truncated forms possessing different activities, and use site-directed mutagenesis to dissect the residues important for different activities, and to identify CDK4 residues important for binding of SEI-1 and different functional domains of SEI-1.