DEREGULATING CYCLIN DEPENDENT KINASE

细胞周期蛋白依赖性蛋白激酶(CDK)在所有真核生物的细胞周期调控中起着至关重要的作用。CDK受细胞周期蛋白结合的严格调控，因此细胞周期蛋白的可获得性是调控细胞周期进程的主要手段之一。这项提议是为了让实验来检验放松对这一体系的管制的后果。在一组实验中，提出了一种策略，用于突变激活萌芽酵母CDK CDC28，而不依赖于细胞周期蛋白结合的要求。实现这一目标(已取得重大进展)将使我们能够剖析周期蛋白在简单酶激活中的作用，而不是将酶定位于特定底物。在第二组实验中，我们建议检查使特定的B型细胞周期蛋白/CDC28复合体Clb5-CDc28的水平和活性在细胞周期中构成的后果。尽管目前的模型预测，由于干扰了DNA复制起始点的功能，这应该会使细胞周期停止在G1期，但我们的结果表明，情况可能并非如此。我们建议通过细胞周期来表征Clb5降解的控制，以及Clb5破坏盒在其降解中的作用。我们提出实验来验证这一假设，即Clb5是S阶段进入的固有专门化，而另一种B型周期蛋白Clb2是进入有丝分裂的固有专门化的。这一假设与目前的模型不同，在现有模型中，所有B型周期蛋白都同样能够对DNA复制进行正向和负向调节。

Cyclin-dependent kinases (Cdk's) are critical in regulation of the cell cycle in all eukaryotes. Cdk's are stringently regulated by cyclin binding, and cyclin availability thus is one of the main means by which cell cycle progression is regulated. This proposal is for experiments to examine the consequences of deregulation of this system. In one set of experiments, a strategy is proposed for mutational activation of the budding yeast Cdk Cdc28 independent of the cyclin binding requirement. Achievement of this goal (towards which significant progress has been made) will allow dissection of the roles of cyclins in simple enzyme activation as opposed to targeting of the enzyme to specific substrates. In a second set of experiments, we propose examining the consequences of making the level and activity of a particular B-type cyclin/Cdc28 complex, Clb5-Cdc28, constitutive through the cell cycle. Although current models predict that this should halt the cell cycle in G1 phase due to interference with function of origins of DNA replication, our results suggest that this may not be the case. We propose to characterize control of Clb5 degradation through the cell cycle, and the role of the Clb5 destruction box in its degradation. We propose experiments to test the hypothesis that Clb5 is intrinsically specialized for S phase entry while another B-type cyclin, Clb2, is intrinsically specialized for entry into mitosis. This hypothesis is in contrast to current models in which all B-type cyclins are equally capable of both positive and negative regulation of DNA replication.