REGULATION OF P27 AND ITS ROLE IN G1 PROGRESSION

描述：这是一份研究CDK的申请的重新提交 抑制物p27。P27最初是由 派是博士后研究员。这笔赠款是基于对 可能解释p27正常作用的三个模型中。合而为一 模型中，p27水平提供了抑制G1期的阈值。要激活 G1CDK，细胞周期蛋白-CDK复合体的水平必须高于 P27在细胞中的阈值。在第二个模型中，P27是一个脚手架 哪些细胞周期蛋白和CDK多肽组装在一起。第三种模式 提示p27结合的不同特异性是通过 各种翻译后修饰语。此应用程序建议 测试这些模型的各个方面。在第一个目标中，PI将测试 门限模型的性质。P27蛋白和p27蛋白表达的时间 RNA的表达将在同步的细胞群体中进行研究。 RNA和蛋白质表达的差异之后将是 测定p27的合成和降解速率。接下来， 调节p27与细胞周期蛋白-CDK复合体的结合 通过分析关联时间、p27的相对水平来确定 与Cyclin-CDK蛋白的比较，以及翻译后的变化 修改。然后调节p27相关蛋白的活性 将会被确定。解释这一活动的激酶，即 活性与组装的时间，以及p27底物的范围 将会被确定。使用这一特征进行比较， 第二个具体目标是分析p27在不同的生长和 差异化系统。国际和平研究所建议研究 蛋白质合成和降解率，如果p27相关的激酶仍然 是否存在于非增殖细胞中，以及p27的特性是否在 对差异化试剂的直接反应。下一个类比 分化和生长停滞程序将通过p27- 空ES单元格，以确定这些更改是否需要p27。在 最终目标是，PI将分析细胞中与p27相关的蛋白质 通过免疫沉淀和双杂交分析。以前的工作已经表明 P27可以在一个大分子量复合体中分离出来。这个 对这种复合体的表征将在增殖和 分化细胞。

DESCRIPTION: This is a resubmission of an application to study the CDK inhibitor p27. p27 was originally identified and characterized by the PI while a post-doctoral fellow. This grant is based on the analysis of three models that might explain the normal role of p27. In one model, p27 levels provide a threshold of inhibitor in G1. To activate a G1 CDK, the levels of cyclin-CDK complexes must increase above the threshold of p27 in the cells. In the second model p27 is a scaffold on which cyclin and CDK polypeptides are assembled. The third model suggests that different specificities of p27 binding are achieved by various post-translational modifications. This application proposes to test various aspects of these models. In the first aim, the PI will test the properties of the threshold model. The timing of p27 protein and RNA expression will be studied in synchronized cell populations. Discrepancies of RNA and protein expression will be followed by determining the rates of synthesis and degradation of p27. Next, the regulation of association of p27 with cyclin-CDK complexes will be determined by analyzing timing of association, relative levels of p27 versus the cyclin-CDK proteins, and changes in post-translational modification. Then the regulation of p27 associated kinase activity will be determined. The kinases that account for this activity, the timing of activity versus assembly, and the range of substrates for p27 will be determined. Using this characterization for comparison, the second specific aim will analyze how p27 changes in different growth and differentiation systems. The PI proposes to study differences in protein synthesis and degradation rates, if p27-associated kinases still exist in non-proliferating cells, and whether p27 properties change in direct response to differentiating agents. Next analogous differentiation and growth arrest procedures will be performed with p27- null ES cells to determine if p27 is required for these changes. In the final aim, the PI will analyze cells for proteins that associate with p27 by immunoprecipitation and two-hybrid analysis. Previous work has shown that p27 can be isolated in a large molecular weight complex. The characterization of this complex will be done in both proliferating and differentiating cells.