Structure-Function Relationship of Tumor Suppressors

描述（由申请人提供）：在上一个资助期内，我们获得了重要的发现，表明四种重要蛋白gankyrin， IkappaBalpha， Tax和SEI-1中的每一种都与周期蛋白依赖性激酶4 （CDK4）相互作用，可能具有不同的机制。下一个授权期的重点是了解CDK4与三种组成表达的人类蛋白gankyrin、IkappaBalpha和SEI-1之间相互作用的结构、生化机制和生物学功能，而病毒蛋白Tax不包括在本次修订的提案中。将使用三种主要方法：(a)使用核磁共振来确定溶液结构；(b)利用合理诱变结合活性测定来剖析特异性的结构基础；(c)利用荧光结合法测定gankyrin、IkappaBalpha和SEI-1与CDK4的Kd。在Specific Aim 1中，我们计划使用位点定向诱变来鉴定gankyrin与CDK4结合的重要关键残基，并且将使用基于荧光的测定来评估gankyrin和突变体的Kd值。随后，通过合理设计生成一组CDK4突变体，用于绘制参与gankyrin结合的CDK4残基。此外，与C. Weghorst合作，将利用核磁共振和诱变数据生成gankyrin/CDK4结构模型，我们将表征从癌症组织中发现的gankyrin突变体。在Specific Aim 2中，我们计划解决IkappaBalpha的cdk4结合域（残基1-206）的溶液结构，这也是一种锚蛋白重复序列蛋白。与gankyrin-CDK4相互作用相同的诱变研究将用于绘制IkappaBalpha和CDK4之间的相互作用。这些结果将允许比较gankyrin， IkappaBalpha和INK4家族的肿瘤抑制因子p16和p18。在Specific Aim 3中，我们计划确定SEI-1和/或其具有不同活性的截断形式的结构，并使用位点定向诱变来解剖对不同活性重要的残基，并鉴定对SEI-1结合和SEI-1的不同功能域重要的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.