Structure-Function Relationship of Tumor Suppressors

描述（由申请人提供）：在上一个资助期间，我们获得了重要的发现，表明四种重要蛋白质gankyrin、IkappaB alpha、Tax和SEI-1中的每一种都与细胞周期蛋白依赖性激酶4（CDK4）相互作用，具有潜在的不同机制。下一个授权期的主要重点是了解CDK4与三种组成型表达的人类蛋白质Gankyrin、IkappaB alpha和SEI-1之间相互作用的结构和生化机制以及生物学功能，而病毒蛋白Tax不在此修订提案中。将使用三种主要方法：（a）使用NMR确定溶液结构;（B）使用合理诱变结合活性测定法来剖析特异性的结构基础;（c）使用基于荧光的结合测定法来确定gankyrin、IkappaB α和SEI-1与CDK 4的Kd。在特定目标1中，我们计划使用定点诱变来鉴定gankyrin的关键残基，这些残基对于其与CDK 4的结合是重要的，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，将通过合理设计产生一组CDK4突变体，并将其用于绘制参与gankyrin结合的CDK4残基。此外，与C. Weghorst，我们将描述从癌症组织中鉴定的Gankyrin突变体。在具体目标2中，我们计划解决IkappaB α（残基1 - 206）的CDK4结合结构域的溶液结构，这也是一种锚定重复蛋白。将使用与针对gankenzyme-CDK4相互作用所述相同的诱变研究来绘制IkappaB α和CDK4之间的相互作用。这些结果将允许比较gankyrin、IkappaB α和肿瘤抑制因子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，并使用定点诱变来解剖对不同活性重要的残基，并鉴定对SEI-1结合重要的CDK4残基和SEI-1的不同功能结构域。

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.