FUNCTIONAL ANALYSIS OF CDK INHIBITOR FROM ARABIDOPSIS THALIANA

植物的发育在很大程度上取决于细胞分裂活动，而细胞分裂活动同时受个体发育的遗传程序和环境条件的控制。真核细胞在分裂时会经历一系列的事件，而细胞分裂的正常进行需要被称为细胞周期蛋白依赖性的丝氨酸/苏氨酸蛋白激酶(CDKs)的协调激活。CDK与调控亚单位细胞周期蛋白结合，细胞周期蛋白推动细胞分裂周期的进程。CDK的活性受多种机制的控制，包括与细胞周期蛋白的结合和CDK激活激酶的磷酸化。此外，一类被称为CDK抑制剂(CKIs)的低分子蛋白质通过与CDK单体或细胞周期蛋白/CDK复合体的物理结合来抑制CDK活性。在哺乳动物中，根据作用方式和序列相似性可以区分两个不同的CKI家族：INK4和KIP/CIP家族。Kip/Cip家族由三个基因产物组成：p21^lt；cip1&gt；，p27^lt；kip1&gt；和p57^lt；kip2&gt；这些CKI结合到细胞周期蛋白/CDK复合体上。在植物中，已发现7个基因，命名为KIP相关蛋白(KRP)，这些基因与哺乳动物的p27^&lt；kip1&gt；序列具有相似性。体外结合实验表明，除KRP7外，所有krp均与细胞周期蛋白/CDK复合体相互作用，但没有一种krp单独与细胞周期蛋白和CDK蛋白相互作用。我们已经从大肠杆菌中纯化了三个重组Krp蛋白。这些蛋白对从昆虫细胞中纯化的拟南芥细胞周期蛋白D2/CDKA复合体的蛋白激酶活性具有不同的抑制作用，且具有不同的剂量依赖性。

The development of plants depends largely on the cell division activity that is simultaneously controlled by the inherited program of ontogenesis and by environmental conditions. Eukaryotic cells undergo the sequential series of events when they divide, and the proper progression of cell division through the cell cycle requires the coordinated activation of serine/threonine protein kinases called cyclin-dependent kinases (CDKs). The CDKs bind to regulatory subunit, cyclins, which drive progression through the cell-division cycle. CDK activity is controlled by a variety of mechanisms, including binding to cyclins and phosphorylation by the CDK-activating kinase. In addition, a family of low-molecular-weight proteins, designated CDK inhibitors (CKIs), inhibits CDK activity by physically association with the monomer of CDK or the cyclin/CDK complexes. In mammals, two different CKI families can be distinguished on the basis of their mode of action and sequence similarity: the INK4 and the Kip/Cip families. The Kip/Cip family comprises three gene products: p21^<cip1>, p27^<kip1>, and p57^<kip2>. These CKIs bind to the cyclin/CDK complexes. In plants, seven genes, designated Kip-related proteins (KRPs), have been found in Arabidopsis and these genes share sequence similarity with the mammalian p27^<kip1>.In this study, the biochemical analysis of KRP proteins was conducted to characterize their functions. With the exception of KRP7, all KRPs interact with cyclin/CDK complexes but none interact with cyclin and CDK protein alone using in vitro binding assay. We have purified three recombinant KRP proteins from E. coli. These proteins have inhibitory property on the kinase activity of Arabidopsis CyclinD2/CDKA complex, which is purified from insect cells, in a different dose-dependent manner.